USF Libraries
USF Digital Collections

Human umbilical cord blood cells migration to stroke cns tissue extracts and the potential cytokines and chemokines involved

MISSING IMAGE

Material Information

Title:
Human umbilical cord blood cells migration to stroke cns tissue extracts and the potential cytokines and chemokines involved
Physical Description:
Book
Language:
English
Creator:
Newman, Mary B
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Ischemic
Chemoattractants
Stroke
Transplantation
Stem cells
Dissertations, Academic -- Psychology -- Doctoral -- USF   ( lcsh )
Genre:
government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Human umbilical cord blood (HUCB) cells consist of a heterogeneous population of cells, rich in hematopoietic stem and progenitor cells. These cells have been used in the treatment of various nonmalignant and malignant hematopoietic diseases. With in the last few years HUCB cells have been used in pre-clinical animal models of brain and spinal cord injuries, in which functional recovery has been shown. The properties of cord blood cells that could be important in cell transplantation (repair or replacement) of CNS injury or disease are currently being evaluated. The major focus of this study was to determine whether HUCB cells would migrate to ischemic tissue extracts. In addition, factors that may be inducing the cells to migrate were examined by identifying the cytokines or chemokines present in the ischemic tissue extracts.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2005.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Mary B. Newman.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 187 pages.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001670344
oclc - 62286362
usfldc doi - E14-SFE0001206
usfldc handle - e14.1206
System ID:
SFS0025527:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam Ka
controlfield tag 001 001670344
003 fts
005 20051216093305.0
006 m||||e|||d||||||||
007 cr mnu|||uuuuu
008 051116s2005 flu sbm s000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0001206
035
(OCoLC)62286362
SFE0001206
040
FHM
c FHM
049
FHMM
090
BF121 (Online)
1 100
Newman, Mary B.
0 245
Human umbilical cord blood cells migration to stroke cns tissue extracts and the potential cytokines and chemokines involved
h [electronic resource] /
by Mary B. Newman.
260
[Tampa, Fla.] :
b University of South Florida,
2005.
502
Thesis (Ph.D.)--University of South Florida, 2005.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
500
Title from PDF of title page.
Document formatted into pages; contains 187 pages.
Includes vita.
3 520
ABSTRACT: Human umbilical cord blood (HUCB) cells consist of a heterogeneous population of cells, rich in hematopoietic stem and progenitor cells. These cells have been used in the treatment of various nonmalignant and malignant hematopoietic diseases. With in the last few years HUCB cells have been used in pre-clinical animal models of brain and spinal cord injuries, in which functional recovery has been shown. The properties of cord blood cells that could be important in cell transplantation (repair or replacement) of CNS injury or disease are currently being evaluated. The major focus of this study was to determine whether HUCB cells would migrate to ischemic tissue extracts. In addition, factors that may be inducing the cells to migrate were examined by identifying the cytokines or chemokines present in the ischemic tissue extracts.
590
Adviser: Paul R. Sanberg.
Co-adviser: Cheryl L. Kirstein
653
Ischemic.
Chemoattractants.
Stroke.
Transplantation.
Stem cells.
690
Dissertations, Academic
z USF
x Psychology
Doctoral.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.1206



PAGE 1

Human Umbilical Cord Blood Cells Migration To Stroke Cns Tissue Extracts And The Potential Cytokines And Chemokines Involved by Mary B. Newman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Psychology College of Arts and Sciences University of South Florida Co-Major Professor: Paul R. Sanberg, Ph.D. Co-Major Professor: Cheryl L. Kirstein, Ph.D. Alison E. Willing, Ph.D. Gary W. Arendash, Ph.D. Thomas H. Brandon, Ph.D. Date of Approval: June 21, 2005 Keywords: ischemic, chemoattractants, stroke, transplantation, and stem cells Copyright 2005 Mary B. Newman

PAGE 2

Dedication Without these three people I would not be who I am today and I would have never completed my Ph.D. For that, I would like to dedicate this dissertation to my father, my mother, and my sister (Joseph, Geraldine, and Cindy Newman). Each of them, in their unique way, has contributed to the completion of this long and sometime tenuous journey. I owe them for their love, patience, support, and understanding especially my father, who taught me at a very early age that you can do and be whatever you want as long as you work hard and love what you are doing. To my mother who has always been there for me, and to my sister, Cindy, there are no words to describe my feeling for you and how you have helped me. I love you and thank you.

PAGE 3

Acknowledgements Several people assisted me in my journey of achieving this Ph.D. I doubt that any one person can perform such a task without any assistance or guidance from other people. Dr. Paul R. Sanberg, my Major Professor, and mentor provided me with the necessary guidance, assistants, and education in neuroscience to acquire my Ph.D. However, his mentorship went far beyond that and I believe he has prepared me to become an excellent independent scientist and administrator. There is a necessity for todays scientist to understand the politics behind the science especially because of the overlap with biotechnical companies and academia along with the governmental budget cuts in research. In addition, he emphasized the importance of making the transition from basic science to clinic, which becomes important when attempting to achieve independence as an investigator and for obtaining funding. There is much more that he willingly taught me, which is difficult to describe in this brief synopsis. However, for everything he has done I wish to personally thank him and let him know what a differences he has made in my life. When it comes to assistance with science and for her continuous encouragement, Dr. Alison E. Willing wins the award. Although she was a committee member, in which I thank her for, she went above and beyond what the University requires of a committee member. She guided me in every stage of the study design, was there to talk

PAGE 4

with when things were not going well (what a surprise), she has probably read through the dissertation more times than she would like to remember, and has provided me with encouragement and sometimes with the necessary kick to complete the studies. To her, I will be forever grateful. Thank you, Alison. Dr. Samuel S. Saporta, in addition to being my Chairperson, was also there for that much needed kick, provided me with scientific knowledge, support, and his friendship every step of the way. Thank you. There is a special past committee member, who was my Major Professor for my Masters that I would like to acknowledge. Dr. Doug R. Shytle has been both a mentor and dear friend who has supported, listened, and assisted me throughout this process. Thank you. Along these same lines, there is a relatively new committee member and my Co-Major Professor who assisted me during a time of great difficulty. Dr. Cheryl Kirstein, graciously volunteered to step-in and become my Co-Major Professor after the death of Dr. Thomas Tighe, who also deserves a special acknowledgement. Those of you who know the responsibilities that come with being a major professor and the problems that often come with finding someone who is willing to take on this responsibility will understand this unselfish act. To Cheryl, thank you and may I someday pass along this goodwill to someone else in need. I would also like to acknowledge and thank, committee member Dr. Gary W. Arendash, who holds the record for being on my committee the longest (Honors Thesis, Master Thesis and now the Dissertation). He has also provided support and scientific guidance to me throughout the years. Dr. Thomas Brandon also deserves acknowledgement and my thanks for his willingness to go outside his area of expertise and for being my one clinical psychology committee member.

PAGE 5

In addition, there are several other people that considerably helped me along the way. John Manresa, who after volunteering and working with me for over 4 years has finally earned his B.A. and B.S. He came to me as an undergraduate who wanted to obtain some research experience, stayed with me, and has been a tremendous help in accomplishing this dissertation. There is also my brain homogenizing assistant Donna Morrison, who is actually the office manger for the Center and Dr. Sanbergs administrative assistant, but has been a dear close friend and did whatever was needed to help me. I thank her for her friendship, love, and dedication. I will never forget you and the assistance you extended to me in completing this dissertation. Finally, I would like to thank Drs. Svitlana Garbuzova-Davis and Paula Bickford, who were my confidants and shared their vast knowledge of science with me.

PAGE 6

Table of Contents List of Tables iv List of Figures v Abstract viii Chapter 1: Umbilical Cord Blood (HUCB) Cells in the Repair of Damage in the CNS 1 1.1 Introduction 1 1.2 Brief History of Stem Cells 3 1.3 Advantages of HUCB Cells 7 1.3.1 Easily Obtainable Sources of Cells 7 1.3.2 Abundant Source of Cryopreservable Cells 8 1.3.3 Low Incidence of GvHD Following Transplantation of Cord Blood Cells and Their Immune Immaturity 10 1.3.4 HUCB Cells Have Been Safely Transplanted Clinically for Over 15 Years 12 1.4 Phenotyping of HUCB Cells 13 1.5 In Vitro Studies of HUCB Cells 16 1.6 Transplantation Studies Utilizing HUCB Cells 18 1.6.1 Normal Developing Brain 21 1.6.2 Stroke 22 1.6.3 Traumatic Brain Injury 26 1.6.4 Spinal Cord Injury 28 1.6.5 Amyotrophic Lateral Sclerosis 29 1.7 HUCB Stem Cells for Gene Therapy 31 1.8 Clinical Studies using HUCB Cells 34 1.8.1 Transplantation of HUCB Cells into Patients with Inborn Errors in Metabolism 34 1.9 Conclusions Thus Far 36 1.10 Explanation of Study and Overall Purpose 36 1.11 References 38 Chapter 2: Chemotactic Assay and Cell Migration 48 i

PAGE 7

2.1 Introduction 48 2.2 Background on Chemotactic Assay and Cell Migration 50 2.3 Migration Induced by Inflammation 52 2.4 Summary 56 2.5 Reference 57 Chapter 3: Progression of the Study Design, Disease Models, and Preliminary Data 61 3.1 Progression of the Study Design 61 3.2 Background and Preliminary Data 64 3.2.1 Migration Apparatus 65 3.2.2 Cell Diameter of HUCB Cells 66 3.2.3 HUCB Cells in Culture 68 3.2.3.1 Preparation of HUCB Cells 68 3.2.3.2 Phenotype of HUCB Cells 68 3.2.3.3 Morphology of HUCB Cells 70 3.2.3.4 Live/Dead Assay of HUCB Cells 72 3.2.3.5 Proliferation Assay of HUCB Cells 74 3.2.4 Tool to Measure Migrated HUCB Cells and Control Selection 75 3.2.5 Protein Assays for Tissue Extracts and Media from HUCB Cells 79 3.3 Migration of HUCB Cells and Animal Models of Disease or Injury 81 3.3.1 Basic Migration Assay Procedure 81 3.3.2 Preparation of Tissue Extracts 82 3.3.3 Animal Models Used for HUCB Cell Migration 83 3.3.3.1 Neonatal Young Rat Model 83 3.3.3.2 Parkinsons Disease 6-OHDA Rat Model 86 3.3.3.3 Sanfilippo Mouse Model 91 3.4 Summary 95 3.5 References 97 Chapter 4: Stroke Induced Migration of Human Umbilical Cord Blood Cells: Time Course and Cytokines 100 4.1 Abstract 100 4.2 Introduction 101 4.3 Materials and Methods 104 4.3.1 Animal Care and Handling 104 4.3.2 Middle Cerebral Artery Occlusion Surgery 104 4.3.2.1 MCAO Behavior Test 105 4.3.3 Preparation of Brain Tissue Extracts 105 4.3.4 Protein Assay 106 4.3.5 Preparation of HUCB Cells 106 4.3.6 Migration Assay 107 4.3.6.1 Controls 107 4.3.6.2 Standard Curves 108 4.3.7 Rat Cytokine Array 108 ii

PAGE 8

4.3.8 ELISAs for RAT GRO/CINC-1 and MCP-1 in Ischemic Tissue Extracts 109 4.3.9 Statistical Analysis 109 4.4 Results 110 4.4.1 Migration of HUCB Cells to Ischemic Tissue Extracts 110 4.4.2 Rat Cytokine Array 116 4.4.3 Rat ELISAs 117 4.5 Discussion 123 4.6 Reference 130 Chapter 5: Cytokines Produced by Cultured Human Umbilical Cord Blood Cells 134 5.1 Abstract 134 5.2 Introduction 135 5.3 Materials and Methods 137 5.3.1 Preparation of HUCB Cells 137 5.3.2 Human Cytokine Array Protocol 137 5.3.2.1 Human Cytokine Array Time Response 138 5.3.2.2 Human Cytokine Array Seeding Density 138 5.3.2.3 Human Cytokine Array Hematopoietic Media and Stimulants 139 5.3.3 ELISA for Human IL-8 and MCP-1 in Culture HUCB Cells 139 5.3.4 Migration Assays 140 5.3.5 Statistical Analysis 141 5.4 Results 142 5.4.1 Human Cytokine Array Time Response 143 5.4.2 Human Cytokine Array Seeding Density 145 5.4.3 Human Cytokine Array Hematopoietic Media and Stimulants 146 5.4.4 ELISA Results for Human IL-8 and MCP-1 in CM From HUCB Cells 149 5.4.5 Migration Assays 151 5.5 Discussion 153 5.6 References 158 Chapter 6: Relevance of Dissertation Studies, Conclusions, and Future Directions 162 6.1 Limitations 168 6.2 Future Directions 169 6.2 References 172 About the Author End Page iii

PAGE 9

List of Tables Chapter 1 Table 1.1 Description of Stem Cells 5 Table 1.2 Significant Published CNS Transplantation Studies Using the Mononuclear Fractions of HUCB Cells 20 Chapter 3 Table 3.1 Phenotype of HUCB Cells at 1 to 14 DIV 70 Chapter 4 Table 4.1 Analysis of Variance for HUCB Cells Migration to Cortex 113 iv

PAGE 10

List of Figures Chapter 1 Figure 1.1 HUCB Cells Transplanted into Stroke Rat Brain 25 Figure 1.2 HUCB Cells Transplanted into Spinal Cord Injury in Rats 29 Figure 1.3 HUCB Cells that are Labeled with Green Fluorescent Protein 32 Chapter 3 Figure 3.1 Dissertation Studies 62 Figure 3.2 Depiction of Migration Chamber 65 Figure 3.3 Average Cell Diameters of HUCB Cells 67 Figure 3.4 HUCB Cells Morphology 71 Figure 3.5 FDA/PI Viability of HUCB Cells 73 Figure 3.6 Proliferation of HUCB Cells in Culture 75 Figure 3.7 HUCB Cells Migrating through Membrane 76 Figure 3.8 HUCB Cells Labeled with DAPI 77 Figure 3.9 Luminescent Standard Curve of HUCB Cells 78 Figure 3.10 Standard Curve for Protein Assays 80 Figure 3.11 Migration of HUCB Cells to Neonatal Rat Brain 85 Figure 3.12 Migration of HUCB Cells to 6-OHDA Toxin and SDF-1 89 Figure 3.13 Migration of HUCB Cells to 6-OHDA Animal Model of PD 90 Figure 3.14 Migration of HUCB Cells to Sanfilippo Tissue Extracts 94 v

PAGE 11

Chapter 4 Figure 4.1 Migrated HUCB Cells in Striatal Ischemic Tissue Extracts 111 Figure 4.2 Migration of HUCB Cells to Stroke Tissue Extracts 114 Figure 4.3 Rat Ischemic Extracts Cytokine Array 116 Figure 4.4 Levels of GRO/CINC-1 in Ischemic Extracts 118 Figure 4.5 Levels of MCP-1 in Ischemic Extracts 121 Chapter 5 Figure 5.1 Cytokine Produced by HUCB Cells 10 and 28 DIV 143 Figure 5.2 Effects of Seeding Density on Cytokine Secretion 145 Figure 5.3 Effects Hematopoietic Media has on Cytokine Secretion 146 Figure 5.4 Effects on Cytokine Secretion by IL-3 and TPO 148 Figure 5.5 ELISA of IL-8 and MCP-1 from CM of HUCB Cells 150 Figure 5.6 Migration Assay 152 vi

PAGE 12

Human Umbilical Cord Blood Cells Migration To Stroke CNS Tissue Extracts And The Potential Cytokines And Chemokines Involved Mary B. Newman ABSTRACT Human umbilical cord blood (HUCB) cells consist of a heterogeneous population of cells, rich in hematopoietic stem and progenitor cells. These cells have been used in the treatment of various nonmalignant and malignant hematopoietic diseases. With in the last few years HUCB cells have been used in pre-clinical animal models of brain and spinal cord injuries, in which functional recovery has been shown. The properties of cord blood cells that could be important in cell transplantation (repair or replacement) of CNS injury or disease are currently being evaluated. The major focus of this study was to determine whether HUCB cells would migrate to ischemic tissue extracts. In addition, factors that may be inducing the cells to migrate were examined by identifying the cytokines or chemokines present in the ischemic tissue extracts. The secondary focus was to establish whether cultured HUCB cells are releasing cytokines and chemokines (in vitro) in response to their environment. The results of these studies showed that HUCB cells migrate to ischemic tissue in a time dependent manner. In which there is a 48 to 72 hour window of opportunity for the delivery of HUCB cells to the ischemic brain. In addition, the cord blood cells were shown to release cytokines and chemokines that may be aiding in the behavioral recovery seen in the transplantation vii

PAGE 13

studies. The results from this study are promising in that the current 3-hour therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of cord blood cell therapy with the peak at 48 hours. viii

PAGE 14

Chapter 1 1.0 Umbilical Cord Blood (HUCB) Cells in the Repair of Damage in the CNS 1.1 Introduction Numerous CNS diseases are characterized by the deterioration of cognitive and motor functions, frequently leading to prolonged periods of increasing incapacity. Among the most problematic and prevalent neurological disorders are those associated with the loss of CNS cells populations, such as Alzheimers (AD), Parkinsons (PD), Huntingtons disease (HD), amyotropic lateral scelerosis (ALS), and stroke. While advances in molecular biology, genetic engineering, proteomics, and genomics will hopefully produce compounds with enormous treatment potential in the future, currently there are no available means of altering the pattern, rate of cell loss, or function in diseased or injured CNS once damage has occurred. For several decades, researchers and clinicians have been exploring the potential of cellular treatment for diseases, disorders, and injuries of the CNS. Fetal neural tissue was initially grafted into the brain in order to study its development and regenerative capacity. These studies quickly revealed the therapeutic potential of tissue grafting in animal models of neurological disorders, which eventually led to pre-clinical and then clinical trials. The outcomes of clinical transplantation studies were generally positive enough to warrant further investigation. However, the lack of significant improvement 1

PAGE 15

reported in the older population or late stage of disease in patients with Parkinsons disease has brought some doubt and uncertainty to the future direction of the neural transplantation field (Newman et al 2003, Sanberg et al 2002) In addition, societal, legal, and ethical issues, along with the limited availability of tissue, complicate the use of fetal tissue. These factors have not only pushed investigators to look for alternative sources of cells, but also alternative ways in which cells may be utilized in cellular therapies. This is supported by the growing consensus that stem cells are obtainable from several sources and can be epigenetically expanded or genetically perpetuated to develop, if needed, into specific cellular phenotypes or to deliver needed substrates/factors from genetically engineered cells. One of the most promising sources for human stem cells is those cells from hematopoietic origin, which include HUCB, adult peripheral blood, and bone marrow. Each has their own sets of distinct advantages and disadvantages and a comprehensive review of each source is beyond the scope of this dissertation. This chapter will focus on the utilization of HUCB cells as a potential source for cellular therapies, the clinical and pre-clinical studies that have been preformed, and a general description of research undertaken for this dissertation. Stem cells derived from hematopoietic sources, such as HUCB, are an emerging and relatively young area of research that may provide the opportunity to further our understanding of the extent and limitations in the use of stem cells in cellular therapy field. 2

PAGE 16

1.2 Brief History of Stem Cells Most stem cell biologists would agree that Artur Pappenheim (1870 1916) was the first to propose the concept of a common ancestral stem cell (Lajtha 1980), 1980). Till and McCulloch (1961) were the first to demonstrate the ability of transplanted bone marrow cells to form colony-forming units (CFU) on the spleen of lethally irradiated mice in vitro. Their initial studies, measured the proliferation potential of bone marrow cell colonies, and led to the developing hypothesis of hematopoietic stem cells (Potten 1983, Till & McCulloch 1961). Since then much confusion has occurred in the meaning and usage of the words stem, progenitor, and precursor cells. In an attempt to unify terminology in this field, a workshop in 1978 comprised mostly of hematologists (Lajtha 1979a, Lajtha 1979b, Schofield & Lajtha 1983), defined stem cells as cells with extensive self-maintaining (self-renewal capacity), extending throughout the whole (or most) of the life-span of the organism. Differentiation potential is a property of some types of stem cells but is not an essential feature of stem-ness. Differentiation has been defined as cells becoming specialized for particular functions (Lackie & Dow, 1999) and as the change in genetic expression patterns (Lajtha 1979a). Therefore, any cell that replicates its own numbers throughout most of the organisms life is considered a stem cell, regardless of whether the cell differentiates further. As Lajtha (Lajtha 1979b) has pointed out, every cell in the body except for the zygote is already differentiated, the question is whether a cell is capable of further differentiation (Lajtha 1979b). More recently, however, other scientists, mostly in the neuroscience and transplantation field, have defined a stem cell as an undifferentiated cell that is capable of self-renewal, proliferation, and can asymmetrically divide to generate differentiated 3

PAGE 17

cells or multipotent cells (Rao & Mattson, 2001; Shihabuddin, et al. 1999; Gage, 2000). At this time there seems to be a consensus regarding the terminology stem cells, although there has been no formal establishment of a nomenclature. Table 1.1 presents a hierarchical listing along with defined terminology of stem cells, which has been incorporated from several scientific publications. Please refer to the following papers for their discussion of this topic: (Blau et al 2001, Marshak et al 2001, National Research Council (U.S.). Committee on the Biological and Biomedical Applications of Stem Cell Research., Rao 2001a, Rao & Mattson 2001, Rao 2001b). Totipotent stem cells are of interest due to their ability to generate any cell type from both ectodermal and extraectodermal tissue. Pluripotent stem cells can give rise to cells derived from the three embryonic germ layers, however, they are more restricted, produce committed progenitor and precursor stem cells of more than one lineage or cell type, and can self-renew. Cell lineage can be described as the embryonic origins of specific cells, which can be recorded through fate maps that trace the cell(s) embryonic origin through developmental stages. Accordingly, a stem cell that is more ancestral or primitive (higher in the hierarchy) is of greater interest due to the cells ability to generate progenitors of more than one lineage. The progenitor and precursor cells are more limited (more restricted) than stem cells in their ability to give rise to cells of a different lineage. 4

PAGE 18

Table 1.1: Description of Stem Cells Primitive Stem Cells Totipotent early blastula stage cells, giving rise to both ectodermal and extraectodermal tissue. Stem Cells A cell that is capable of proliferation, self-renewal, self-maintaining during the life of the organism is a stem cell. The cell may or may not produce cells that are further differentiated. Ancestral Cell stem cells that yield further differentiated descendants. Pluripotent Stem Cells These cells originate from the inner cell mass after development of the blastula, they give rise to cells from the three embryonic germ layers (mesoderm, endoderm and ectoderm). However, they cannot give rise to the tissue of the placenta (this is restricted to cells from the trophectoderm). They can differentiate into germinal and somatic tissues. Multipotent, Bipotent and Unipotent these are used to describe stem cells that are further differentiated daughter cells of pluripotent or totipotent stem cells. They may produce daughter cells that are further differentiated. However, they are restricted in the ability to give rise to other cells of different lineages. Progenitor/Precursor Cells These are the progeny of stem cells, and have a much more restricted differentiation potential with limited self-renewal capacity, and a limited lifespan. These cells cycle more than stem cells and provide the necessary cells for self-maintenance of the organism, thus conserving the stem cell. Table 1.1 Presents an interpretation of the hierarchy and increased complex terminology of stem cells. The definitions are a compilation from several sources as referenced in the paper. Both the progenitor and precursor cells (also referred to as transit amplifying cells) are the progeny of more ancestral stem cells and seem to be responsible for the ongoing regeneration of the given tissue in which they reside; allowing the more 5

PAGE 19

primitive stem cells to proliferate only as necessary (Marshak et al 2001). This concept is in agreement with the slow cycling observed in more primitive stem cells, which has been proposed as a way to protect the integrity of the genome (Cairns 1975, Potten 1983). Thus, the fewer cell cycles, the less chance there is of disrupting or damaging the genome. The endogenous factors that trigger proliferation and differentiation of stem cells and their progeny are just beginning to be resolved. The proliferation, expansion, and differentiation of stem cells are regulated through intrinsic cellular factors, extrinsic cellular factors (cytokines/growth factors, cell adhesion molecules), and cell-to-cell interaction (Rao & Mattson 2001). For example, embryonic stem cells can be maintained in vitro in the presence of Leukemia Inhibitory Factor (LIF) and these cells will produce neural stem cells when supplemented with fibroblast growth factor (FGF) in culture (Tropepe et al 2001). Both epidermal growth factor (EGF) and basic fibroblast growth factors (FGF2) will keep neural stem cells in a proliferative state (Gritti et al 1999), while brain derived neurotrophic factor (BDNF) will direct neuronal progenitors to differentiation (Eaton & Whittemore 1996). Some stem cells have been reported to transdifferentiate; that is to give rise or transit to a cell with properties distinct from the germinal layer in which they originated. Kondo and Raff (2000) have shown that oligodendrocyte progenitor cells may transdifferentiate to pluripotent stem cells and give rise to neurons(Kondo & Raff 2000). However, caution should be employed when determining the ability of a cell to transdifferentiate. Progenitor cells that were obtained from muscle and thought to be of muscle origin were reported to transdifferentiate into hematopoietic stem cells. Upon further investigation, these stem cells were, in fact, 6

PAGE 20

determined to be hematopoietic stem cells residing within the muscle (Ogawa et al 2002). While it is possible to direct the differentiation of pluripotent stem cells and lineage restricted progenitor cells in vitro, we are only at the initial stages of learning how to direct stem cells to phenotypes that might be useful in vivo. 1.3 Advantages of HUCB Cells Many fundamental observations have already been made in the use of cord blood cells as transplantable cells for treating CNS diseases. Those observations are discussed here. 1.3.1 Easily Obtainable Source of Cells HUCB is a well-established, reliable source of a heterogeneous population of cells, which is known to be rich in hematopoietic stem and progenitor cells (Broxmeyer 1996, Mayani & Lansdorp 1998). Additionally, millions of healthy babies are born each year leading to virtually an unlimited supply of HUCB cells that can be easily preserved and available for transplantation at any time. Cord blood is easily obtained and routinely collected without jeopardizing the mother or infant (National Center For Farmworkers 2002). Cord blood can be collected either in utero or ex utero. Typically, cord blood is collected after the placenta has been delivered (ex utero which does not endanger mother or infant) in a closed system to prevent contamination, which utilizes either placental collection bags or blood collection bags that contain the anticoagulants. The placenta is suspended in a specially designed scaffold enabling the cord to hang down and causing enlargement of the cord. This permits easier access in which to cannulate the cord vein and allow the blood to drain. Standard procedures are used to process and 7

PAGE 21

test the isolated cord blood to ensure the absence of infectious agents such as HIV and hepatitis prior to cryopreservation. In addition, cord blood cells are available from every ethnic group for human leukocyte antigen (HLA) matching. The cells that are isolated from the cord blood have the advantage of being immunologically immature (discussed below) and thus are less likely to rejected by the host.. Obviously, the ethical issues associated with using cells or tissues from embryos or aborted fetuses are obviated, removing the arguments that a potential life is being sacrificed for the advancement of science or that the use of aborted fetuses influences or justifies the decision to have an abortion. 1.3.2 Abundant Source of Cryopreservable Cells The yield of hematopoietic stem cells is relatively high with a greater potential for proliferation and expansion when compared to those of adult bone marrow (Cardoso et al 1993a, Cardoso et al 1993b, Hows et al 1992). For example, a higher percentage of cluster differentiation (CD)34 + CD38 cells are present in HUCB; 4% fraction when compared 1% bone marrow (Broxmeyer et al 1992b, Cardoso et al 1993b, Metcalf 1984). In addition, cord blood contains CD133 positive cells, which are a subpopulation of CD34 cells and are believed to be more primitive stem cells (Miraglia et al 1997, Potgens et al 2001, Yin et al 1997) (see section on phenotypes for further discussion). The advantages of a high yield of stem and progenitor cells dovetails nicely with the ability to cryopreserve cord blood cells for indefinite period, which is an essential prerequisite for any cell-based therapy. With proper handling and processing cryopreservation does not materially affect the viability of the stem or progenitor cells obtained from HUCB. Cells 8

PAGE 22

from cord blood frozen maintain their viability and proliferation capacities for as long as 15 years and are no different than HUCB cells stored for shorter periods (Broxmeyer & Cooper 1997, Broxmeyer et al 2003). For example, cells that were magnetically separated with CD34 antibody and cryopreserved for 15 years were infused into irradiated NOD/SCID mice. After 11-13 weeks in vivo the cells expressed multi-lineage phenotypes that were comparable to those obtained using freshly harvested cord blood (Broxmeyer et al 2003). More importantly, cryopreserved cord blood stored up to 4 years has been transplanted successfully in allogeneic sibling that had malignant or non-malignant diseases (Broxmeyer 1998b, Wagner et al 1992). Cryopreservation may actually confer certain advantages, in gene therapy, relative to fresh, non-cryopreserved cord blood. Cord blood cells that have been cryopreserved transduce retroviral vectors more easily than fresh cells, and this has been suggested to be the result of higher expression of amphotropic retrovirus receptor mRNA in the cryopreserved cord blood cells (Orlic et al 1999a, Orlic et al 1999b). The easy transduction of retroviral vectors and the high expression of amphotropic retrovirus receptor on HUCB cells may suggest a role for these cells in gene therapy, especially since, amphotropic retrovirus receptors are the most widely used receptors for the transduction of gene vectors currently. This area is further discussed in section HUCB Stem Cells for Gene Therapy. 9

PAGE 23

1.3.3 Low Incidence of GvHD Following Transplantation of Cord Blood Cells and Their Immune Immaturity In children that were transplanted with HUCB cells from related or unrelated donor with one HLA-mismatch there was a low incidence of graft-vs-host disease (GvHD) (Gluckman 2001, Rocha et al 2001, Rocha et al 2000; Wagner et al 1995). This suggests that the host antigen-presenting cells did not recognize these transplanted cells as foreign. The immaturity of cord blood cells has been postulated as the reason for this low rejection rate. Several converging lines of evidence support this notion. One way to study this area of immune immaturity is to compare the phenotypes and functionality of T-lymphocytes in cord blood to that of mature T-cells such as those from adult blood (for review on this subject see Fallen & Cohen 2000). Most T cells from cord blood express CD45RA (nave isoform) with a few expressing CD45RO (mature memory isoform), whereas in adult blood the opposite of this is true with few CD45RA and more CD45RO cells (Beck & Lam-Po-Tang 1994, Han et al 1995). Also in cord blood there are fewer T cells that express CD29 compared to adult blood T cells (Rabian-Herzog et al 1993). In the adult, CD29 recognizes T cells (CD4 positive memory cells) that have been stimulated by specific antigen, therefore the lack of CD29 expression in cord blood T cells is further evidence of their nave state. In addition, the expression of the receptor subunit for IL (CD25), a potent cytokine that acts as a major T cell growth factor, is markedly lower in cord blood cells than in adult blood cells (Cohen et al 1999). Moreover, in cord blood the functional response of T cells is less than those cells in the adult blood. For instance, approximately 30% of CD45RA T cells from neonates produce CD40 ligand (CD40L) whereas over 80% of T cells in adult blood have been 10

PAGE 24

shown to produce this ligand (Fallen & Cohen 2000, Nonoyama et al 1995). CD40L plays a role in the maturation of B cells and it has been proposed that the lack of CD40L production in T cells contributes to the immaturity of B cells in cord blood. This coincides with B cell production of IgG or IgA, which is less in the blood of newborns compared to adult blood (Miyawaki et al 1981, Nagaoki et al 1981). Furthermore, the production of certain cytokines, such as IL-2, IL-3, and IFN, is less in T cells from cord blood than adult blood (for review see Fallen, 2000). A second line of evidence is based on telomeric analysis of HUCB cells. Cellular division is associated with a sequential loss of telomeric DNA leading to cellular senescence. Adult bone marrow cells have inherently shorter telomeres than cord blood cells, providing circumstantial evidence that HUCB cells are immature(Vaziri et al 1994). Together this evidence strongly supports the proposition that HUCB cells are immune-immature and further suggests the immune cells within cord blood are functionally and phenotypically at an early stage of development. If additional studies confirm that HUCB cells are indeed in an immature state at the time of delivery and shortly thereafter, this raises the clinically important possibility that immunosuppressive therapy may not be necessary at the time of transplantation. The ability to avoid immunosuppression is especially important for children who are victims of stunted growth, when have increased risk of malignancy resulting from systemic immunosuppressant therapy. 11

PAGE 25

1.3.4 HUCB Cells Have Been Safely Transplanted Clinically for Over 15 Years Systemic administration of HUCB cells has a long standing and successful history in the hematopoietic field and is commonly used to treat various nonmalignant and malignant hematopoietic diseases (for review see Broxmeyer 2000, Lu et al 1996). The first documented HUCB transplant with an HLA-matched sibling was performed in 1988 on a child with Fanconis anemia. The procedure was well tolerated and, at last report, the patient had survived for more than nine years without disease reoccurrence (Broxmeyer 2000, Gluckman et al 1989). Concurrent pre-clinical studies demonstrated that the bone marrow of lethally irradiated adult mice could be fully rescued/reconstituted by injections of cord blood from pre-term and term deliveries (Broxmeyer 1998a, Broxmeyer et al 1991). Since these initial reports, over 850 cord blood transplants have been performed in children (NMDP, 2003) using either complete or partial HLA-antigen matches for the treatment of nonmalignant and malignant hematopoietic diseases (for reviews see Broxmeyer 1998a, Lu et al 1996). HUCB has also been successfully transplanted into adult recipients although the number of transplants are fewer than those performed on children (Gluckman et al 1997, Laughlin et al 2001). Fewer adult transplants have been performed because of the open question concerning the appropriate dosage of HUCB cells. It is unknown whether a single collection or sample of cord blood contains enough stem cells to repopulate the adult hematopoietic system. Despite the need for continued clinical evaluation to address this issue, HUCB cells from a single donor have been successfully used. Interestingly, the number of cord blood cells infused in these studies has often been less 12

PAGE 26

than the number of bone marrow cells typically used (Gluckman et al 1999a, Gluckman et al 1999b) further supporting the supposition that the number of hematopoietic stem cells residing in cord blood is sufficient for improved clinical outcome. These suppositions from the clinical data are supported by quantitative in vitro evidence in which the number of myeloid progenitor cells supplemented with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs) were analyzed. The result suggested that a single collection of cord blood contains a sufficient number of cells for engraftment and repopulation of the adult hematopoietic system (for review see Broxmeyer et al 1992a). 1.4 Phenotyping of HUCB Cells Careful and accurate phenotyping of cells within a heterogeneous substrate such as cord blood is essential for full realization of the clinical potential. Clonogenic assay, flow cytometry, and the expression of CD antigens coupled with self-renewal proliferation assays have historically been used to delineate hematopoietic stem and progenitor cells from the other cellular components of cord blood. Clonogenic assays have identified several colony-forming units, such as, erythroid burst forming units (BFUe), granulocyte-macrophage colony-forming cells (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony forming units (CFU-GEMM) (Broxmeyer & Carow 1993, Broxmeyer et al 1989). Phenotypic markers that recognize cell surface components, such as CD34 (a type I transmembrane glycophosphoprotein), are routinely used to identify and sort cells. CD34 is believed to identify hematopoietic stem cells, but the expression of this protein alone does not reliably distinguish hematopoietic 13

PAGE 27

stem cells from progenitor cells. For example, the heterogeneous population of cells in cord blood consist of several lineage committed cells that express CD34: myeloid lineage cells that consist of mononuclear phagocytes and granulocytes co-express CD34 and CD33 (marker for myeloid progenitor cell); erythroid lineage cells co-express CD34 and CD71 ( a proliferating cell marker); lymphoid lineage cells can co-express CD34 with either CD19 (B cells) or CD7 (T cells); and megakaryocyte lineage cells co-express CD34 and CD61 (platelets, megakaryocytes) (for review see Kinniburgh & Russell 1993). A more reliable discriminatory technique to isolate and identify hematopoietic stem cells uses combinations of cell surface markers, CD antigens. For example, cells that are CD34 + CD38 CD90 low CD117 low CD135 + or HLA-DR are younger cells and are higher in the hierarchy of hematopoietic stem and progenitor cells (Broxmeyer 1995, Cardoso et al 1993b, Mayani & Lansdorp 1998, Rappold et al 1997, Traycoff et al 1994). The converse of this is also true CD38 + cells that also co-express CD34 are more committed progenitor cells (erythroid and granulocyte)(Mitsui et al 1993), and CD34 and HLA-DR are shown to be expressed on mature dendritic cells, B-cells, and erythroid progenitors (Kempuraj et al 1999). In addition, cells that are CD34 + and Thy-1 + (CD90 progenitor cell subset) express low levels of CD117 (c-Kit tyrosine kinase receptor). The ligand for c-Kit is steel factor, also known as stem cell factor, which is believed to play a vital role in the proliferation and viability of hematopoietic cells. CD34 + cells that are negative for c-Kit have been shown to give rise to long-term culture-initiating cells (LTC-IC), while in those high in c-Kit expression, LTC-IC were almost absent. This pattern correlates with telomerase activity; in cells that are CD34 + CD38 or CD34 + 14

PAGE 28

CD117 have less mitotic activity, thus conserving telomeres, compared to CD38 + or CD117 high and low expressing cells (Sakabe et al 1998a, Sakabe et al 1998b). The authors in this study, suggested cells that are CD34 + and CD38 or c-kit negative are more quiescent and therefore probably more primitive. Although CD34 has been extensively used to identify hematopoietic stem and progenitor cells, studies have suggested there may be subset of stem and progenitor cells within cord blood that are CD34 and lineage negative (Goodell et al 1997, Osawa et al 1996). However, the importance of this cell type in relation to cell transplantation requires further investigation. Within the hematopoietic stem cell population there is a subset of CD133 + (originally called AC133) cells that strongly co-express with CD34 bright cells. There are two forms of the antigen that have been identified; CD133/1 is a cell surface glycoprotein and CD133/2 an intracellular protein (perinuclear label) (for review see Miraglia et al 1997, Yin et al 1997, for CD133/1 and Potgens et al 2001, for CD133/2). These antigens appear to be present on more primitive stem cells, in that, CD133/1 is not recognized in cultured human umbilical vein endothelial cells (HUVECs) or peripheral blood cells, but is expressed in human cord blood, fetal and adult bone marrow, and fetal liver cells (Yin et al 1997). CD133/2 also shows no reaction with cultured HUVECs, but is expressed in placental tissue, specifically trophoblast cells(Potgens et al 2001). This provides circumstantial evidence that CD133 + cells may be more a primitive stem cell than the CD34 + lineage cells of HUCB. However, further investigation into their proliferation and expansion capacity is needed before their clinical relevance is understood. 15

PAGE 29

CD markers have also been used to assist in identifying neural stem cells. CD133 is consistently found in 90-95% of neurosphere cells whereas cells of the neurosphere express neither CD34 nor CD45. Nor can CD34 and CD45 (common leukocytes antigen) positive cells form neurospheres (Uchida et al 2000). Cells that are CD133 + CD34 CD45 and CD24 -/lo (B cell marker) that were isolated from human fetal brain and spinal cord (week 12) differentiated into neurons and glia, generated neurospheres, and had self-renewal properties (Tamaki et al 2002, Uchida et al 2000). Additional markers are available to aid in identifying neural stem cells (for review see Newman et al 2003). Using these tools, in vitro studies have clearly demonstrated the expression of neural phenotypes in a subset of mononuclear cells within HUCB. 1.5 In Vitro Studies of HUCB Cells Sanchez-Ramos and colleagues (2001) initially cultured HUCB cells for 7 days in a proliferation media supplemented with N2 (neural proliferating component), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Additional cells were cultured in differentiation medium consisting of the proliferation medium with the addition of all-trans-retinoic acid (Yamaguchi et al) and nerve growth factor (NGF). Immunocytochemical analysis revealed an increased expression of Musashi-1 (an early marker of neural precursors), -tubulin III (a specialized tubulin found in neurons TuJ1) and glial fibrillary acidic protein (GFAP a marker for astrocytes) in HUCB cells that were cultured in retinoic acid (RA) and NGF. RT-PCR analysis confirmed the presence of Musashi-1, the mRNA for neuronal markers, nestin and necdin, GFAP in RA and NGF treated cord blood cells (Sanchez-Ramos 2002, Sanchez-Ramos et al 2001). 16

PAGE 30

Since then, studies have confirmed and extended these results. -mercaptoethanol has been shown to differentiate cultured HUCB cells towards a neural phenotype, which was confirmed by immunocytochemical expression of NeuN, neurofilament, and GFAP and by RT-PCR mRNA for nestin, neurofilament and MAP2 (Ha et al 2001). A multipotent HUCB cell subset that does not express CD14, CD34, or CD45 was identified by Bicknese et al., (2002). These cord blood cells when plated in a low glucose and more acidic media and supplemented with bFGF and hEGF differentiated and expressed both GFAP and TuJ1 after 7 day in culture. Interestingly, both hematopoietic progenitors and osteoclast-like cells were eliminated in this media condition (Bicknese et al 2002). Finally, some of the most elegant work elucidating the differentiation of HUCB cells into neural cells has been conducted by Buzanska et al., (2002). Using a magnetic cell sorter, isolated CD34 + and CD45 + cells were used to provide a clonogenic cell subfraction of the mononuclear heterogeneous fraction of cord blood cells. Following culturing in DMEM with selected growth factors the remaining cells showed a very high commitment (30-40%) to neuronal, astrocyte fates, and a modest proportion of oligodendrocytes (11%). These clone-forming cells expressed nestin, but did not produce hematopoietic colonies. Cells were immuno-positive for TuJ1 (a low level was present in untreated cells), MAP2, GFAP, and GalC (oligodendrocyte marker) after exposure to RA and brain-derived neurotrophic factor (BDNF). While BDNF seemed to increase the number of astrocytes, RA treated cells alone had more neuronal expression. In addition, when HUCB selected cells were co-cultured with rat cortical cells for 4 days all three types of neural progeny were present (Buzanska et al 2002). 17

PAGE 31

These same investigators recently reported that after 2 years in culture, HUBC-derived stem cells continue to display normal chromosomal patterns, proliferation, and self-renewal properties (Buzanska et al 2003a). EGF and FGF receptor profiles appeared normal and both early and late passage cultures expressed internexin, Glutamate Receptor-2 (GluR2), Glutamic acid decarboxylase (GAD 65), GAD 67, and tyrosine hydroxylase (TH). HPLC analysis confirmed that the HUCB stem cells produced and secreted serotonin (5-HT) and dopamine (DA) metabolites. Differentiated HUCB stem cells also exhibit electrophysiological properties indicative of neurons including hyperpolarization-activated inward and outward currents (Buzanska et al 2003b). Missing from this area of research is the long-term potential and expansion capabilities of these cells. More than likely the establishment of a neural stem cell line derived from HUCB cells that is both homogenous and reliable will be the next research step. At this time, it remains unknown whether it is necessary to establish such a cell line in order to repair CNS diseases or disorders. However, such a cell line would allow researchers to test the hypothesis of cell replacement to treat certain neurodegenerative and neurological disorders more easily. 1.6 Transplantation Studies Utilizing HUCB Cells To date, there are comparatively few published papers demonstrating that HUCB cells express neural phenotypes after transplantation (Table 1.2). Nonetheless, the accumulating in vivo data is quite consistent with the available in vitro data and points to the possibility that HUCB transplants may provide a means of ameliorating the deficits associated with a wide range of CNS diseases. In addition, the in vivo evidence suggest 18

PAGE 32

that there are alternative mechanisms, besides cell replacement, in which transplanted HUCB cells are delivering therapeutic benefits in animal models of injury and disease. These mechanisms include but are not limited to the release of trophic and neurotrophic factors by HUCB cells and the attraction of endogenous factors or stem cells by cord blood cells to the site of injury/damage. These alternative possibilities are discussed below with the appropriate study(s). 19

PAGE 33

Table 1.2 Significant Published CNS Transplantation Studies Using the Mononuclear Fractions of HUCB Cells Work Cited Animal Model & Cell Treatment Route & Time of Delivery/ Concentration of Cells for Transplant Behavioral Effects Immunocytochemistry Results Zigova et al., 2002 Cell Transplantation, Vol. 11, pp 265-274 Developing rat brain Sprague-Dawley rats Cells treated with: 1. DMEM, FBS + RA & NGF 2. DMEM, FBS only *SVZ injection of cells in 1 day old neonates 6 X 10 4 No behavioral measures were taken. No detrimental or adverse effects noted. One month PT*: Both treatment cell groups: ~2% positive for GFAP, with less than 0. 2% of positive for TuJ1 Chen et al., 2001 Stroke, Vol. 32, pp 2682-2688 Transient MCAO Adult male Wistar rats HUCB cells used immediately after thawing Intravenous tail vein injection 24 hrs or 7 days after MCAO 3 X 10 6 Motor neurological severity scores improved: 24 hr & 7 day groups. Rotarod improvements: 24 hr group only 14 and 35 day PT More cells located in injured tissues in 24hr group. Both groups had some positive cells for NeuN, MAP-2, GFAP, & FVIII* Willing, et al., 2003a J of Neuroscience Research, Vol. 37, pp 296-307 Permanent MCAO Adult male Sprague-Dawley rats HUCB cells used immediately after thawing 1. Intravenous femoral vein 2. Intrastriatal injection In both groups cells delivered 24hrs after MCAO at concentrations of 1 X10 6 or 2.5 X 10 5 Both groups had functional improvements on a number of measures. 1mo PT both groups improved on elevated body swing test. 2mos PT femoral group showed improved motor asymmetry on step test. 2mos PT more cellular debris in area of infarct of striatal group compared to femoral delivery where no cellular infiltration was noted. Human nuclear staining did not clearly demonstrate survival of HUCB cells in injured area. Willing, et al., 2003b Cell Transplantation Vol. 12, pp 449-454 Permanent MCAO Adult male Sprague-Dawley rats HUCB and PBPC* cells were used immediately after thawing. Intravenous femoral vein Cells were injected 24hrs after MCAO 1 X10 6 From 2 to 4 weeks PT HUCB treated rats increased in spontaneous activity and motor asymmetry was prevented in both groups measured on elevated body swing test. No immunocytochemistry or cell analysis performed. Vendrame et al., 2004 Stroke, Vol 35 (10) pp 2390-2395 Permanent MCAO Adult male Sprague-Dawley rats. HUCB cells were used immediately after thawing. HUCB cells intravenous infused. From 10 4 to 5 X 10 7 cells injected. 10 6 or more cells used significant behavioral improvement in step test and elevated body swing. A reduce infarct size was also reported. Lu, et al., 2002 Cell Transplantation Vol. 11, pp 275-281 TBI* Wistar rats HUCB cells were used immediately after thawing. Intravenous tail vein injection Cells were injected 24hrs after TBI 2 X 10 6 At days 14 and 28 PT HUCB treated rats had improvements on rotarod test and on neurological severity scores. 28 days PT Large number of human nuclei cells found in the brain and some double labeled for NeuN, MAP-2, and GFAP. Most found in boundary zone of injured area. Table 1.2 (Continued) 20

PAGE 34

Table 1.2 (Continued) Work Cited Animal Model & Cell Treatment Route & Time of Delivery/ Concentration of Cells for Transplant Behavioral Effects Immunocytochemistry Results Saporta, et al., 2003 J of Hematotherapy & Stem Cell Research Vol. 12, pp 271-278 SCI* Hemicompression Adult male Sprague-Dawley rats HUCB cells were used immediately after thawing. Intravenous tail vein injection 1. 1 day after injury 2. 5 days after injury 1 X 10 6 Open field locomotor activity (Basso, Beattie, Bresnehan -BBB) after 3weeks: 5 day group had improved score compared to 1 day group. 4 weeks PT HUCB cells were found only within the area of SCI. More cells were found in 5 day group than 1 day group. (Identification was difficult due to endogenous fluorescence.) Garbuzova-Davis et al., 2003 J of Hematotherapy & Stem Cell Research Vol. 12, pp 255-270 ALS Transgenic male mice B6SJL-TgN (SOD-G93A) 1GUR ~9.5 weeks of age. HUCB cells were used immediately after thawing. Intravenous jugular vein injection Cells were delivered to pre-symptomatic G93A mice at 66.2 + 0.75 days of age. 1 X 10 6 Delayed progression of ALS for 2-3 weeks and increased lifespan of treatment group. At 17 weeks of age PT HUCB treated mice finished footprint test faster compared to controls. 10 to 12 weeks PT cells were found in the brain, spinal cord, and other organs. HUCB cell in CNS were identified with HuNu and double labeled for CD45, Nestin, TuJ1, and GFAP. PT = post transplantation, PBPC = peripheral blood progenitor cells, FVIII = coagulation factor VIII, TBI = traumatic brain injury, and SCI = spinal cord injury, SVZ = Subventricular Zones. 1.6.1 Normal Developing Brain Prior to initiating studies in animal models of CNS disease, mononuclear HUCB cells were transplanted into the subventricular zone of neonatal rat pups (1 day old) (Zigova et al 2002). Given that the subventricular zone is an area known to promote stem cell survival and provide instructive signals for stem and progenitor cells, it offers a logical starting point to examine the engraftment and differentiation potential of HUCB cells into neural cells. The mononuclear fraction of HUCB cells were cultured in either DMEM with fetal bovine serum or Neurobasal medium with RA and NGF. Rat pups were transplanted with the cultured cells (60,000) and 30 days afterwards were sacrificed for immunohistochemical analysis of transplanted cells that survived and the 21

PAGE 35

corresponding phenotype. Under both culture conditions, cells were found proximal to the transplantation site and in the overlying cortex and corpus callosum. There was a greater distribution and survival rate of cells cultured with DMEM and FBS than with RA and NGF. Double-labeling experiments confirmed the neural nature of at least some of the human transplanted cells that were labeled with either GFAP or TuJ1. Qualitative analysis suggested that the majority of cells possessed an astrocytic-like phenotype and morphology. The study described above suggests that HUCB cells are capable of surviving and migrating within the developing, normal brain. Future studies will certainly need to continue to explore the developmental biology and neural stem cell properties of these cells when grafted into the developing and aging brain. However, the potential use of HUCB cells to treat the damaged brain is among the most exciting contemporary research areas. Despite being a completely new field there are initial results demonstrating the functional effects of HUCB cells transplants in several different animal models of acute and chronic disease. 1.6.2 Stroke Stroke is the third leading cause of death in the majority of developed countries and is a leading health care burden in all developed countries(American Heart Association, 2005). Even though the incidence of stroke has declined over the past decades, progress in treating stroke has been disappointing. Despite our continued understanding of the pathological underpinnings of cell death in stroke, there are no effective treatments for mitigating the loss of neurons in patients once that pathological cascade has been initiated. Cell transplantation has been proposed as one means of 22

PAGE 36

repairing the stroke ravaged brain and recent studies suggest that systemic infusion of HUCB cells produces potent behavioral effects in animal models. Indeed, recent data indicate that intravenously administered HUCB cells can survive, migrate to the site of injury, and provide functional recovery in a rat model of cerebral ischemia. In a pioneering study, Wistar rats received a transient (2 hour) middle cerebral artery occlusion (MCAO) followed by a tail vein injection of HUCB cells (3 x 10 6 as a bolus injection) either 24 hours or 7 days after stroke (Chen et al 2001). Animals were sacrificed at 14 and 35 days after MCAO and all animals showed significant lesions. However, no reduction in lesion volume was observed at either transplant time. Detailed histological analysis revealed grafted HUCB cells within the ipsilateral cortex, subcortex, and striatum. In addition, neuronal markers NeuN, astrocytic marker GFAP, and the endothelical cell marker, FVIII, were shown in animals transplanted at 24 hours after MCAO. Associated with the survival of HUBC cells were improvements in behavioral indices. A battery of tests including the rotarod motor test and the modified neurological severity score test (MNSS) was conducted at 1, 7, 14, 21, 28, and 35 days after MCAO. Rats that received HUCB cells 24hrs post stroke showed significant performance improvements on both tests, whereas treatment at 7 days produced less robust recovery with improved function seen only on the MNSS. Therefore, this data suggest that a time dependent mechanism may be involved in delivery of the cells. We examine this issue in more detail in Chapter 4. Subsequent studies have begun to examine whether the route of administering HUBC cells following MCAO, impacts behavioral recovery. Sprague-Dawley rats received permanent MCAO and 24 hours later HUCB cells were injected either into the 23

PAGE 37

femoral vein (1 x 10 6 cells) or directly into the striatum (250,000 cells) (Willing et al 2003a). Behavioral tests conducted 1-2 months later included measures of locomotor activity, elevated body swing test, step test, and passive avoidance. The pattern of behavioral recovery was dependent to some degree on the route of cell delivery. Motor activity and passive avoidance testing improved in both treatment groups while performance on the step task (a measure of forelimb dexterity) improved in only those animals receiving HUBC cells via the femoral artery. While it is tempting to speculate that the femoral route of administration was more effective than direct striatal injection, several outstanding issues remain including: the lack of definitive identification of HUCB cells in the brain (Figure 1.1), the differences in cell numbers transplanted in the two treatment groups, and the potential role of the immune/inflammatory response produced by the MCAO. This last possibility is especially intriguing and the authors raise the possibility that the immune cells of the transplanted cord blood are attracted to the site of injury, which could help induce endogenous signals for the recruitment of stem cells within the animal or possibly the release of trophic factors from the HUCB cells themselves. This could aid in the repair and the eventual behavioral improvements seen in these stroke animals. In a second study, Willing, et al. (2003b), observed similar behavioral results in MCAO induced rats as in their first study. This study also showed that human granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells delivered by femoral vein (1 x 10 6 cells) improved behavioral deficits seen in MCAO rats(Willing et al 2003b). If a non-specific immunological effect does contribute to the behavioral recovery, it is of interest because in both studies cyclosporine was co-administered (to minimize graft rejection), and yet it did not alter the behavioral 24

PAGE 38

effects of HUCB cell transplants in rats following MCAO. These results provide compelling support for the potential use of HUCB cells in treating stroke. However, because the survival of HUCB cells at the site of damage appears to be relatively low (Figure 1.1) and the behavioral recovery, although present, is only partial, strategies are being developed for further improvement in the beneficial effects of HUCB transplants. Figure 1.1: HUCB Cells Transplanted into Stroke Rat Brain Figure 1.1 Fluorescence photomicrograph of striatum. Converted into black and white (A) Cellular debris in lateral striatum associated with permanent MCAO (MCAO-only group). DAPI was used to label cell nuclei within the striatum. With this method, a cellular infiltrate in the region of the infarct can be seen. These are small cells characteristic of either microglia or neutrophils in the same section as shown in A. (C,D) Higher magnification views from selected areas of the image in A. (E) Cellular debris in striatum in an animal from the femoral group. (F) Striatal group. (G) Normal contralateral striatum from the same animal as shown in E. Scale bar in B = 300 m; for A,B; bar in D = 100 m for C,D; bar in G = 50 m for EG. With permission from J Neurosci Res 73(3), 296-307, (2003) Willing, et al. 25

PAGE 39

Not only have functional improvements in behavioral deficits of MCAO been seen, but reduction in the volume of the infarct have been reported(Vendrame et al 2004). In this study, MCAO rats were intravenously infused with 10 4 to 5 X 10 7 HUCB cells 24 hours after surgery. At 4 weeks after delivery of HUCB cells at the 10 6 or higher dose, the rats with MCAO showed significant improvements in behavioral measures. More importantly, MCAO rats that were treated with 10 7 cells showed a reduced infarct volume, and cells were localized either to the infarct brain or in the spleen. Several important points are clear from theses stroke studies. First, the behavioral improvements are consistent and contribute to a growing literature suggestive of the functional benefits of HUCB cell transplants. Secondly, the study by Vendrame et al., provides the first clear evidence that transplanted HUCB cells can minimize the structural consequences of ischemic damage. Third, the dose range of cells required for behavioral improvements and reduced infarct volume have been determined. Fourth and last, proof of principle for both the behavioral improvement seen in the use of HUCB cells in the treatment of ischemic stroke and the intravenous delivery method has been established. 1.6.3 Traumatic Brain Injury No class of CNS injuries is more steeped in history and frustration than traumatic brain injuries (TBIs). Neurosurgery began with the treatment of head injuries as evidenced by what we know of medical practice in ancient civilizations. Despite this long history, TBIs are much like stroke, in that, we have an enhanced understanding of the pathology associated with injury and have continually refined our principles of patient management. In addition, TBI, similar to stroke, has no effective treatment. While cell 26

PAGE 40

therapy offers theoretical promise for a wide range of CNS diseases, its promise is especially true for trauma if a non-invasive means of cell delivery could be developed. The exact nature of the localized trauma often times prohibits additional surgical intervention at the site until some time after the initial damage has occurred. Unfortunately, much of the potential benefit of an intervening cell therapy may be lost during this time. In contrast, delivery of cells via the circulatory system would bypass these obstacles perhaps permitting sufficient cell delivery to the injured region resulting in anatomical and/or functional repair processes. To evaluate the potential of HUCB transplants in traumatic brain injury, Lu et al., (2002) examined the neurological benefits afforded by intravenous administration of HUCB cells in rats with contusive brain injury. HUCB cells were injected into the tail vein 24 hours after injury and the rats were sacrificed 28 hours later for histological analysis of the migration of cells to the site of trauma. The injected cells were distributed in the brain, heart, lung, kidney, liver, spleen, bone marrow, and muscle as demonstrated by immunohistochemical staining and laser confocal microscopy. It would seem that there was preferential entry and migration of HUCB cells into the parenchyma of the injured brain. The transplanted cells expressed the neural markers, NeuN and MAP-2, and the astrocytic marker, GFAP. Some HUCB cells integrated into the vascular walls within the boundary zone of the injured area. Significant functional improvements were also produced as assessed by both the Rotarod test and the NSS (Lu et al 2002). 27

PAGE 41

1.6.4 Spinal Cord Injury Similar behavioral benefits of cord blood cells were observed in animals with spinal cord injury (SCI) (Saporta, et al., 2003). SCI was induced by unilateral hemicompression with an aneurysm clip for 1 minute and HUCB cells (pre-labeled with FITC-conjugated cholera toxin) were intravenously injected either 1 or 5 days later. Behavioral recovery was determined using open field behavior at 1, 2, and 3 weeks after treatment. Rats receiving HUCB cells at 5 days, but not 1 day after surgery, had gradual and significant improvements on the locomotor tests that were seen over the course of the study. Histological analysis of these animals confirmed the presence of HUCB cells at and around the site of injury, but not in areas of necrosis or in the areas of intact spinal cord (Figure 1.2). Consistent with the pattern of behavioral recovery, nearly twice the number of viable HUCB cells were present within the damaged spinal cord when transplanted at 5 days versus 1 day after surgery (Saporta et al 2003). This is the first study to report that HUCB cells migrate specifically to the site of injury in the spinal cord and not to the contralateral side and the first to show that HUCB cells can improve motor behavior deficits in rat SCI model. 28

PAGE 42

Figure 1.2: HUCB Cells Transplanted into Spinal Cord Injury in Rats Figure 1.2 HUCB cells in the injured spinal cord 4 weeks after intravenous injection. Endogenous fluorescence was quenched with Pontamine Sky Blue (A) Fluorescent photomicrograph through the area of compression from a SCI only animal. No fluorescent HUCB-like cells were visible in these sections. Fluorescent photomicrograph of a section of spinal cord from a laminectomy-only animal that also received an intravenous injection of HUCB cells. No FITC-prelabeled HUCB cells are visible in these sections. (C) FITC-labeled HUCB cells (arrows) in a section from the spinal cord of a rat that received HUCB 5 days after compression injury within the area of compression. (D) Fluorescent photomicrograph through the area of compression of an animal that received HUCB 1 day after compression injury. FITC + HUCB cells similar to those seen in C were seen in these sections (arrows). Calibration bar: A, B, and D, 50 m; C, 100 m. With permission from J Hematother Stem Cell Res, 12(3), 271-278, Saporta et al., (2003). 1.6.5 Amyotrophic Lateral Sclerosis 29 Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is caused by a progressive degeneration of motor neurons. In the United States, 1 to 2 people per 100,000 develop ALS each year with men affected slightly more often than women(NINDS & NIH 2003). Although ALS may occur at any age, it is most commonly diagnosed in middle-aged and older adults. Over a period of months or years, ALS causes increasing muscle weakness, inability to control movement, and problems with speaking, swallowing, and breathing. In general, weakness progresses steadily with no periods of improvement or stability leading to death, usually within 3 to 6 years. There is no cure for ALS at this time. Current treatment for ALS focuses on helping the

PAGE 43

person cope with symptoms and avoiding complications for as long as possible. Cell therapy has been proposed as a possible replacement/repair strategy for the lost motor neurons. However, the diffuse nature of the disease, a significant number of cells are required for transplantation, although in theory, cell replacement via stem cells injected into the circulatory system could be beneficial if the cells could migrate into the region proximal to the motor neurons. The mouse SOD-1 model of ALS provides a useful means of evaluating cell-based neurorepair and neuroprotective strategies. In a recent study 1 x 10 6 mononuclear cells from HUCB were administered intravenously (via the jugular) into G93A transgenice male mice at 9.5 weeks of age (prior to the onset of significant behavioral impairments)(Garbuzova-Davis et al 2003). Significant benefits were observed in hindlimb extension and gait. Grafted animals lost less weight over the course of the study and, most impressively, lived a significantly longer (2-3 weeks) than controls. A detailed analysis of the survival and distribution of the injected HUCB cells revealed cells in the parenchyma of the brain, spinal cord, spleen kidneys, liver, lungs and heart. Some HUCB cells were immuno-positive for neural and astrocytic markers (TuJ1 and GFAP) within the parenchyma of the brain. This study was the first to report that HUCB cells are capable of migrating to a progressive brain and spinal cord disease without occurrence of an acute traumatic injury. Future studies should examine this approach in other progressive degenerating diseases such as PD, HD, and AD. Although there have been studies demonstrating longer life span expectancy in animal models of degenerative disorders treated with cord blood cells (Ende, Weinstein, Chen, & 2000; Ende, Chen, & Ende-Harris, 2001; Ende & Chen, 2002), they did not examine the size of lesion, grafted 30

PAGE 44

cell survival, location of the cells, immuno-markers, or optimal time periods in which to administer the cells. 1.7 HUCB Stem Cells for Gene Therapy Gene therapy, or the manipulation of human genetic material to produce a therapeutic effect, is a maturing scientific field and hematopoietic stem cells are promising targets for gene therapy. Like other areas of gene therapy, the use of modified hematopoietic cells suffers from low and variable transfection efficiency, uncontrolled production of foreign gene products, and poor safety profiles. Nonetheless, significant progress has been made in transfecting hematopoietic stem cells using both viral (sendai virus, simian foamy virus, human foamy virus, lentivirus, and adenovirus) and non-viral (centrifugal enhancement and particle-mediated gene delivery) methods (Buchet et al 2002, Falk et al 2002, Hughes et al 2002, Mitta et al 2002, Rosenqvist et al 2002, Watson et al 2003). In addition, cryopreserved HUCB cells may offer a certain advantage in the field of gene therapy research (as discussed early) in that they express a high level of amphotropic retrovirus receptors and amphotropic retrovirus receptors are widely used for transduction of gene vectors (Orlic et al 1996). Figure 1.3 represents one of the viral methods using green fluorescent protein (GFP). 31

PAGE 45

Figure 1.3: HUCB Cell that are Labeled with Green Fluorescent Protein Figure 1.3Green fluorescent protein labeling of HUCB. Using the lentiviral vector approach (provided by Dr. Didier Trono), HUCB cells (A, phase contrast) exhibit the green fluorescent protein (GFP) in vitro(B, FITC fluorochrome). In addition, following transplantation into the striatum, HUCB cells retain GFP epifluorescence at least up to 6 months of graft maturation (C, magnified in D). Figure kindly provided by Dr. Didlier Trono and with permission from Newman et al., (2004) Expert Opin Biol Ther, 4(2), 121-130. The results obtained, using Sendai viral mediated transfection, are particularly promising. Very high transfection of GFP has been achieved by using CD34 + HUCB or BM cells (>80%) and other HUCB progenitor cells (Woods et al 2001, Woods et al 2003). Efficient gene transfer was independent of cell cycle and in vitro cell differentiation studies confirmed that gene transfer occurred in progenitor cells of multiple lineages (Luther-Wyrsch et al 2001) indicating that this system provides very high gene transfer rates while maintaining the cells ability to reconstitute the normal compliment of hematopoietic lines. In theory, stem/progenitor cells within the mononuclear fraction of HUCB offer yet another unexplored means of enabling gene therapy for the CNS. Cell-based gene therapy is hampered by the lack of an abundant, safe, and immunologically acceptable source of tissue. Conceivably, HUCB stem cells could overcome this limitation because 32

PAGE 46

of their ability to evade detection by the host immune system and continue to secrete a therapeutic compound. As described above, several of in vivo studies have confirmed that HUCB cells survive following engraftment into the brains of xenogeneic hosts (i.e. human to rat). The continued survival of HUCB cells in these studies cannot be solely attributed to the adjunctive pharmacological regimen, such as cyclosporine, but rather it reflects some inherent aspect of the transplanted cells. A recent study by Weiss and colleagues (2003), clearly shows that a subset of umbilical cord matrix cells from pigs differentiate into glia or neurons and survive following transplantation in rat brain for more than six weeks without triggering any recognition by the immune system (Weiss et al 2003). The authors suggest that the in vivo differentiation of cord blood cells into neurons might provide a unique approach to treating CNS diseases. This may be the case; however, this also provides a new opportunity to engineer and transplant stem cells from HUCB that could produce a desired therapeutic protein such as dopamine or GDNF for the treatment of PD. If longer-term studies confirm the stable transfection and longer-term survival of HUCB cells following transplantation this may create a new arm of xenotransplantation research that could potentially obviate many of the issues associated with tissue rejection. 33

PAGE 47

1.8 Clinical Studies using HUCB Cells 1.8.1 Transplantation of HUCB cells into Patients with Inborn Errors in Metabolism As mentioned HUCB cells, along with bone marrow cells have been used to treat various nonmalignant and malignant hematopoietic diseases, such as juvenile chronic myelogenous leukemia, Fanconi's anemia, and in patients with neuroblastoma that have varying clinical outcomes depending on the stage of the disease at time of transplantation and the disease itself. In addition, HUCB cells have been used to treat lysosomal/enzymatic disorders, such as Krabbe disorder and Maroteaux-Lamy syndrome. Krabbe disease is caused by a deficiency in galactocerebrosidase (GALC), which results in insufficient myelin formation in the peripheral and CNS. The majority of infants diagnosed with this disorder die before reaching 2 years of age, while the prognoses in adults is quite variable. Hematopoietic stem cells have been used to treat patients with Krabbe disease (Wenger et al 2000), and although this treatment decreases the symptoms, it does not eliminate or cure it. Infants with this disorder have been shown to have a longer lifespan if they receive hematopoietic stem cells within the first two weeks of life. Snyder and colleagues recently presented their findings, at the America Society of Neural Transplantation and Repair annual conference (2003), on a 21-month-old female infant suffering from Krabbe leukodystrophy, who received HUCB cells of male origin, and survived for a year after transplantation (Kosaras et al 2003). This study showed that upon postmortem examination of the brain, by FISH and 34

PAGE 48

immunocytochemistry analyzes, the transplanted HUCB cells were located in both white and gray matter, the ventricular ependyma, and within blood vessels throughout the host brain. Furthermore, HUCB cells were determined to differentiate into microglia, but not neural cells and the authors proposed that these cells could be producing GALC. This finding directly supports the pre-clinical studies discussed here that suggest that transplanted HUCB cells are acting through other mechanisms besides the differentiation of these cells into neural phenotypes to produce the improved behavioral results observed in animal models of injury. The FDA has already approved clinical trials using HUCB cells for hematologic malignancies, aplastic anemia, immunodeficiency diseases, and metabolic disorders (Thomson et al 2000, Wagner et al 1995). While these clinical trials are not for neurological disorders, the level of comfort that regulatory agencies already have with this general approach together with the data collected from ongoing trials (related to dosage, GVHD, cell survival, safety of the HUCB cells, and their efficacy) will facilitate entry into clinical trials for cellular transplantation therapies. With over 1500 cord blood transplants performed worldwide, a strong safety and efficacy database is being compiled (Gluckman et al 2001, Laughlin et al 2001). Although no official clinical trials for neurological disorders are in progress, various media sources have reported that HUCB cell transplants for ALS, stroke, and Sanfilippo Syndrome have been performed. However, without corroborating scientific data to review: these reports are insufficient to determine the effects of the HUCB cells on the treated patients. 35

PAGE 49

1.9 Conclusions Thus Far Cell transplantation for CNS diseases has always held tremendous medical potential. Nevertheless, despite the generally promising clinical results of numerous strategies, widespread clinical evaluation has been difficult. The most obvious example of this is the use of fetal neural tissue. Even though fetal grafts, survived and produced functional benefits in pre-clinical models, and have been suggested to be beneficial in small clinical trials, the limited tissue availability and ethical concerns have seriously hampered development of this approach. Described here is an alternative approach to the traditional problems of cell therapy that focuses on the use of HUCB as a rich and transplantable source of non-embryonic or adult stem cells that appear to be capable of exerting pronounced effects even when administered systemically. 1.10 Explanation of Study and Overall Purpose The studies within this manuscript were preformed to answer questions that will advance the potential of HUCB cells in the field of cell therapy and repair. In the research that was presented, in this chapter, three areas clearly required further exploration; the ability of HUCB cells to migrate to the injured tissue, the chemoattractants (if any) within the damaged tissue and the factors responsible for the improvement in behavior. We examined basic characteristics that were needed to proceed with the planned studies, such as, the average diameter of the cells, their phenotypes in culture, the ability of these cells to proliferate in culture, their ability to 36

PAGE 50

migrate to known chemoattractants along with the optimal time, testing of several assays in which to measure migrating cells, protein assay to standardized protein amount in migration studies, and migration to the young rat brain, which we believed at the time could be use as a positive control. These findings are presented in Chapter 3. The ability of HUCB cells to migrate to injured tissue, which is especially important when using systemic administration, and the optimal time after injury to deliver these cells was examined. Initially experiments were designed to address the cells ability to migrate in a variety of animals models of disease or injury of the CNS. After the initial migration experiments with several animal models of injury (Chapter 3) one in particular (ischemic rat) showed significant clinical and basic science relevance, and this study is present in Chapter 4. The ischemic rat migration study was expanded to determine some of the cytokines or chemokines present in the ischemic tissue (Chapter 4), and the ability of HUCB cells to produce cytokines/chemokines, these data are presented in Chapter 5. Compared to the number of cells that were injected, in the HUCB cells transplantation studies, few cells were found at the site of injury. This is very interesting due to the significant improvements in behavior that were presented in the animal models of injury therefore; we felt exploration in this area was essential. These experiments and results are present in Chapter5. In addition, the relevance to clinical and basic sciences from these studies is discussed in Chapter 6. 37

PAGE 51

1.11 References AHA American Heart Association. 2005. Heart Disease and Stroke Statistics. Dallas, Texas: American Heart Association Beck R, Lam-Po-Tang PR. 1994. Comparison of cord blood and adult blood lymphocyte normal ranges: a possible explanation for decreased severity of graft versus host disease after cord blood transplantation. Immunol Cell Biol 72: 440-4 Bicknese AR, Goodwin HS, Quinn CO, Henderson VC, Chien SN, Wall DA. 2002. Human umbilical cord blood cells can be induced to express markers for neurons and glia. Cell Transplant 11: 261-4 Blau HM, Brazelton TR, Weimann JM. 2001. The evolving concept of a stem cell: entity or function? Cell 105: 829-41 Broxmeyer HA, ed. 1998a. Cellular characteristics of cord blood and cord blood transplantation. Bethesda: AABB Press Broxmeyer HE. 1995. Cord blood as an alternative source for stem and progenitor cell transplantation. Curr Opin Pediatr 7: 47-55 Broxmeyer HE. 1996. Primitive hematopoietic stem and progenitor cells in human umbilical cord blood: an alternative source of transplantable cells. Cancer Treat Res 84: 139-48 Broxmeyer HE. 1998b. Cord Blood Transplantation Study Standard Operating Procedures: an evolving document will improve cord blood unit quality. J Hematother 7: 479-80 Broxmeyer HE. 2000. Introduction: Cord blood transplantation looking back and to the future. In Cord Blood Characteristics: Role in Stem Cell Transplantation., ed. SB Cohen, E Gluckman, P Rubinstein, JA Madrigal. London: Martin Dunitz Broxmeyer HE, Carow CE. 1993. Characterization of cord blood stem/progenitor cells. J Hematother 2: 197-9 Broxmeyer HE, Cooper S. 1997. High-efficiency recovery of immature haematopoietic progenitor cells with extensive proliferative capacity from human cord blood cryopreserved for 10 years. Clin Exp Immunol 107 Suppl 1: 45-53 Broxmeyer HE, Cooper S, Yoder M, Hangoc G. 1992a. Human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells. Curr Top Microbiol Immunol 177: 195-204 38

PAGE 52

Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, et al. 1989. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A 86: 3828-32 Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, et al. 1992b. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 89: 4109-13 Broxmeyer HE, Kurtzberg J, Gluckman E, Auerbach AD, Douglas G, et al. 1991. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 17: 313-29 Broxmeyer HE, Srour EF, Hangoc G, Cooper S, Anderson SA, Bodine DM. 2003. High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years. Proc Natl Acad Sci U S A 100: 645-50 Buchet D, Serguera C, Zennou V, Charneau P, Mallet J. 2002. Long-term expression of beta-glucuronidase by genetically modified human neural progenitor cells grafted into the mouse central nervous system. Mol Cell Neurosci 19: 389-401 Buzanska L, Machaj EK, Zablocka B, Pojda Z, Domanska-Janik K. 2002. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci 115: 2131-8 Buzanska L, Stachowiak E, Stachowiak M, Domanska-Janik K. 2003a. Neural stem cell line derived from human umbilical cord blood morphological and functional properties. Journal of Neurochemistry 85: 33 Buzanska L, Sun W, Salvi RJ, Domanska-Janik K, Stachowiak MK. 2003b. Changing electrophysiological properties in differenitating human cord blood-derived neural stem cells. Journal of Neurochemistry 85: 33 Cairns J. 1975. Mutation selection and the natural history of cancer. Nature 255: 197-200 Cardoso AA, Li ML, Batard P, Hatzfeld A, Brown EL, et al. 1993a. Release from quiescence of CD34+ CD38human umbilical cord blood cells reveals their potentiality to engraft adults. Proc Natl Acad Sci U S A 90: 8707-11 Cardoso AA, Li ML, Batard P, Sansilvestri P, Hatzfeld A, et al. 1993b. Human umbilical cord blood CD34+ cell purification with high yield of early progenitors. J Hematother 2: 275-9 39

PAGE 53

Chen J, Sanberg PR, Li Y, Wang L, Lu M, et al. 2001. Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 32: 2682-8 Cohen SB, Perez-Cruz I, Fallen P, Gluckman E, Madrigal JA. 1999. Analysis of the cytokine production by cord and adult blood. Hum Immunol 60: 331-6 Eaton MJ, Whittemore SR. 1996. Autocrine BDNF secretion enhances the survival and serotonergic differentiation of raphe neuronal precursor cells grafted into the adult rat CNS. Exp Neurol 140: 105-14 Falk A, Holmstrom N, Carlen M, Cassidy R, Lundberg C, Frisen J. 2002. Gene delivery to adult neural stem cells. Exp Cell Res 279: 34-9 Fallen P, Cohen SBA. 2000. Cord blood T-cell immunobiology. In Cord Blood Characteristics: Role in Stem Cell Transplantation., ed. SB Cohen, E Gluckman, P Rubinstein, JA Madrigal, pp. 39-60. London: Martin Dunitz Ltd Garbuzova-Davis S, Willing AE, Zigova T, Saporta S, Justen EB, et al. 2003. Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J Hematother Stem Cell Res 12: 255-70 Gluckman E. 2001. Hematopoietic stem-cell transplants using umbilical-cord blood. The New England Journal of Medicine 344: 1860-1 Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, et al. 1989. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321: 1174-8 Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, et al. 1997. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 337: 373-81 Gluckman E, Rocha V, Chastang C. 1999a. Peripheral stem cells in bone marrow transplantation. Cord blood stem cell transplantation. Baillieres Best Pract Res Clin Haematol 12: 279-92 Gluckman E, Rocha V, Chastang CL. 1999b. Umbilical cord blood hematopoietic stem cell transplantation. Eurocord-Cord Blood Transplant Group. Cancer Treat Res 101: 79-96 40 Gluckman E, Rocha V, Chevret S. 2001. Results of unrelated umbilical cord blood hematopoietic stem cell transplant. Transfus Clin Biol 8: 146-54

PAGE 54

Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, et al. 1997. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3: 1337-45. Gritti A, Frolichsthal-Schoeller P, Galli R, Parati EA, Cova L, et al. 1999. Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 19: 3287-97 Ha Y, Choi JU, Yoon DH, Yeon DS, Lee JJ, et al. 2001. Neural phenotype expression of cultured human cord blood cells in vitro. Neuroreport 12: 3523-7 Han P, Hodge G, Story C, Xu X. 1995. Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation. Br J Haematol 89: 733-40 Hows JM, Marsh JC, Bradley BA, Luft T, Coutinho L, et al. 1992. Human cord blood: a source of transplantable stem cells? Bone Marrow Transplant 9 Suppl 1: 105-8 Hughes SM, Moussavi-Harami F, Sauter SL, Davidson BL. 2002. Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol Ther 5: 16-24 Kempuraj D, Saito H, Kaneko A, Fukagawa K, Nakayama M, et al. 1999. Characterization of mast cell-committed progenitors present in human umbilical cord blood. Blood 93: 3338-46 Kinniburgh D, Russell NH. 1993. Comparative study of CD34-positive cells and subpopulations in human umbilical cord blood and bone marrow. Bone Marrow Transplant 12: 489-94 Kondo T, Raff M. 2000. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289: 1754-7 Kosaras B, Kurtzberg J, Sidman RL, Wenger D, Bianchi D, Snyder EY. 2003. Human umbilical cord cells (UCCS) distribute themselves throughout the degenerating huma brain but do not transdifferentiate into neural cells. Exp Neurol 181: 96 Lajtha LG. 1979a. Haemopoietic stem cells: concept and definitions. Blood Cells 5: 447-55 Lajtha LG. 1979b. Stem cell concepts. Nouv Rev Fr Hematol 21: 59-65 Lajtha LG. 1980. Bone marrow: The seedbed of blood, in Wintrobe MM (ed): Blood, Pure and Eloquent. New York: McGraw Hill. p 57. 41

PAGE 55

Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, et al. 2001. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. The New England Journal of Medicine 344: 1815-22 Lu D, Sanberg PR, Mahmood A, Li Y, Wang L, et al. 2002. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 11: 275-81 Lu L, Shen RN, Broxmeyer HE. 1996. Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: current status and future application. Crit Rev Oncol Hematol 22: 61-78 Luther-Wyrsch A, Costello E, Thali M, Buetti E, Nissen C, et al. 2001. Stable transduction with lentiviral vectors and amplification of immature hematopoietic progenitors from cord blood of preterm human fetuses. Hum Gene Ther 12: 377-89 Marshak DR, Gardner RL, Gottlieb D, eds. 2001. Stem cell biology., Vols. Monograph 40. Cold Spring Harbor: Cold Spring Harbor Laboratory Press Mayani H, Lansdorp PM. 1998. Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells 16: 153-65 Metcalf D. 1984. Clonal culture of hemopoietic cells: techniques and applications. The Netherlands: Elsevier Science Publishers B.V. 3 pp. Miraglia S, Godfrey W, Yin AH, Atkins K, Warnke R, et al. 1997. A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood 90: 5013-21 Mitsui H, Furitsu T, Dvorak AM, Irani AM, Schwartz LB, et al. 1993. Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand. Proc Natl Acad Sci U S A 90: 735-9 Mitta B, Rimann M, Ehrengruber MU, Ehrbar M, Djonov V, et al. 2002. Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. Nucleic Acids Res 30: e113 Miyawaki T, Moriya N, Nagaoki T, Taniguchi N. 1981. Maturation of B-cell differentiation ability and T-cell regulatory function in infancy and childhood. Immunol Rev 57: 61-87 42

PAGE 56

Nagaoki T, Miyawaki T, Ciorbaru R, Yachie A, Uwadana N, et al. 1981. Maturation of B cell differentiation ability and T cell regulatory function during child growth assessed in a Nocardia water soluble mitogen-driven system. J Immunol 126: 2015-9 National Center For Farmworkers INCFH. 2002. National Vital Statistics Reports Centers for Disease Control and Prevention. National Research Council (U.S.). Committee on the Biological and Biomedical Applications of Stem Cell Research. 2002. Stem cells and the future of regenerative medicine. Washington, D.C.: National Academy Press. xv, 94 pp. Newman MB, Freeman TB, Davis Sanberg C, Sanberg PR. 2003. Neural Stem Cells for Cellular Therapy in Humans. In Neural stem cells : development and transplantation, ed. JE Bottenstein, pp. 379-13 p. Boston: Kluwer Academic Publishers NINDS NIoNDaS, (NIH) NIoH. 2003. Amyotrophic Lateral Sclerosis Fact Sheet. Nonoyama S, Penix LA, Edwards CP, Lewis DB, Ito S, et al. 1995. Diminished expression of CD40 ligand by activated neonatal T cells. J Clin Invest 95: 66-75 Ogawa Y, Sawamoto K, Miyata T, Miyao S, Watanabe M, et al. 2002. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 69: 925-33 Orlic D, Bock TA, Kanz L, New York Academy of Sciences. 1999a. Hematopoietic stem cells : biology and transplantation. New York, N.Y.: New York Academy of Sciences. x, 405 pp. Orlic D, Girard LJ, Anderson SM, Barrette S, Broxmeyer HE, Bodine DM. 1999b. Amphotropic retrovirus transduction of hematopoietic stem cells. Ann N Y Acad Sci 872: 115-23; discussion 23-4 Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP, Bodine DM. 1996. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci U S A 93: 11097-102 Osawa M, Hanada K, Hamada H, Nakauchi H. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273: 242-5 43

PAGE 57

Potgens AJ, Bolte M, Huppertz B, Kaufmann P, Frank HG. 2001. Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast. Placenta 22: 639-45 Potten CS, ed. 1983. Stem Cells: Their identification and characterisation. New York: Churchill Livigstone. 1 pp. Rabian-Herzog C, Lesage S, Gluckman E, Charron D. 1993. Characterization of lymphocyte subpopulations in cord blood. J Hematother 2: 255-7 Rao MS. 2001a. Stem cells and CNS development. Totowa, N.J.: Humana Press. x, 370 pp. Rao MS, Mattson MP. 2001. Stem cells and aging: expanding the possibilities. Mech Ageing Dev 122: 713-34 Rao SG. 2001b. Stem cells and their therapeutic potential. Indian J Exp Biol 39: 1205-6 Rappold I, Ziegler BL, Kohler I, Marchetto S, Rosnet O, et al. 1997. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase. Blood 90: 111-25 Rocha V, Cornish J, Sievers EL, Filipovich A, Locatelli F, et al. 2001. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 97: 2962-71 Rocha V, Wagner JE, Jr., Sobocinski KA, Klein JP, Zhang MJ, et al. 2000. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 342: 1846-54 Rosenqvist N, Hard Af Segerstad C, Samuelsson C, Johansen J, Lundberg C. 2002. Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation. J Gene Med 4: 248-57 Sakabe H, Kimura T, Zeng Z, Minamiguchi H, Tsuda S, et al. 1998a. Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: comparison with stem cell factor. Eur J Haematol 60: 297-306 44 Sakabe H, Yahata N, Kimura T, Zeng ZZ, Minamiguchi H, et al. 1998b. Human cord blood-derived primitive progenitors are enriched in CD34+c-kitcells: correlation

PAGE 58

between long-term culture-initiating cells and telomerase expression. Leukemia 12: 728-34 Sanberg PR, Willing AE, Cahill DW. 2002. Novel cellular approaches to repair of neurodegenerative disease: from sertoli cells to umbilical cord blood stem cells. Neurotoxicity Research 4: 95-101 Sanchez-Ramos JR. 2002. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 69: 880-93 Sanchez-Ramos JR, Song S, Kamath SG, Zigova T, Willing A, et al. 2001. Expression of neural markers in human umbilical cord blood. Experimental Neurology 171: 109-15 Saporta S, Kim JJ, Willing AE, Fu ES, Davis CD, Sanberg PR. 2003. Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior. J Hematother Stem Cell Res 12: 271-8 Schofield R, Lajtha LG. 1983. Determination of the probability of self-renewal in haemopoietic stem cells: a puzzle. Blood Cells 9: 467-83 Tamaki S, Eckert K, He D, Sutton R, Doshe M, et al. 2002. Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. J Neurosci Res 69: 976-86 Thomson BG, Robertson KA, Gowan D, Heilman D, Broxmeyer HE, et al. 2000. Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation. Blood 96: 2703-11 Till JE, McCulloch EA. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14: 213 Traycoff CM, Abboud MR, Laver J, Clapp DW, Hoffman R, Srour EF. 1994. Ex vivo expansion of CD34+ cells from purified adult human bone marrow and umbilical cord blood hematopoietic progenitor cells. Prog Clin Biol Res 389: 385-91 Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D. 2001. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30: 65-78 Uchida N, Buck DW, He D, Reitsma MJ, Masek M, et al. 2000. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 97: 14720-5 45

PAGE 59

Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM. 1994. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci U S A 91: 9857-60. Vendrame M, Cassady CJ, Newcomb J, Bulter T, Pennypacker KR, et al. 2004. Infusion of human unbilical cord blood cells in rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35: 1-6 Wagner JE, Broxmeyer HE, Byrd RL, Zehnbauer B, Schmeckpeper B, et al. 1992. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 79: 1874-81 Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E. 1995. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 346: 214-9 Watson DJ, Longhi L, Lee EB, Fulp CT, Fujimoto S, et al. 2003. Genetically modified NT2N human neuronal cells mediate long-term gene expression as CNS grafts in vivo and improve functional cognitive outcome following experimental traumatic brain injury. J Neuropathol Exp Neurol 62: 368-80 Weiss ML, Mitchell KE, Hix JE, Medicetty S, El-Zarkouny SZ, et al. 2003. Transplantation of porcine umbilical cord matrix cells into the rat brain. Exp Neurol 182: 288-99 Wenger DA, Rafi MA, Luzi P, Datto J, Costantino-Ceccarini E. 2000. Krabbe disease: genetic aspects and progress toward therapy. Mol Genet Metab 70: 1-9 Willing AE, Lixian J, Milliken M, Poulos S, Zigova T, et al. 2003a. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res 73: 296-307 Willing AE, Vendrame M, Mallery J, Cassady CJ, Davis CD, et al. 2003b. Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant 12: 449-54 Woods NB, Mikkola H, Nilsson E, Olsson K, Trono D, Karlsson S. 2001. Lentiviral-mediated gene transfer into haematopoietic stem cells. J Intern Med 249: 339-43 Woods NB, Muessig A, Schmidt M, Flygare J, Olsson K, et al. 2003. Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis. Blood 101: 1284-9 46 Yamaguchi M, Sayama K, Yano K, Lantz CS, Noben-Trauth N, et al. 1999. IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid

PAGE 60

mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release. J Immunol 162: 5455-65 Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, et al. 1997. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90: 5002-12 Zigova T, Song S, Willing AE, Hudson JE, Newman MB, et al. 2002. Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 11: 265-74 47

PAGE 61

Chapter 2 2.0 Chemotactic Assay and Cell Migration 2.1 Introduction Cell migration is an essential component of embryonic development, adult maintenance (such as inflammatory response and wound healing), and tumor metastasis. Additionally, cell migration is of critical importance in the field of cell transplantation. Stem cell replacement therapy is one of the more promising treatments for certain neurodegenerative disease and traumatic insults such as Parkinsons disease and ischemia. Scientific studies are rapidly being published regarding the capability of transplanted stem cells to regenerate, integrate, and/or supply trophic factors to the target tissue. However, a major dilemma within this field is the migratory behavior of cells once transplanted, whether locally in the target tissue (e.g. intrastriatial) or distally (e.g. intravenous injection). In addition, if cells migrate to areas beside the target tissue this could cause problems, therefore it is essential that these cells are tracked and safety studies performed. 48 There are real limitations in cell migration research. In vivo, it is a slow and labor-intensive process to track cell migration and the migrated cells are only identified after histological examination. There is the possibility of using magnetic resonance

PAGE 62

imaging (MRI) system to track labeled cells. However, MRI systems are rather costly and not many researchers have access to these systems. In addition, without disturbing or invading the brain, .there is no in vivo method to study the possible factors that induce the migration of cells to the injured tissue in the brain. Endogenous microenvironment factors at the site of injury or damage may serve as chemoattractants for the migration of the transplanted cells or may inhibit the migration. Again, only by invading the brain or after tissue processing are those factors possibly identified. Furthermore, both the disruption of the brain and the tissue processing may interfere with obtaining reliable results. The homing or migration of stem cells to a chemoattractant(s) has been determined in bone marrow transplantation. Hematopoietic stem cells once transplanted will home to the bone marrow, leave the peripheral circulation, become stable and proliferate in extravascular space of the bone marrow cavity, thus serving to reconstitute the hematopoietic system (Whetton & Graham, 1999). Yet, even in this area of research, the migration and trafficking of cells and the molecular mechanisms regulating these interactions are poorly understood. There are a number of studies that have reported the migration of stem cell and neural stem cells to the sites of degenerative or injured tissue (reviews see (Keirstead, 2001; Zigova, Snyder, & Sanberg, 2002). Moreover, when compared to our knowledge of cellular migration during development, inflammatory response and wound healing, which is limited, our understanding of cell migration from allografts or xenografts to injured tissues is in infancy. A further understanding of the intrinsic and extrinsic 49

PAGE 63

mechanisms and signals involved in the migration of cells to injured or damaged tissue is certainly warranted. However, the first step is to determine systematically whether a given population of cells will migrate to the damaged tissue. The second step would be to determine whether there is an optimal time at which to administer the cells. In addition, the third step would be possible chemoattractant factors in the damaged tissue, and to identify the cytokines or chemokines that may be released by the transplanted cells. 2.2 Background on Chemotactic Assay and Cell Migration The chemotactic assay Boyden Chamber (Boyden, 1962) was originally developed for and is now a standard method in the area of cancer and inflammatory response research. This type of transmigration assay has been widely used to study leukocyte, fibroblast, and endothelial cell migration to chemoattractants, chemotactic and adhesion stimulants, and/or chemokines. Although the methodologies and types of chambers have advanced, the basic function remains the same. In addition, these types of chambers or apparatus allow control over the chemoattractants, stimulants, and type of migration. There are three basic types of cell movement/migration. Chemotaxis, in general, refers to the movement of leukocytes or cells induced by a chemotactic stimulus and typically, the cells are attracted in a positive gradient (those moving towards the agent) or negative gradient (those moving away from the agent). Chemokinesis is the 50

PAGE 64

migration of cells in a random direction by a chemoattractant (stimulant to move), and random migration refers to undirected and spontaneous migration of cells. The process of cell migration is closely coupled to the presence of chemokines (chemoattractants), which belong to the super family of cytokines; these are also referred to as chemotactic cytokines. Chemokines in the range of 8 to 17 kD molecular mass have been shown to be a chemoattractant selective for leukocytes, the reason for this is still unclear (Benveniste, 1998). Recently several studies have examined factors influencing the migration of hematopoietic progenitor cells (HPC) in the bone marrow environment. HPCs are known to migrate to bone marrow during transplantation and fetal development. These cells also migrate from bone marrow to the peripheral blood in response to cytokines (Imai et al., 1998; Kim, Pelus, White, & Broxmeyer, 1998). The mobilization of stem cells within the stromal layer of bone marrow and peripheral blood is a complex interaction of numerous cytokines (for review see Horuk, 2001; Pelus, Horowitz, Cooper, & King, 2002). Stromal cell-derived factor-1 (SDF-1) has been shown to be a strong chemoattractant for HPCs and has induced migration of CD34 + cells from HUCB (Kim & Broxmeyer, 1998) using chemotactic assays, and is believed to play a vital role in the homing of HPC to bone marrow (Voermans, Anthony, Mul, van der Schoot, & Hordijk, 2001). SDF-1 is classified as a CXC-chemokine and is a chemoattractant for monocytes and lymphocytes. The receptor for SDF-1 is CXCR4 (also called Fusin and LESTR), which is a G-protein coupled receptor, typical for chemokines. Another chemoattractant, somatostatin (SST), which is not a chemotactic cytokine, but a regulatory peptide produce by neuroendocrine and immune cells, has been reported to attract immature hematopoietic cells (CD34 + CD117 + ) in a transwell 51

PAGE 65

migration assay (Oomen, Hofland, van Hagen, Lamberts, & Touw, 2000). Interestingly, the attractant and homing molecule for leukocytes, L-selectin, was reported not to attract hematopoietic stem cells (CD34 + ) (de Boer et al., 2002). The use of the in vitro migration assay is a well-established method for screening a select cell population to chemoattractants, and the proven chemoattractant, SDF-1, for hematopoietic stem and progenitor cells was used in this study as a positive control. 2.3 Migration Induced by Inflammation The migration of white blood cells in inflammatory and wound response is well documented. In brief, the inflammation response occurs by a number of separate and simultaneous events. There is an acute vascular response, in which the blood vessels widen to increase blood flow to the inflamed area, leading to an increase in vascular permeability allowing diffusible substances to enter, and an acute cellular infiltration occurs through chemotaxis or movement of the proinflammatory cells. The hallmark of inflammation is the appearance of neutrophils in the tissue and they migrate directly through the blood vessel walls to the site of injury. There is also a change in biosynthesis profiles of many organs and the release of several substances including cytokines. Lastly, there is the activation and migration of immune cells to the inflamed site. The migration of cells to an injured area is regulated by a variety of signaling molecules that are produced locally by mast cells, nerve endings, and platelets in which there is a selective production of adhesion, chemokines, and inflammatory molecules (cytokines) (Johnston & Butcher, 2002). Initially, leukocytes are adherent to endothelial cells by L52

PAGE 66

selectin (CD62L), which is expressed by leukocytes. When cytokines are activated, endothelial cells produce E-selectin (CD62E), and the receptors for this adhesion molecule are expressed on neutrophils, monocytes, eosinophils, and subsets of lymphocytes. Only after cytokine activation, is P-selectin (CD62P) released from granules of platelets or Weibel-Palade bodies of endothelial cells and the target cells are the same as E-selectin (Lorant et al., 1995; Shah et al., 2002; Smith, Kunjummen, Kishimoto, & Anderson, 1992; Stanimirovic, Wong, Shapiro, & Durkin, 1997). The integrins, which are expressed by leukocytes, monocytes, macrophages, granulocytes and a variety of other cell types, allow stronger adherence of neutrophils to the endothelium, where they crawl and migrate to the tissue in response to a chemoattractant gradient (Johnston & Butcher, 2002). Other molecules also act as chemoattractants for specific types of white blood cells, escaping into the blood and inducing the production of leukocytes within the bone marrow (Alberts et al., 1994) Little is known about the mechanisms involved in stem cell migration. We know now that stem and progenitor cells migrate from organ to organ during embryogenesis through adulthood (Broxmeyer, 1998), and we are just beginning to understand the factors and circumstances that drive or direct these cells to their targets. The process of cell migration itself is complex and varies in accordance with the conditions of migration. For example, during embryonic development cellular migration is conceptualized within the context of tissues and organ formation. The migrating cell and its resulting phenotype are exclusively dependent upon the three germ layers formed at gastrulation. Embryonic stem cells proliferate and their progeny undergo a process of progressive lineage restriction finally generating differentiated cells that form mature tissue. For 53

PAGE 67

example, cells from the endoderm layer will migrate and give rise to the digestive tract and its associated components and glands (e.g. the esophagus, stomach, salivary glands, liver, lungs etc), while the mesoderm layer cells give rise to the supporting muscular and fibrous elements. The ectoderm layer cells, which form the epidermis, will give rise to the entire nervous system through neurulation. The involvement of the extracellular matrix (EMC) is vital for cellular migration, along with the complex signaling of several cytokines, integrins, selectins, and other adhesion molecules and receptors that create a cascade of intracellular and extracellular events that participate in the directing of embryonic cell migration. For instance, cell adhesion to the ECM is essential; however, there is a necessary loss of cell-cell contact when cells move through the ECM during mesoderm and neural crest formation (Perris & Perissinotto, 2000). In addition, cells acquire migratory properties when they split off (delaminate) from the ectodermal layer and transit from the epithelial to the mesenchymal layer (Locascio & Nieto, 2001; Perris & Perissinotto, 2000). In the development of the CNS, stem cells reside, proliferate, and migrate from two germinal matrices. The neuroepithelium (also called ventricular zone and ependymal layer) is the primary source of neurons and is derived from the neuroectoderm of the neural plate. The second germinal matrices consist of the subventricular zone, which is close to the ventricle and the external germinal/granular layer, which is farther away (Committee., 1969). Stem cells in this region give rise to mostly granular cells. The neuroblast cells (precursors of neurons) migrate away from the matrices, stop proliferating, and differentiate (Altman & Bayer, 1995). The stem cell proliferation, differentiation, and migration are regulated by a strict timetable. Neurogenesis is known 54

PAGE 68

to continue in the postnatal period, mainly in the subventricular zone, the external granular layer of the cerebellar cortex and perhaps in the dentate gyrus of the hippocampus (Altman & Bayer, 1995; Peretto, Merighi, Fasolo, & Bonfanti, 1999). Until recently, only embryonic stem cells were thought to be pluripotent since they must form all tissue types. However, a certain degree of plasticity is now recognized with adult stem cells, in that, some adult stem cells are capable of acting and expanding outside where they reside and assist in regeneration of distal tissue (Blau, Brazelton, & Weimann, 2001). Moreover, adult neural stem cells that reside within the subventricular zone (subependymal layer) of the forebrain, migrate a great distance within the rostral migratory stream to reach the olfactory bulb and regenerate this population of cells (reviews see Gritti, Vescovi, & Galli, 2002; Peretto et al., 1999). The external granular proliferative layer in the dentate gyrus of the hippocampus is also known to give rise to astrocytes and neurons (Kuhn, 1996; Okano, Pfaff, & Gibbs, 1993). With global ischemia (Liu, Solway, Messing, & Sharp, 1998) and induced seizure, this area showed an increase in neurogenesis. Additionally, in neuronal injury models, transplanted adult neural stem cells have been shown to migrate to the site of damage (Akiyama et al., 2001; Herrera, Garcia-Verdugo, & Alvarez-Buylla, 1999; Kurimoto et al., 2001). 2.4 Summary Very little is known about the properties involved in the migration of HUCB cells. We do know HUCB cells will migrate towards ischemic brain tissue extract (Chen et al., 2001). When administered intravenously to rats that have undergone traumatic brain injured or ischemia, HUCB cells will survive and migrate to the damaged brain area 55

PAGE 69

(Chen et al., 2001; Lu et al., 2002). Together the results suggest that some signal may be attracting and/or inducing these cells to migrate towards these tissue extracts. From what has been presented so far it is plausible that HUCB cells, especially the stem and progenitor cells, would migrate to the developing, degenerating, and/or injured brain. Chapter 4 and 5 will present our findings on the probable cytokines and chemokines that might induce the migration of HUCB cells to injured or damaged tissues and those the could be produced by these cells once at the site of injury, which could aid in the behavioral recovery that has been seen in the animal models of injury. 56

PAGE 70

2.5 References Akiyama, Y., Honmou, O., Kato, T., Uede, T., Hashi, K., & Kocsis, J. D. (2001). Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord. Exp Neurol, 167(1), 27-39. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., & Watson, J. D. (Eds.). (1994). Molecular biology of the cell. (Third ed.). New York: Garland Publishing, Inc. Altman, J., & Bayer, S. A. (1995). Atlas of prenatal rat brain development. Boca Raton, Fla.: CRC Press. Benveniste, E. N. (1998). Cytokine actions in the central nervous system. Cytokine Growth Factor Rev, 9(3-4), 259-275. Blau, H. M., Brazelton, T. R., & Weimann, J. M. (2001). The evolving concept of a stem cell: entity or function? Cell, 105(7), 829-841. Boyden, S. (1962). The chemotactic effect of nixtures of antibody and antigen on polymorphonuclear leucocytes. Journal of Experimental Medicine, 115, 453-466. Broxmeyer, H. A. (Ed.). (1998). Cellular characteristics of cord blood and cord blood transplantation. Bethesda: AABB Press. Chen, J., Sanberg, P. R., Li, Y., Wang, L., Lu, M., Willing, A. E., et al. (2001). Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 32(11), 2682-2688. Committee., B. (1969). Embryonic vertebrate central nervous system: Revised terminology. Anatomy Recieved, 166, 257-261. de Boer, F., Kessler, F. L., Netelenbos, T., Zweegman, S., Huijgens, P. C., van der Wall, E., et al. (2002). Homing and clonogenic outgrowth of CD34(+) peripheral blood stem cells: a role for L-selectin? Exp Hematol, 30(6), 590-597. Gritti, A., Vescovi, A. L., & Galli, R. (2002). Adult neural stem cells: plasticity and developmental potential. J Physiol Paris, 96(1-2), 81-90. 57

PAGE 71

Herrera, D. G., Garcia-Verdugo, J. M., & Alvarez-Buylla, A. (1999). Adult-derived neural precursors transplanted into multiple regions in the adult brain. Ann Neurol, 46(6), 867-877. Horuk, R. (2001). Chemokine receptors. Cytokine Growth Factor Rev, 12(4), 313-335. Imai, T., Chantry, D., Raport, C. J., Wood, C. L., Nishimura, M., Godiska, R., et al. (1998). Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4. J Biol Chem, 273(3), 1764-1768. Johnston, B., & Butcher, E. C. (2002). Chemokines in rapid leukocyte adhesion triggering and migration. Semin Immunol, 14(2), 83-92. Keirstead, H. S. (2001). Stem cell transplantation into the central nervous system and the control of differentiation. J Neurosci Res, 63(3), 233-236. Kim, C. H., & Broxmeyer, H. E. (1998). In vitro behavior of hematopoietic progenitor cells under the influence of chemoattractants: stromal cell-derived factor-1, steel factor, and the bone marrow environment. Blood, 91(1), 100-110. Kim, C. H., Pelus, L. M., White, J. R., & Broxmeyer, H. E. (1998). Differential chemotactic behavior of developing T cells in response to thymic chemokines. Blood, 91(12), 4434-4443. Kuhn, K. H. (1996). Mitotic activity of the hemocytes in the tick Ixodes ricinus (Acari; Ixodidae). Parasitol Res, 82(6), 511-517. Kurimoto, Y., Shibuki, H., Kaneko, Y., Ichikawa, M., Kurokawa, T., Takahashi, M., et al. (2001). Transplantation of adult rat hippocampus-derived neural stem cells into retina injured by transient ischemia. Neurosci Lett, 306(1-2), 57-60. Liesveld, J. L., Rosell, K., Panoskaltsis, N., Belanger, T., Harbol, A., & Abboud, C. N. (2001). Response of human CD34+ cells to CXC, CC, and CX3C chemokines: implications for cell migration and activation. J Hematother Stem Cell Res, 10(5), 643-655. Liu, J., Solway, K., Messing, R. O., & Sharp, F. R. (1998). Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci, 18(19), 7768-7778. 58

PAGE 72

Locascio, A., & Nieto, M. A. (2001). Cell movements during vertebrate development: integrated tissue behaviour versus individual cell migration. Curr Opin Genet Dev, 11(4), 464-469. Lorant, D. E., McEver, R. P., McIntyre, T. M., Moore, K. L., Prescott, S. M., & Zimmerman, G. A. (1995). Activation of polymorphonuclear leukocytes reduces their adhesion to P-selectin and causes redistribution of ligands for P-selectin on their surfaces. J Clin Invest, 96(1), 171-182. Lu, D., Sanberg, P. R., Mahmood, A., Li, Y., Wang, L., Sanchez-Ramos, J., et al. (2002). Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant, 11(3), 275-281. Okano, H. J., Pfaff, D. W., & Gibbs, R. B. (1993). RB and Cdc2 expression in brain: correlations with 3H-thymidine incorporation and neurogenesis. J Neurosci, 13(7), 2930-2938. Oomen, S. P., Hofland, L. J., van Hagen, P. M., Lamberts, S. W., & Touw, I. P. (2000). Somatostatin receptors in the haematopoietic system. Eur J Endocrinol, 143 Suppl 1, S9-14. Pelus, L. M., Horowitz, D., Cooper, S. C., & King, A. G. (2002). Peripheral blood stem cell mobilization. A role for CXC chemokines. Crit Rev Oncol Hematol, 43(3), 257-275. Peretto, P., Merighi, A., Fasolo, A., & Bonfanti, L. (1999). The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain. Brain Res Bull, 49(4), 221-243. Perris, R., & Perissinotto, D. (2000). Role of the extracellular matrix during neural crest cell migration. Mech Dev, 95(1-2), 3-21. Shah, A., Unger, E., Bain, M. D., Bruce, R., Bodkin, J., Ginnetti, J., et al. (2002). Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia. Int J Hyperthermia, 18(6), 534-551. Smith, J. B., Kunjummen, R. D., Kishimoto, T. K., & Anderson, D. C. (1992). Expression and regulation of L-selectin on eosinophils from human adults and neonates. Pediatr Res, 32(4), 465-471. 59

PAGE 73

Stanimirovic, D. B., Wong, J., Shapiro, A., & Durkin, J. P. (1997). Increase in surface expression of ICAM-1, VCAM-1 and E-selectin in human cerebromicrovascular endothelial cells subjected to ischemia-like insults. Acta Neurochir Suppl, 70, 12-16. Voermans, C., Anthony, E. C., Mul, E., van der Schoot, E., & Hordijk, P. (2001). SDF-1-induced actin polymerization and migration in human hematopoietic progenitor cells. Exp Hematol, 29(12), 1456-1464. Whetton, A. D., & Graham, G. J. (1999). Homing and mobilization in the stem cell niche. Trends Cell Biol, 9(6), 233-238. Zigova, T., Snyder, E. Y., & Sanberg, P. R. (Eds.). (2002). Neural stem cells for brain repair. Totowa, N.J.: Humana Press. 60

PAGE 74

Chapter 3 3.0 Progression of the Dissertation Studies, Disease Models, and Preliminary Data 3.1 Progression of the Dissertation Studies The use of HUCB cells as a possible treatment for certain CNS diseases or injuries is becoming increasingly feasible. However, as discussed in Chapter 1 and 2 there are many issues to address for progress to continue in this area of research. The study presented in this and the next two chapters addresses some of those issues: 1) Whether HUCB cells have the ability to migrate to injured brain. 2) The optimal time or window of opportunity at which to administer the cells based on the migration data. 3) The identification of chemokines and chemoattractants in tissue extract from stroke brain (that may have induced HUCB cells to migrate). 4) And the numerous cytokines produced by HUCB cells in response to their microenvironment are identified. Figure 3.1 is a flow chart of the Dissertation Studies. 61

PAGE 75

Figure 3.1 Dissertation Studies 8 Migration Assays Young Rat Sanfilippo Mouse 6-OHDA Model of MCAO Rat Parkinsons Disease Model of Stroke Migration Assays Overtime 4 hours to 1 week Ischemic Striatal Tissue Cytokine Arrays Ischemic Tissue ELISAs Overtime 4 hours to 1 week For MCP-1 and GRO/CINC-1 (IL-8) IN ADDITION Human Cytokine Arrays Condition Media from HUCB Cells Cultured Under Various Conditions ELISAs for Human Chemokines ILand MCP-1 Migration Assay HUCB Cells to MCP-1, IL-3, TPO 62

PAGE 76

Initially, the study was designed to investigate the migration of HUCB cells to several models of injury or disease (Parkinsons Disease, Stroke, Sanfilippo Syndrome and aging brain). The cell migration experiments using tissue extracts from these animal models were accomplished for all models except aging brain. The aging brain was not studied, because of the long wait period and the expense in obtaining aged rats. However, instead the migration of HUCB cells to the young rat brain was performed, results of which are presented in preliminary data section of this chapter. In addition, during the initial migration experiments, one of the animal models in particular became of great interest middle cerebral artery occlusion (MCAO) rat model of stroke. The preliminary migration assay data from this model showed a time dependent factor indicating there could be a window of opportunity for delivery of these cells after ischemic stroke that could result in the most cells migrating to the site of injury. Therefore, in order to tease apart this phenomenon, the migration of cord blood cells to rat ischemic tissue extracts, at several time points after the induction of stroke (4 hours to 1 week), and for three areas of the brain (striatum, hippocampus, and the frontal cortex surrounding the striatum) was performed (see Chapter 4). The next logical step was to identify the mechanism(s) that may be responsible for inducing migration of these cells. Therefore, the chemokine(s) or chemoattractant(s) present in the striatal ischemic rat tissue extracts were determined. This data is presented in Chapter 4. Also of importance was to determine whether the cord blood cells were producing cytokines/chemokines that may be aiding in the functional recovery that has previously been reported when HUCB cells are transplanted into MCAO rats (reviewed in Chapter 1) (Chen et al., 2001; Vendrame et al., 2004; Willing, Lixian et al., 2003; Willing, 63

PAGE 77

Vendrame et al., 2003). The cells were cultured in various conditions to determine how microenvironments might influence their cytokine/chemokine production and these cytokine profiles are presented in Chapter 5. Further characterization, by performing ELISAs on the most significant chemokines in condition media of cultured HUCB cells (Chapter 5) and those from the striatal extracts were performed (Chapter 4). Lastly, to characterize these cells further, migration assays of cord blood cells to monocyte chemoattractant protein-1, (MCP-1), (which was present in both the tissue extracts and condition media) and the stimuli IL-3 and thrombopoietin (TPO) (which were added to the cells in culture), were completed. In concentrating on one model of injury (stroke), the study developed further than the several migration assays alone, and the results provide data that have true clinical relevance for the utilization of HUCB cells in the treatment of human injuries and/or diseases. 3.2 Background and Preliminary Data Several issues needed to be address before beginning the migration and cytokine assays, such as the mean diameter of cord blood cells, basic culturing and proliferation ability, general morphology and phenotypes of the cells, develop and optimize the migration assay, and perform protein assays to standardize tissue extracts. These are presented in the following sections of this chapter. The preliminary experiments that were required in order for the main studies to occur and the experimental results from the other animal models used are discussed. The methods for each are presented along with the findings. 64

PAGE 78

3.2.1 Migration Apparatus Until recently, the most common way of performing migration assays was to use Transwell plate (Corning) or the traditional Boyden Chamber. However, these apparatus proved to very expense due to the small number of samples the plates hold. Furthermore, the Transwell did not allow for true gradient migration, because the top well sit within the media or the chemoattractant. The decision was made, after extensive testing, to use the 96-Chemotx Chamber plate (Neuro Probe, Inc.). This plate allows 96 samples to be run simultaneously, and the cells must be attracted or stimulated to migrate due to the design of the plate (Figure 3.2). Figure 3.2 Depiction of Migration Chamber A. B. Figure 3.2 Illustration of 96-Chemotx Chamber from Neuro Probe, Inc. (A.) This diagram shows a single top well with a porous membrane and a hydrophobic coating surrounding it. (B.) A diagram of top well (side profile) representing how cell suspension is kept in place. Cell concentrations are placed on the top well and tissues extracts are placed in the bottom well. (C.) Shows the 96-well chamber fully assembled. 65

PAGE 79

3.2.2 Cell Diameter of HUCB Cells One study previously explored the migration of HUCB cells to ischemic brain tissue extract (Chen et al., 2001). However, in their study a membrane with 8m pore size was used. The preliminary data present here shows that the average size of HUCB cells at 1 day in vitro (DIV) and 3 DIV to be between 6 and 8m, depending on the sample (Figure 3.3). This finding suggests that the pore size used by Chen et al. (2001) allowed cells to drop through the membrane instead of truly migrating to the extract. Therefore, the all migration experiments in this study used a pore size of 5m on the top membrane. There were no significant differences between the diameters of the cells at 1 DIV compared to those at 3 DIV or between the samples. Typically, in our hands, HUCB cells began to differentiate at day 4 in culture in which distinct morphologies are apparent by 7 DIV. 66

PAGE 80

Figure 3.3 Average Cell Diameters of HUCB Cells 1 DIV 3 DIV 10 9 8 Mean Diameter of 500 Cells /Sample in m 7 6 5 4 3 2 1 0 1 2 3 4 5 HUCB Cell Sample Numbers Figure 3.3 The graph represents the average cell diameter of HUCB cells.Different lots of HUCB cells, 5 lots total, were cultured in DMEM, with 5% FBS and Gentamicin (50 g/mL) for either 1 or 3 DIV. Initial seeding density was 10 6 /cm 2 and 500 cells were randomly measured per sample and day. The cells measured on day 1 were not from the same plate as those on day 3. Live cells were measured for their diameter with an image analysis program, ImagePro-Plus (Version 5.0, Media Cybernetics, San Diego, CA). Data is presented as mean + SEM. There were no significant differences in the diameter of the HUCB cells between lots or days in culture. (Students T-test, p <0.05) 67

PAGE 81

3.2.3 HUCB Cells in Culture To establish the general characteristic of the cord blood cells and to ensure that they would sustain and grow, in our hands, the cells were cultured for different periods and proliferation and live/dead assays were performed. 3.2.3.1 Preparation of HUCB Cells Cryopreserved mononuclear fractions of HUCB cells were obtained from Saneron CCEL Therapeutics, Inc. Cells were thawed and pipetted into 9 mL of media [clear Dulbeccos modified Eagles medium (DMEM, Invitrogen, Carlsbad) with 10% FBS and 1 L/1mL of Gentamicin (Sigma, St. Louis)]. Cells were centrifuged at 400 g for 15 minutes, the supernatant was removed, cells were resuspended in 1 mL of media, and viability assessed using the trypan blue dye exclusion method as described previously (Zigova & Newman, 2002). Cord blood cells were then plated in either 8-well chamber slides, 35 mm dishes (Nunc, Naperville), or low adherence 6-well culture dishes (Corning, Corning) at a seeding density of 10 5 /cm 2 and placed in water jacket incubator set at 37 0 C and with 5% CO 2.. 3.2.3.2 Phenotype of HUCB Cells 68 To determine phenotype and morphology HUCB cells were cultured from 1 to 14 days in 8-well chamber slides, as described above. The media was changed very 3 days until cells were fixed. Phenotyping of the cell used immunocytochemistry methods. Media was removed and cell cultures were fixed in 4% paraformaldehyde (pH 7.4) and then thoroughly washed (3 x 10 min each) in 0.1 M phosphate-buffered saline (PBS). Then the cultured cells (slides) were incubated for 1 hr at room temperature in 10% normal goat serum and 0.03% Triton X-100 in 0.1 M PBS for blocking. Slides were then

PAGE 82

incubated for 24 hrs at 4C with the primary antibody directed against one of several hematopoietic cell markers with PBS containing 0.03% Triton X-100 and 2% normal goat serum. The control slides were handled in the same manner except that the primary antibody was omitted. Primary antibodies used were CD45 leukocyte common antigen (BD PharMingen; 1:500), CD133/1 (AC133) cell surface antigen present on hematopoietic stem and progenitor cells (MACS; 1:50), and CD34 (TUK3) transmembrane glycoprotein expressed on hematopoietic progenitor cells (Santa Cruz; 1:50). Rhodamine or fluorescein conjugated secondary antibody were used against primary at 1:500 800 (Molecular Probes, Inc.). Slides were then rinsed in 0.1 M PBS, 3 x 10 minutes, coverslipped with either 95% glycerol or Vectashield mounting medium with DAPI (Vector Laboratories) to help visualize cells and quantify immunostaining, and then examined by standard epi-illumination fluorescence microscopy. Results are presented in Table 3.1 and were quantified by counting cells in 80 random fields for 8 slides per antibody by investigators blind to the conditions. Scores were then tabulated as percentage of total cells. In Table 3.1 the stem and progenitor cells showed a decrease in number that corresponded to the length of time the cells were kept in culture. These results are in agreement with the current literature, and because cells were allowed to differentiate and not kept in a proliferation media the percentage of stem and progenitor cells should decrease the longer the cells are kept in culture. Depending on the culturing conditions (media, growth factors, etc...) the cells can be directed to keep proliferating or to differentiate towards specific phenotypes. 69

PAGE 83

Table 3.1 Phenotype of HUCB Cells at 1 to 14 DIV 1 DIV 2 4 DIV 5 10 DIV 11 14 DIV CD45 80 90% 90 95% 90 95% 95 99% CD34 4 5% 1 4% < 1% 0 CD133 1 4% <1 2% 0 0 Table 3.1 HUCB cells immunostaining results for hematopoietic cell markers. Cord blood cells from 5 different donors where used. The total number of cells and those immuno-positive for specific markers were counted and averaged over 80 fields. Data are expressed as the percentage range of phenotypes from donor cells. 3.2.3.3 Morphology of HUCB Cells In order to evaluate morphology of the HUCB cells, slides were stained with Giemsa (Sigma) counterstain to visualize the cell bodies and processes. After cell were prepared and cultured as described above, the cells were in fixed 4% paraformaldehyde (pH 7.4) and then thoroughly washed (3 x 10 min each) in 0.1 M phosphate-buffered saline (PBS). Giemsa stain (100 L) was applied to single wells in the 8-well chamber slides for 5 minutes, then rinsed with tap water 3 times, and coverslipped 95% glycerol. Figure 3.4 shows cells cultured until day 14 and the five distinct cell types are represented. Cord blood cells started differentiating at 4 DIV and by 14 DIV most of the small around cells from days 1-3 had either differentiated or died. 70

PAGE 84

Figure 3.4 HUCB Cells Morphology Figure 3.4 Photomicrographs demonstrating morphology of HUCB cells. Cells were stained with Giemsa after 14DIV. The morphology of the cord blood cells shows 5 distinct cell types which are numbered 1 5. (1) Depicts cells that have a small cell body with single long process. (2) Show cells with large cytoplasm and nucleus. (3) Represent cells with small cytoplasm and multiple processes. (4) These cells are similar to 2, but appear to have a smaller cell body and nucleus. (5) These cells appear to be forming bipolar cell morphology or they could not yet be fully differentiated. (20x objective) 71

PAGE 85

3.2.3.4 Live/Dead Assay of HUCB Cells Part of the procedure in characterizing the population of HUCB cells was to determine their viability while in culture. The viability of HUCB cells in suspension was determined by a double-labeling procedure using the fluorescein diacetate (FDA, Molecular Probes) and propidium iodide (PI, Sigma) method (Zigova & Newman, 2002). This method allows identifying the viable, FDA-labeled cells fluorescing bright green while dead cells, stained by PI, are bright red. Cells were periodically assayed throughout the 14 DIV. FDA/PI solutions were prepared as follows: FDA was prepared from stock solution of 5 mg of FDA in 1 mL of acetone from which a working dilution of 5 L of stock FDA in 1 mL of 0.1 M phosphate buffer was made prior to staining. PI stock solution was 1 mg of PI in 50 mL of 0.1 M phosphate buffer (kept refrigerated). FDA/PI solution was made be adding 100 L of working FDA stock solution to 30 L of PI stock solution, which is enough to cover the bottom of one well in the 8-well chamber slides. Solution was made from stock as needed. Media from the well was removed and FDA/PI pipetted on top of the cells. Chamber slides were then placed in the refrigerator for about 5 minutes. Cells were counted under dual fluorescent filter (FITC and rhodamine) and living (green) and dead (red) cells were visualized (Figure 3.5). The FDA/PI live/dead assays showed that the HUCB cells, in our hands, could be maintained in culture. In addition, the initial viability and cell seeding density of the cord blood cells influenced their long-term viability. The cells have better long-term viability when initial viability is high (75% or greater) and with seeding density of at least 10 6 /cm 2 or greater. 72

PAGE 86

Figure 3.5 FDA/PI Viability of HUCB Cells Figure 3.5 Fluorescent photomicrographs of HUCB cells immunolabeled with FDA/PI However, the photomicrograph has been changed to black and white: denotes red cells that are dead; all other cells are alive (stained green). (A, B, & C) Demonstrates that the initial sample viability and cell number are critical to the long-term cultures of these cells. (A) Cells at 1 DIV show high viability of sample. (B) Photomicrograph of HUCB cell at 14 DIV shows low viability. At time of plating, this sample had low cell number and viability (64%). (C) Photomicrograph of HUCB cells at 14 DIV shows high viability (all green cells) and at time of plating the cell number and viability (96%) was high. In this photograph the bottom layer of cells are adherent to the plate and differentiated, while the top layer of cell are small, floating and have not differentiated. (10x objective) 73

PAGE 87

3.2.3.5 Proliferation Assay of HUCB Cells To determine whether HUCB cells proliferate in culture, bromodeoxyuridine (BrdU), a thymidine analog, that is incorporated during the S (synthetic)-phase of the cell cycle (Gratzner, 1982) was used. An antibody against BrdU was used to detect the cells that have incorporated this analog. HUCB contains a heterogeneous population of cells in which there are approximately 4% stem/progenitor cells that are positive for CD34, and less than 1% positive CD133 (Cohen, Gluckman, Rubinstein, & Madrigal, 2000). This is in agreement with our results in Table 3.1. The ability to expand these cells in culture could be of importance in the cell transplantation field, especially if more cells from one donor are needed due to its unique properties, such as, its HLA antigens. This assay will help determine whether there are stem/progenitor cells that can be expanded in culture. HUCB cells were prepared as describe above except they were plated in 96-well plates at a seeding density of either 1 or 2 x 10 6 per well. Cells were exposed to BrdU (10 M) in DMEM cell culture media at 6, 24, or 48 hours before fixing the cells to detect BrdU labeling. Controls used were DMEM and cells plated 1 hour before fixation at the initial seeding densities but not exposed to BrdU. Figure 3.6 shows the proliferation of cord blood cells throughout the 48-hour period. Samples and controls were ran in triplicate. BrdU assay was performed according to the manufacturers instruction (Chemicon, International) and the number of proliferating cells was determine using a microplate reader (BioTek) set to detect luminescence with wavelength of 450/550 nm. Results showed that HUCB cell in these culture conditions do proliferate and that higher concentrations of cells at plating will increase the rate of proliferation. 74

PAGE 88

Figure 3.6 Proliferation of HUCB Cells in Culture 6900 100,000cells/well 200,000cells/well 5900 Luminescene Expression (RLUs) 4900 3900 2900 1900 900 -100 Figure 3.6 BrdU labeled HUCB Cells. Cord blood cells were pulsed at 6, 24, and 48 hours. Cells proliferated in culture at both seeding density. The results suggest the cord blood cells can be grown and expanded in culture and that higher seeding densities result in an increased rate of proliferation. Shows significantly more cells in these conditions compared to either control cells conditions. (Students T-test, p < 0.05). 3.2.4 Tool to Measure Migrated HUCB Cells and Control Selection Several initial steps were taken in order to ensure a reliable and verifiable migration assay. The primary step was to establish a way to quantify the number of migrating cells without relying on manual cell counting, as this technique is highly subjective. Initially the HUCB cells were labeled with calcein AM (Invitrogen), which fluorescently labels the cell bodies and appears red to orange in color. Figure 3.7 show the cord blood cell migrating through the membrane. While the calcein AM labeled all BrdU Pulsing 100,000cells/well 0.125 Assa y Control 1024 Control Cells 1523 6 Hrs 1687 24 Hrs 3464 48 Hrs 1920 2230 5346 4149 -0.25 200,000cells/well 75

PAGE 89

cells, there was a large amount of fluorescent background, which interfered when cell plates were read in the automatic plate reader (BioTek). The measurements were not reliable, which was noticeable in the standard curves. The same was true for cholera toxin subunit B conjugated to FITC (Alexa Fluor 488, Molecular Probes). While it labeled the nucleus of cells very well, either the background was high or because the plate was clear, the fluorescence marker was interfering with neighboring wells. Hoechst 33342, DAPI, fluorescent ester dye labeled the cells nucleus, however, many of the cells, presumably the stem and progenitor cells, exocytosed the dye very quickly (Figure 3.8). Figure 3.7 HUCB Cells Migrating through Membrane Figure 3.7 HUCB cells migrating through the top we l membrane to neonatal rat brain tissue extract. ( A Photomicrograph taken under bright-field showing pores omembrane (dark round shapes) and migrating cells (whit e irregular shapes). (B) Fluorescent photomicrograph of sam e membrane. HUCB cells were pre-labeled with calcein A M white denotes cells (5 M solution in 1 mL of media). (20 x objective) 76

PAGE 90

Figure 3.8 HUCB Cells Labeled with DAPI Figure 3.8 Photomicrographs of HUCB cells in culture at 1DIV. (A) Cells labeled with DAPI (Hoechst 33342) fluoresced blue under the DAPI filter, but are depicted here in black and white. (B) Same field taken under bright-field optics. Note not all cells are labeled with DAPI (10x objective). Because of the high background in the fluorescent labels, a chemoluminescent cell viability assay kit (CellTiter-Glo, Promega) was tried. This assay determines the number of viable cells based on the presence of adenosine 5 triphosphate (ATP), which is present in metabolically active cells. The luminescent signal is produced by a luciferase reaction and the amount of ATP expressed is proportional to the number of viable cells in the culture. The results from the replication of three standard curves using HUCB cells are shown in Figure 3.9. 77

PAGE 91

Figure 3.9 Luminescent Standard Curve of HUCB Cells 0.0050.00100.00200.00250.00300.00350.00400.00450.00500.0001005001000500010,00015,00020,00025,00030,00035,00040,00045,00050,00055,00060,00065,00070,00075,00080,000 150.00 ATP Luminescence in RLUs Concentration of HUCB Cells Figure 3.9 HUCB cells were assayed after migration using a luminescent cell viability kit (Celltiter-Glo, Promega). Standard curves were established for each assay and for determining sensitivity of the plate reader (BioTech, Inc.). For standard curves, a serial dilution of cells, at a concentration running from 0 to 80,000 cells/300 L of media, was performed in quadruplicate, and pipetted directly into the bottom well of a 96-well plate. RLUs = relative light units. Graph shows the mean of three standard curves for HUCB cells. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of live cells present. The graph contains the mean data points of three standard curves. The results from the standard curves and from initial studies proved the CellTiter-Glo cell viability assay to be very reliable and accurate each time. 78

PAGE 92

In addition to finding a reliable tool to measure the cells that had migrated, there was need for a positive control. The known chemoattractant Stromal Derived Factor (SDF-1) was chosen and both time and dose response curve were determined. SDF-1 is a strong chemoattractant for CD34 + cells from cord blood and bone marrow. The optimal dose of SDF-1 to induce migration in HUCB cells was 100 ng/mL and optimal time was between 4 and 6 hours after which there were no significant increase in the number of cells migrating (data not shown). Additional controls used in migration assay were the cell culture media DMEM and PBS. Both served as negative controls. 3.2.5 Protein Assays for Tissue Extracts and Media from HUCB Cells Bicinchoninic acid (BCA) protein assays (Promega, Madison, WI) were used to determine the total amount of protein in the tissue extracts for each condition and for both the positive and negative controls used in the migration assay. Protein assays were performed twice and each unknown (tissue extracts and controls) or standard were ran in triplicate (three wells per assay for 6 data points) according to the manufacturers instructions. The standard curves were performed in the same 96 well plate and the assay had a working range of 20 to 2,000 g/mL of protein. The plates were read in an automated plate reader (Synergy, BioTek, Winooske, VT) set to absorbance with wavelength of 562 nm. After measuring the protein concentration for tissue extracts, the extracts were standardized to 500 g/mL for use in migration, cytokine, and ELISA assays. Figure 3.10 represent the mean of 3 standard protein curves. 79

PAGE 93

Figure 3.10 Standard Curve for Protein Assays 2500 2000 Net Absorbance (562nm) 1500 1000 500 0 0 25 1000 1500 125 250 500 750 2000 Protein Standards ug/mL -500 Figure 3.10 BCA protein assay standard curve. The standards were performed by serial dilution, ran in triplicate, and according to manufacturers instructions (Promega). The diluent for the standards was clear DMEM, which was the same dilute for the tissue extracts used in all migration assays. 80

PAGE 94

3.3 Migration of HUCB Cells and Animal Models of Disease or Injury In this section, the migration of HUCB cells to neonatal rat brain, the 6-OHDA rat model of Parkinsons Disease (PD), and Sanfilippo mouse model are presented along with the methods and materials used. The stroke model is presented in Chapter 4. The disease or injury models that were used in this study were based on the prevalence and impact they have in the general population of the U.S., and our expertise in performing these experiments. 3.3.1 Basic Migration Assay Procedure The following methods were used for all migration assays. HUCB cells were prepared as described in section 3.2.3.1, except they were plated in low adherence 6-well culture dishes (Corning, Corning, NY) for 24 hrs in a water jacket incubator set at 37 0 C and with 5% CO 2 which allowed cells to adjust to the environment before beginning the migration assay. After 24 hrs cells were lifted by gentle pippetting, placed in a 15 mL tube, centrifuged, resuspended in 1 mL of media, and viability was assessed using the trypan blue dye exclusion method. Only HUCB cells with 80% or greater viability were used and cell concentration was adjusted to 62,000 cells/25 L of media. The basic procedures for the migration assay in all models are similar. The main difference in the procedures between models was the selection of brain tissue to use. The assay uses a 96-well chamber plate consisting of bottom wells that hold the samples or chemical attractants and a top plate, which is a polycarbonate membrane with 5 m pore size and is where the cells are placed. Tissue extracts (300 L) were pipetted into the bottom wells of the 96-well plates. The top plate was placed on top of the bottom well 81

PAGE 95

plate and HUCB cells were directly pipetted into the top well at a concentration of 62,000 cells per 25 L. The migration chambers were then placed in a water-jacket incubator at 37C with 5% CO 2 for 4 hours. SDF-1 was used for positive control and DMEM used as negative control. For each assay sample, control and standard were performed in triplicate. After 4 hours, the top well plates were removed. The bottom plates were then centrifuged; half the media (150 L) was removed, and Celltiter-Glo cell viability assay was performed (according to manufacturers instructions). The plates were then read in an automatic plate reader (BioTek) set to luminescence to determine the number of cells that migrated into the lower chamber. In all migration studies, the plate reader was set to a sensitivity of 100. 3.3.2 Preparation of Tissue Extracts Animals were sacrificed by exposure to CO 2. Brains were removed within two minutes, immediately frozen on dry ice and then stored at 0 C until needed. The frozen brains were semi-thawed, kept cold, and the brain areas of interest were dissected from both hemispheres. Tissue from each of the conditions were pooled, and kept on ice in clear DMEM. The preliminary findings showed that fetal bovine serum attracts HUCB cells, therefore this was not added to the homogenate media (data not shown). Tissue was homogenized at 150 mg of tissue per 1mL of media. The homogenates were centrifuged (400g for 20 minutes), the tissue extracts (supernatant) collected, filtered (0.22 m, Millipore, Bedford, MA), and stored at -80 0 C until used. 82

PAGE 96

3.3.3 Animal Models Used for HUCB Cell Migration The three animal models that follow used the same methods and materials described above. The unique methods or materials that are relevant to each model is discussed along with a brief background for each model, which has been incorporated to provide the reader with the necessary information to comprehend the premise of the animal models selected. 3.3.3.1 Neonatal Young Rat Model Cell migration is an essential component of embryonic development as mentioned in Chapter 2. During embryonic genesis in central nervous system, there is a tremendous amount of stem cell proliferation, migration, and differentiation. Stem cells reside, proliferate, and migrate from two germinal matrices. The neuroepithelium (also called ventricular zone and ependymal layer) is the primary source of neurons and is derived from the neuroectoderm of the neural plate. The second germinal matrices consist of the subventricular zone (which is close to the ventricle) and the external germinal/granular layer (Boulder Committee, 1969). Stem cells in this region give rise to mostly granular cells. The neuroblast cells (precursors of neurons) migrate away from the matrices in several manners. They will migrate in both short and long distances in various directions and go through dense brain tissues and over the surface of the brain. Once the cells reach their destination, they stop proliferating and differentiate (Altman & Bayer, 1995). The stem cell proliferation, differentiation, and migration are regulated by a strict timetable. Neurogenesis is known to continue into the postnatal period, mainly in the external granular layer with respect to the cerebellar cortex and the dentate gyrus of the hippocampus (Altman & Bayer, 1995; Peretto, Merighi, Fasolo, & Bonfanti, 1999). 83

PAGE 97

In the rat, the CNS in not fully developed until at least postnatal day 14 (Clancy, Darlington, & Finlay, 2001), which enables the study of stem cells attraction to the embryonic and postnatal brain across a developmental period. In addition, cell transplantation studies in the developing rodent brain have shown that this environment supports the survival and migration of embryonic human neural stem cells (Flax et al., 1998), neuronal restricted precursors (Yang et al., 2000), as well as less differentiated progenitor cells derived from the neonatal subventricular zone (Brustle et al., 1998; Zigova et al., 1996). Following transplantation into germinal matrices/zones embryonic stem cells were able to participate in normal development including migration along established migratory routes and to differentiate into site-specific cell types. Also, studies have reported that the rodent adult brain can support the transplantation of subventricular zone derived human embryonic progenitor cells (Fricker et al., 1999; Lois & Alvarez-Buylla, 1993). These findings suggest that the developing brain is a favorable environment for studying stem cell proliferation and migration. Therefore, the neonatal rat brain was used to investigate the migration of HUCB cells to these tissue extracts (Figure 3.11). Neonatal Sprague-Dawely rat pups (male and female) were sacrificed (anesthetized with Equithesin, 3.5 mL/kg and heads decapitated) at postnatal days 2 and 7 (n = 10 per condition). The cerebellum and brain stem were dissected from the brain and both the cerebellum and whole brain were used for the tissue extracts from the 2 day old rat pups. The cortex, hippocampus, midbrain and cerebellum were dissected from 7 day old rats. Tissue was prepared as described above. 84

PAGE 98

Figure 3.11 Migration of HUCB Cells to Neonatal Rat Brain 020406080100120140Tissue ExtractsATP Luminescence (RLUs) 2 Day Brain 2 Day Cerebellum 7 Day Cortex 7 Day Hippocampus 7 Day Cerebellum 7 Day Midbrain SDF-1 DMEM ** ** * * Figure 3.11 The bar graph represents the migration of HUCB cells to neonatal rat brain tissue extracts. Tissue extracts from 2 and 7 day old rat pups were used. The CellTiter-Glo luminescent viability assay kit was used to measure the amount of ATP expressed in live cells. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of live cells present. In all brain tissue extract conditions (*) significantly more cord blood cells migrated to these conditions when compared to controls (media DMEM). The 2 day brain and 7 day hippocampus conditions had (**) significantly more HUCB cells that migrated than the other extracts conditions and more than the positive control SDF-1. [ANOVA One-way between groups: F = 2.42, df = 7, 31 with p < 0.0001 followed by planned comparisons using Dunns Bonferroni (corrected) test with p < 0.05]. 85

PAGE 99

The results from this experiment showed that the neonatal brain extracts strongly attracts HUCB cells. Most striking were the 2 day old brain and the 7 day hippocampal tissue extracts, which had significantly more cord blood cells migrating compare to the other extract conditions. The results suggest that there are areas within the neonatal brain that contain signals, chemoattractants, to induce HUCB cells to migrate. Because the brain is still developing at this time and numerous migration signals are present this model was chosen to be the first. This allowed the techniques and procedures to be determined before performing the migration assay on injured or disease models. 3.3.3.2 Parkinsons Disease 6-OHDA Rat Model 86 Parkinsons Disease is a widespread neurodegenerative disorder first described by James Parkinson in 1817 (Parkinson, 1817) and is characterized by a gradual degeneration of the dopamine (DA) nigrostriatal system. Approximately 40,000 people in the United Stated are diagnosed with PD every day, and over 1 million Americans have this disease. The daily cost is approximately $66 million that include direct and indirect costs such as loss of earnings due to inability to work, social security, medical care, and long term care (PDF, 2005). Symptoms start to appear when at least 50% of the dopamine (DA) producing neurons in the substantia nigra are lost, which produces a 70 80% reduction in the DA content of the caudate and putamen. The most widely used treatment is L-dopa, a DA precursor that crosses the blood brain barrier. L-dopa improves symptoms of PD, but this is not a cure and effectiveness is diminished after long period of administration (C. R. Freed, Breeze, & Fahn, 2000; W. J. Freed, 2000). The transplantation of human embryonic ventral mesencephalic (VM) tissue has had encouraging results in patients with PD (Isacson, van Horne, Schumacher, & Brownell,

PAGE 100

2001; Olanow, Kordower, & Freeman, 1996; Piccini et al., 1999). However, there are some critical limitations with the use of fetal donor tissue. Only a small percentage of VM cells are dopaminergic (<10%) and of that only 1% will survive transplantation, therefore, up to 15 fetal VM may be required per patient (Isacson, Bjorklund, & Pernaute, 2001). Perhaps concerns that are more critical are the ethical and availability issues of not only fetal VM tissue but also any other fetal derived cell (stem and progenitor). In light of this, HUCB cells may be one possible alternative to the use of fetal tissue/cells. The 6-OHDA experimental model is one of the most commonly used in PD research. 6-OHDA is a neurotoxin that hydroxylates analogues of the endogenous neurotransmitter dopamine resulting in nigral degeneration both in vitro and in vivo (for review see (Blum et al., 2001; Blum, Torch, Nissou, Benabid, & Verna, 2000). Senoh and colleagues (1959) were the first to report its isolation (Senoh, Creveling, Udenfriend, & Witkop, 1959; Senoh, Witkop, Creveling, & Udenfriend, 1959), and then later Porter and colleagues (1963) showed that 6-OHDA depleted noradrenaline in the heart (Porter, Totaro, & Stone, 1963). The presence of this neurotoxin has been reported in the human and rat brain, suggesting it may be an endogenous substance (Curtis & Johnston, 1974). 6-OHDA is typically injected unilaterally into the striatum, substantia nigra or the ascending medial forebrain bundle, because it cannot cross the blood brain barrier. This induces asymmetric degeneration of the substantia nigra pars compacta and quantifiable motor behavior after administration of DA receptor agonists. Several populations of cells have been experimentally transplanted into the PD animal model, with some success. One of the more interesting was the use embryonic stem cells, which were reported to proliferate and differentiate into DA neurons within 87

PAGE 101

the striatum (Bjorklund et al., 2002). Another study using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD demonstrated that the intrastriatal transplantation of bone marrow stromal cells did improve behavioral deficits seen in mice (Y. Li et al., 2001). Both pre-clinical and clinical studies have shown that certain stem cells migrate to the degenerative PD brain. Therefore, it seemed probable that HUCB cells would migrate toward 6-OHDA lesion brain extracts. However, it was also probable that 6-OHDA toxin would inhibit cell migration. The results are presented in Figure 3.12. Additionally, the migration of HUCB cells to 6-OHDA lesioned brain extracts are presented in Figure 3.13. In this experiment adult male Sprague-Dawley rats were lesioned with 6-OHDA. The rats (n = 10) were anesthetized with Equithesin (3.5 mL/kg) and unilateral injections of 6-OHDA (Sigma; 2.5 L, 3.6 g/L in 2% ascorbic acid in saline) was made at 4.4 mm posterior to bregma, -1.2 mm laterally and .8 mm ventral to dura with the toothbar set at .4 mm and delivered at a rate of 1 L/min. Five minutes after each injection the needle was slowly withdrawn and incision sutured. Other rats (n = 10) received sham injection (2% ascorbic acid in saline). Animals were sacrificed 3 weeks after 6-OHDA lesion or sham surgery. Tissue extract preparation was performed as describe above, except that the left and right substantia nigra and the striatum were dissected. The preparation of the cord blood cells and migration assay methods were identical to those described above. 88

PAGE 102

Figure 3.12 Migration of HUCB Cells to 6-OHDA Toxin and SDF-1 0.0050.00100.00150.00200.00250.00300.00350.00400.001 microL2 microL4 microL10 microL50ng/mL75ng/mL100ng/mLDMEMPBS 6-OHDA SDF-1ATP Luminescence (RLUs) * Figure 3.12 Migration of HUCB cells to the toxin 6-OHDA and to the chemoattractant SDF-1. 6-OHDA had an inhibitory effect of the migration of these cells in a dose dependent manner. The opposite was seen with SDF-1 in which cord blood cells significantly migrated to this chemoattractant in a dose dependent manner. All conditions of SDF-1 induced (*) significantly more cells to migrate compared to all other conditions. 6-OHDA was not different from controls. The CellTiter-Glo luminescent viability assay kit was used to measure the amount of ATP expressed in live cells. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of live cells present. [ANOVA One-way between groups: F = 2.30, df = 8, 35 with p < 0.0001 followed by planned comparisons using Dunns Bonferroni (corrected) test with p < 0.05] 89

PAGE 103

Figure 3.13 Migration of HUCB Cells to 6-OHDA Animal Model of PD 050100150200250SubstantiaNigra StriatumSubstantiaNigra StriatumPlainMediaPBS Sham Sham 6-OHDAPD6-OHDAPDControls ATP Expressed by Migrated HUCB Cell (in RLUs) * Figure 3.13 Migration of HUCB cells to tissue extracts from rat 6-OHDA model of PD. Sham is equal to sham surgery and injected with vehicle instead of 6-OHDA and 6-OHDA PD equal lesion animals that have been injected with 6-OHDA. Tissue extracts from the substantia nigra and the striatum of unilateral 6-OHDA lesion brains showed significantly more migration of HUCB cells compared to the controls and their respective sham surgery conditions. Significantly different from sham surgery and from plain media and PBS controls. [ANOVA One-way between groups: F = 2.38, df = 5, 59 with p < 0.0001 followed by planned comparisons using Dunns Bonferroni (corrected) test with p < 0.05] 90

PAGE 104

These results showing migratory behavior of the HUCB cells in this PD model suggest that there are endogenous signals generated by pathology that are capable of attracting these cells to the area of injury. The toxin 6-OHDA actually inhibited the cord blood cells from migrating (Figure 3.12). The typical amount of 6-OHDA used in rat models of PD ranges between 3 to 10 g, which is usually dependent on the injection site. In this experiment, the rats received at total of 9 g. However, the brains of these animals were not taken until 3 weeks after administration of toxin, which should be enough time to reduce the amount of 6-OHDA level directly at the site of the lesion. The sham surgery tissue extracts (Figure 3.13) were significantly different from the toxin 6-OHDA. This suggests that in addition to the 6-OHDA in the tissue extracts some other factor(s) may be inducing the migration of these cells, such as, chemokines or neurotrophic factors. It also seems likely that the trafficking of these mononuclear cells to the site of injury in an in vivo animal model of PD would be a function of several factors, including blood brain barrier breakdown, chemokines, and inflammatory response. 3.3.3.3 Sanfilippo Mouse Model Sanfilippo syndrome type B or mucopolysaccharidosis type III (MPS III) is a group of hereditary lysosomal storage disorders resulting from a failure to break down glycosaminoglycan, heparan sulfate, within lysosomes. Sanfilippo syndrome or each of the four subtypes (A, B, C, and D) is an autosomal recessive disorder caused by the deficiency of a different enzyme in the degradative pathway of heparan sulfate: A) heparan-N-sulfatase, B) -N-acetylglucosaminidase (Naglu), C) acetyl-CoA N91

PAGE 105

acetyltransferase, and D) N-acetylglucosamine-6-sulfatase. The absence of any one of these enzymes leads to undegraded heparan sulfate accumulation in lysosomes and then it is excreted in urine. MPS III is caused by a deficiency of enzyme alpha-N-acetylglucosaminidas (Naglu), as a result of a mutation on chromosome 17q21 in the gene encoding Naglu. Premature death occurs one hundred percent of the time (usually occurs between 11-20 years of age) with devastating clinical abnormalities, severe central nervous system involvement and somatic disease. Sanfilippo syndrome has no cure and affects approximately 1 in 25,000 births in the United States (MPS, 2003) A knockout mouse model of MPS III B, demonstrating biochemical abnormalities similar to the human disease, has been created by disrupting the Naglu gene responsible for heparan sulfate degradation (H. H. Li et al., 1999). In this mouse model, biochemical abnormalities have been report to be similar to those in human patients (no Naglu activity and accumulation of heparan sulfate in many organs). As the mouse ages, pathological changes are observed and become more prominent, which include brain disruptions in the neurons. Visible symptoms start to occur at 6 months; these symptoms include abdominal distention, weight loss, and distended urinary bladder. Behaviorally the mice are hypoactive when tested in open field (Gografe et al., 2003). All animals used in the study were obtained (a gift) from Dr. Svitlana Garbuzova-Davis established colony of Naglu mice, at the University of South Florida, which was developed from three heterozygous breeding pairs resulting is strain B6.129S6-Naglutm1Efn mouse (The Jackson Laboratory, Bar Harbor, Maine). This was the only disease model that was used in the migration studies and there was preliminary data from our laboratory (Dr. Garbuzova-Davis) suggesting the HUCB cells (when injected into the 92

PAGE 106

Naglu mice at one month of age) may increase the life span of these animals. Therefore, the migration study would determine whether HUCB cells would migrate to brain areas in this animal model. Mice [10 Wild Type (WT) and 10 mutant (MT)] were sacrifice near end of life (between 1 to 3 days before death, between 11 to 12 months old), which was determined by physical examination of known signs that are exhibited by this mouse population. Tissue extracts were prepared as describe above and cerebellum and brain stem were dissected from brain this area was labeled whole brain (WB). The graph (Figure 3.14) represents one of three migration assays that were performed for this disease model. As a result of the nature of these findings, we felt there may have been interference with either the protein or some other factor in the tissue extracts. The protein concentration for mutant and wild type mouse extracts ranged from 1900 to 2100 g/mL, which is in the high range of the protein assay (working range of 20 to 2,000 g/mL). Therefore, two more assays were performed, with further diluted extracts, 100 L or 10 L of extract diluted with 200 L or 290 L of media, respectively. These assays did not show a pattern of migration and were not similar to the initial migration assay using extracts that were standardized for protein concentration (protein 500 g/mL) for each condition. Although the statistical analysis showed significantly more cells migrating to the WT cerebellum extract this finding did not match with diluted assays findings. In fact, in the other assays more cells were found migrating to the all tissue extract conditions. Overall, the data for this model is not conclusive. 93

PAGE 107

Figure 3.14 Migration of HUCB Cells to Sanfilippo Tissue Extracts 0.00100.00200.00300.00400.00500.00600.00700.00800.00900.001000.00 Wild WBWild Stem Wild CB Mutant WBMutant Stem Mutant CBBlanksMediaATP Luminescent (RLUs) Tissue Extracts Figure 3.14 Migration of HUCB cells to Sanfilippo tissue extracts. The WT stem cerebellum extract was the only condition that showed a significant difference from media control. Each unknown (the tissue extracts), standards, and controls were performed in triplicate and the migration assay was performed twice for a total of 6 data points per condition. Significance was set at p<0.05. (Students T-test). WB = whole brain, CB = cerebellum and Stem = brain stem. 94

PAGE 108

3.4 Summary The results from the preliminary data have shown that HUCB cells (in vitro) will survive for an extended period and that they are capable of proliferating. After testing several labeling methods, to determine the number of migrated cells, the CellTiter-Glo luminescent assay was determined to be the most reliable method. HUCB cells were shown to be attracted to the neonatal brain extracts from rats. A significant number of HUCB cells were shown to migrate to the 2 and 7 day old whole brain tissue extracts and to hippocampal extract of 7 day old rats. This suggests that the developing brain does contain chemoattractants that are signals to HUCB cells to migrate. It may also be that as the brain develops the chemoattractant signaling may become more restricted to certain brain regions as indicated by the 7 th day. The 6-OHDA rat model of PD, showed the migration of HUCB cells to the substantia nigra and striatum extracts of lesioned rats. In addition, HUCB cells, in vitro, were shown to be inhibited from migrating to the toxin 6-OHDA at doses from 1 to 10 L, which suggest that there are endogenous signals generated by pathology of this model that are capable of attracting these cells to the area of injury. This is an intriguing possibility and other models of PD should be investigated for a chemoattractant property and compared to the 6-OHDA model 95

PAGE 109

The data from the Sanfilippo mouse model was unclear and we could not confirm that HUCB cells migrate to tissue extracts from this disease model. The next two chapters present the data on stroke animals, the cytokines produce in the ischemic tissue extracts, and the cytokines or chemokines present in condition media from HUCB cells in various culturing conditions. 96

PAGE 110

3.5 References Bjorklund, L. M., Sanchez-Pernaute, R., Chung, S., Andersson, T., Chen, I. Y., McNaught, K. S., et al. (2002). Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci U S A, 99(4), 2344-2349. Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A. L., Sadoul, R., et al. (2001). Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease. Prog Neurobiol, 65(2), 135-172. Blum, D., Torch, S., Nissou, M. F., Benabid, A. L., & Verna, J. M. (2000). Extracellular toxicity of 6-hydroxydopamine on PC12 cells. Neurosci Lett, 283(3), 193-196. Boulder Committee, T. (1969). Embryonic vertebrate central nervous system: Revised terminology. Anatomy Recieved, 166, 257-261. Brustle, O., Choudhary, K., Karram, K., Huttner, A., Murray, K., Dubois-Dalcq, M., et al. (1998). Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol, 16(11), 1040-1044. Chen, J., Sanberg, P. R., Li, Y., Wang, L., Lu, M., Willing, A. E., et al. (2001). Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 32(11), 2682-2688. Clancy, B., Darlington, R. B., & Finlay, B. L. (2001). Translating developmental time across mammalian species. Neuroscience, 105(1), 7-17. Cohen, S. B., Gluckman, E., Rubinstein, P., & Madrigal, J. A. (Eds.). (2000). Cord blood characteristics: Role in stem cell transplantation. London: Martin Dunitz Ltd. Curtis, D. R., & Johnston, G. A. (1974). Amino acid transmitters in the mammalian central nervous system. Ergeb Physiol, 69(0), 97-188. Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., et al. (1998). Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol, 16(11), 1033-1039. Freed, C. R., Breeze, R. E., & Fahn, S. (2000). Placebo surgery in trials of therapy for Parkinson's disease. N Engl J Med, 342(5), 353-354; author reply 354-355. Freed, W. J. (2000). Neural transplantation : an introduction. Cambridge, Mass.: MIT Press. 97

PAGE 111

Fricker, R. A., Carpenter, M. K., Winkler, C., Greco, C., Gates, M. A., & Bjorklund, A. (1999). Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci, 19(14), 5990-6005. Gografe, S. I., Garbuzova-Davis, S., Willing, A. E., Haas, K., Chamizo, W., & Sanberg, P. R. (2003). Mouse model of Sanfilippo syndrome type B: relation of phenotypic features to background strain. Comp Med, 53(6), 622-632. Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo and 5-iododeozyuridine: a new reagent for detection of DNA replication. Science, 218, 474-475. Isacson, O., Bjorklund, L., & Pernaute, R. S. (2001). Parkinson's disease: interpretations of transplantation study are erroneous. Nat Neurosci, 4(6), 553. Isacson, O., van Horne, C., Schumacher, J. M., & Brownell, A. L. (2001). Improved surgical cell therapy in Parkinson's disease. Physiological basis and new transplantation methodology. Adv Neurol, 86, 447-454. Li, H. H., Yu, Y. H., Rozengurt, N., Zhao, H. Z., Lyons, K. M., Anagnostaras, S., et al. (1999). Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding a-N-acetylglucosaminidase. Proc Natl Acad Sci (USA), 96, 14505-145010. Li, Y., Chen, J., Wang, L., Zhang, L., Lu, M., & Chopp, M. (2001). Intracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Neuroscience Letters, 316(2), 67-70. Lois, C., & Alvarez-Buylla, A. (1993). Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A, 90(5), 2074-2077. MPS. (2003, 2003). MPS Disorder Fact Sheet. Retrieved 5-10-2005, 2005, from http://www.mpssociety.org/mps3.html Olanow, C. W., Kordower, J. H., & Freeman, T. B. (1996). Fetal nigral transplantation as a therapy for Parkinson's disease. Trends Neurosci, 19(3), 102-109. Parkinson, J. (1817). An Essay on the Shaking Palsy. London: Sherwood, Neely and Jones. PDF. (2005). Parkinson's diseae fact sheet. New York: Parkinson's Disease Foundation. 98

PAGE 112

Piccini, P., Brooks, D. J., Bjorklund, A., Gunn, R. N., Grasby, P. M., Rimoldi, O., et al. (1999). Dopamine release from nigral transplants visualized in vivo in a Parkinson's patient. Nat Neurosci, 2(12), 1137-1140. Porter, C. C., Totaro, J. A., & Stone, C. A. (1963). Effect of 6-hydroxydopamine and some other compounds on the concentation of norepinephrine in the hearts of mice. Journal of Pharmacology & Experimental Theraputics, 140, 308-316. Senoh, S., Creveling, C. R., Udenfriend, S., & Witkop, B. (1959). Chemical, enzymatic and metabolic studies on teh mechanism of oxidation of dopamine. Journal of American Chemicals Society, 81, 6236-6240. Senoh, S., Witkop, B., Creveling, C. R., & Udenfriend, S. (1959). 2,4,5-tri-hydroxyphenetylamine, a new matbolite of 3,4-dihydroxyphenetylamine. Journal of American Chemicals Society, 81, 1768-1769. Vendrame, M., Cassady, C. J., Newcomb, J., Bulter, T., Pennypacker, K. R., Zigova, T., et al. (2004). Infusion of human unbilical cord blood cells in rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke, 35, 1-6. Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., et al. (2003). Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res, 73(3), 296-307. Willing, A. E., Vendrame, M., Mallery, J., Cassady, C. J., Davis, C. D., Sanchez-Ramos, J., et al. (2003). Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant, 12(4), 449-454. Yang, H., Mujtaba, T., Venkatraman, G., Wu, Y. Y., Rao, M. S., & Luskin, M. B. (2000). Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation. Proc Natl Acad Sci U S A, 97(24), 13366-13371. Zigova, T., Betarbet, R., Soteres, B. J., Brock, S., Bakay, R. A., & Luskin, M. B. (1996). A comparison of the patterns of migration and the destinations of homotopically transplanted neonatal subventricular zone cells and heterotopically transplanted telencephalic ventricular zone cells. Dev Biol, 173(2), 459-474. Zigova, T., & Newman, M. B. (2002). Transplantation into neonatal rat brain as a tool to study properties of stem cells. Methods Mol Biol, 198, 341-356. 99

PAGE 113

Chapter 4 4.0 Stroke Induced Migration of Human Umbilical Cord Blood (HUCB) Cells: Time Course and Cytokines 4.1 Abstract There is often a significant delay from the onset of ischemia to the time when therapy is initiated in stroke patients. Thus, there is a critical need to extend the therapeutic window regardless of the treatment regime. Previously, we have shown significant improvements in the behavioral defects of rats that received HUCB cells twenty four-hours after a middle cerebral arterial occlusion (MCAO). While the delivery of HUCB cells at 24 hours was effective, the optimal time to administer these cells has not been established. Thus, this study investigated the migration of HUCB cells to ischemic tissue extracts over several time points with an in vitro model. A forty eight-hour window of opportunity was established for the delivery of HUCB cells to ischemic brain. The migrated HUCB cells formed cell aggregates in the ischemic extracts, suggesting cell-to-cell signaling occurs and the expression of cytokines/chemokines in the ischemic extracts suggests their participation in HUCB cell migration. The results from this study are promising in that the current 3-hour therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of cord blood cell therapy with the peak at 48 hours. 100

PAGE 114

4.2 Introduction Stroke is a leading cause of disability worldwide and in the United States affects approximately 700,000 (~500,000 first and 200,000 recurrent stroke) people annually (AHA 2005). Although, in recent years there has been an increase in stroke survivors, there is still a need to develop more effective treatments and rehabilitative therapies that will allow fuller recovery than what is presently seen. The only Food and Drug Administration (FDA) approved drug treatment for acute ischemic stroke is the thrombolytic tissue-Plasminogen Activator (t-PA), which acts by breaking down the blood clot in the vessel, thus allowing the blood to flow through. However, t-PA must be administered within the first three hours after the onset of acute ischemia and the effectiveness of t-PA decreases with time (NINDS, 1995). Unfortunately, approximately 40% of stroke patients do not reach a medical establishment within the 3-hour treatment window, and the possibility of intracranial hemorrhage must be ruled out by CT thus furthering the delay in treatment. Effective treatments with a longer window of opportunity that increases survival rate and improves subsequent recovery are desirable. This could also lower the cost of on-going and long-term care. Our research group, demonstrated that transplanted HUCB cells significantly improve behavioral defects in animal models of stroke (J. Chen et al., 2001; Vendrame et al., 2004; Willing, Lixian et al., 2003; Willing, Vendrame et al., 2003), spinal cord injury (Saporta et al., 2003), and traumatic brain injury (TBI) (Lu et al., 2002). Further, HUCB cells lengthen life span in mouse model of amyotrophic lateral sclerosis (ALS) (Chen & Ende, 2000; Garbuzova-Davis et al., 2003). In addition, others have shown functional 101

PAGE 115

recovery in a mouse stroke model after delivery of CD34 + HUCB cells; this functional recovery was accompanied with neovascularization and neuroangiogenesis at the infarcted site (Taguchi et al., 2004). Thus, the improved behavioral defects in damaged CNS animals utilizing HUCB cells is well recognized and has been extensively reviewed (Newman, Davis, Borlongan, Emerich, & Sanberg, 2004; Newman, Davis, Kuzmin-Nichols, & Sanberg, 2003; Newman, Emerich, Borlongan, Davis Sanberg, & Sanberg, 2004). However, several factors pertaining to the HUCB cell treatment model of induced MCAO in animals require additional investigation. Some of the most critical factors that require further analysis are the dose of HUCB cells required, location site(s) of infusion, delivery time of the cells after induction of MCAO and possible cytokine/chemokines that may attract cells to the infarct area or produce the improvements seen in behavioral deficits. Vendrame et al., (2004), studied the number of HUCB cells required in MCAO rats, and showed that the HUCB cells dose dependently reduced the infarct volume of ischemic rats. The optimal dose was 10 7 cells; at this dose, infarct volume was 11.46 + 4.13% of the total hemisphere while control rats (media injected) had an infarct volume of 33.15 + 4.29%. Particularly important to these findings was the observation that the number of cells that migrated to the site of injury was far less than the number of cells that were administered. Similar results were also obtained in spinal cord injury (Saporta et al., 2003), and TBI models (Lu et al., 2002). The location in which to delivery these cells was addressed in a study by Willing et al., (2003). They showed that intravenous delivery of HUCB cells to stroke rats resulted in significantly enhanced 102

PAGE 116

behavioral improvements compared to intrastriatal injection. However, the time at which to administer these cells has not been addressed until now. Typically, HUCB cells have been administered 24 hours after inducing MCAO in the rats, and the evolving damage and death of cells, from the ischemic insult have been reported to continue up to 21 days post-ischemia (Butler, Kassed, Sanberg, Willing, & Pennypacker, 2002). The loss of blood flow induces an ischemic cascade of events that begins within seconds with the accumulation of Na + in neurons leading to the shortage of ATP and general loss of ionic homeostasis. The immediate and long-term tissue damage depends on the total reduction in cerebral blood flow (CBF), the amount of time the tissue is ischemic, the presence of residual flow and the amount of CBF that returns (Maxwell, 1999). In addition to the primary insult, an inflammatory response starts within hours and can last for days, which can lead to further damage from pro-inflammatory cytokines (i.e. tumor necrosis factor-alpha (TNF-), IL-1 and IL-6, (see Vila et al., 1999), free oxygen radicals, and nitric oxide (Liu et al., 2001). Therefore, in the treatment of stroke there may be on optimal therapeutic window in which to deliver HUCB cells that will assist in preventing or reducing the inflammatory insult. Thus, this study systematically addressed the optimal period to administer HUCB cells, by determining when enhanced migration of HUCB cells to ischemic tissue extracts is observed in vitro. In addition, the second part of this study addressed the cytokines contained within ischemic tissue extracts. The results will help determine whether a critical window of opportunity exists to administer these cells and provide insight into the possible chemokines/cytokines involved in ischemia and the migration of HUCB cells to the infarct site. 103

PAGE 117

4.3 Material and Methods 4.3.1 Animal Care and Handling Adult male Sprague-Dawley rats (Harlan) weighing 200 to 300g were used for this study (n = 6 per condition for a total of 54 rats). Animals were housed in pairs in a temperature-controlled room (22 + 3 0 C) with free access to food and water, and kept on a 12 hour light /dark cycle. The rats were handled and monitored daily for two weeks before MCAO surgery and afterward until they were sacrificed. All experimental procedures were approved by the USFs Institutional Animal Care and Use Committee and comply with the National Institute of Health, Guide for the Care and Use of Laboratory Animals. 4.3.2 Middle Cerebral Artery Occlusion Surgery Animals were anesthetized with 5% Isoflurane in 1L O 2 /min and maintained in 1.0 to 2.0% Isoflurane in 1L O 2 /min throughout surgery. Anesthetized rats were placed in a supine position and a neck incision was made to expose the junction between the right external and internal carotid arteries. The right common carotid, external carotid, internal carotid, and pterygopalatine arteries were isolated using blunt dissection techniques. The common carotid was dissected free of the vagus nerve. The external carotid artery was permanently tied in two positions rostrally and a cut was made between the two sutures. After clamping the common and internal carotid arteries, a 26-gauge needle was inserted into the lumen of the external carotid artery and an embolus (a nylon filament 40 mm in length) was inserted through the lumen of the internal carotid 104

PAGE 118

artery to the origin of the middle cerebral artery (approximately 25 mm from point of entry). The filament was tied to produce a permanent stroke and the neck incision was closed. Body temperature was kept within normal limits by use of heating pads until the rat recovered. All animals received Baytril 0.5 ml/kg s.c. and Ketofen 5 mg/kg i.m. for 3 days after surgery and were monitored continuously until sacrificed. 4.3.2.1 MCAO Behavior Test To insure MCAO surgery induced ischemia the Postural Tail-Hang test (also known as The Forelimb Akinesia test) was performed on rats anywhere between 3 to 24 hours after surgical recovery. Briefly, the rats were held by the base of the tail and the positions of the forelimbs were recorded. Rats in which stroke was induced demonstrated a characteristic posture with the left forelimb flexed to their chest and with the right forelimb stretched out. Only the rats that met criteria with complete flexure of the forelimb were included in the study. 4.3.3 Preparation of Brain Tissue Extracts Animals were sacrificed by exposure to CO 2 at 4, 6, 12, 24, 48, 72 hours or 1 week after MCAO. Brains were removed within two minutes, immediately flash-frozen and then stored at 0 C until needed. The frozen brains were semi-thawed, kept cold, and the striatum, hippocampus, and the cortex were dissected from both the ipsilateral and contralateral sides to the occlusion. The same dissections were performed for sham surgery and normal rats. Tissue from each of the conditions were pooled, and kept on ice in clear Dulbeccos modified Eagles medium (DMEM, Invitorgen, Carlsbad). Tissue was homogenized at 150 mg of tissue per 1mL of media. The homogenates were 105

PAGE 119

centrifuged (4000g for 20 minutes), the tissue extracts (supernatant) collected, filtered (0.22 m, Millipore, Bedford, MA), and stored at -80 0 C until used. 4.3.4 Protein Assay Bicinchoninic acid (BCA) protein assays (Promega, Madison, WI) were used to determine the total amount of protein in the tissue extracts for each condition and for both the positive and negative controls used in the migration assay. Protein assays were performed twice and each unknown (tissue extracts and controls) or standard were ran in triplicate (three wells per assay for 6 data points) according to the manufacturers instructions. Briefly, 25 L of tissue extracts or controls were pipetted directly into the wells of a 96-well plate, 200 L of working reagent was added, and the plate was placed on a shaker for 30 seconds, covered, and incubated at 37 0 C for 30 minutes. The standard curves were performed in the same 96 well plate and the assay had a working range of 20 to 2,000 g/mL of protein. The plates were read in an automated plate reader (Synergy, BioTek, Winooske, VT) set to absorbance with wavelength of 562 nm. After measuring the protein concentration for rat ischemic tissue extracts they were standardized to 500 g/mL for use in migration, cytokine, and ELISA assays. 4.3.5 Preparation of HUCB Cells Cryopreserved mononuclear fraction of HUCB cells were obtained from Saneron CCEL Therapeutics, Inc. Cells were thawed and pipetted into 9 mL of media [clear DMEM with 5% FBS and 1 L/1mL of Gentamicin (Sigma, St. Louis)]. Cells were then centrifuged at 400 g for 15 minutes, the supernatant was removed, and the cells 106

PAGE 120

resuspended in 1 mL of media. Cord blood cells were then plated in low adherence 6 well culture dishes (Corning, Corning, NY) for 24 hrs in a water jacket incubator set at 37 0 C and with 5% CO 2.. After 24 hrs cells were lifted by gentle pippetting, placed in a 15 mL tube, centrifuged, resuspended in 1 mL of media without FBS, and viability assessed using the trypan blue dye exclusion method. Only HUCB cells with 80% or greater viability were used and cell concentration was adjusted to 62,000 cells/25 L of media. 4.3.6 Migration Assay The tissue extracts (supernatant) of each brain area (striatum, hippocampus, and cortex) from both the ipsilateral and contralateral sides and for each time period (4, 6, 12, 24, 48, 72 hours, or 1 week after stroke) were pooled together per condition. The 96-Chemotx Chambers (Neuro Probe, Gaithersburg) were used for these migration assays. The chamber is a 96-well plate consisting of bottom wells that hold the unknowns or chemoattractant and a top plate, which has a polycarbonate membrane with 5 m pore size. Either 300 L of the tissue extracts (unknowns), standards, or controls were pipetted into the bottom wells, in triplicate, after which the top plate was securely attached to the bottom plate. The HUCB cells were pipetted directly into the top well at a concentration of 62,000 cells per 25 L. The migration chambers were then placed in a water-jacket incubator at 37C with 5% CO 2 for 4 hours. The top plates were removed and the bottom plates were then centrifuged (300g for 10 minutes) so that all migrating cells would be forced to the bottom. Half the media (150 L) was then removed and a cell viability assay (CellTiter-Glo Kit, Promega), which is based on the incorporation of adenosine 5 triphosphate (ATP) into live cells, was used to determine the number of 107

PAGE 121

cells that had migrated. The migration plate was read in an automated plate reader (BioTek) set to luminescence. 4.3.6.1 Controls Stromal cell-derived factor 1 (SDF-1, 100 ng/mL, Serologicals, Norcross), a chemoattractant known to induce the migration of HUCB CD34 + cells, was used in this study as a positive control. Clear DMEM, which was used as the diluent in the tissue extracts and the media used to culture HUCB cells, was used as a negative control in the migration and protein assays. 4.3.6.2 Standard Curves Standard curves were established for the migration assays and for determining sensitivity of the plate reader. A serial dilution of cells, at a concentration running from 0 to 80,000 cells/300 L of media, was performed in triplicate, and pipetted directly into the bottom wells of a 96-well plate. The CellTiter-Glo cell viability assay was performed according to the manufacturers instructions. The amount of ATP expressed was directly proportional to the number of cells present in each well. 4.3.7 Rat Cytokine Array The rat cytokine antibody array kit (RayBiotech, Inc) simultaneously profiles 19 cytokines from rat brain tissue extracts. Array membranes were placed in the tray provided 2 mL of blocking buffer was added and incubated for 30 minutes at room temperature. After washing [3 times with 2 mL of Wash Buffer I (provided), then 2 times with Wash Buffer II (provided) on shaker for 5 minutes], 1 mL of standardized striatal tissue extracts (12, 48 hours, 1-week MCAO and non-MCAO extract) was added and incubated for 2 hours with the membrane to allow cytokine binding to immobilized 108

PAGE 122

antibodies on the arrays. Membranes were washed again as described above and 1 mL of diluted biotin-conjugated antibodies (4 L in 996 L of blocking buffer) was added, incubated for 2 hours, and washes were repeated. After which 2 mL of diluted HRP-conjugated streptavidin (2 L in 1998 L of blocking buffer) were incubated on membranes for 60 minutes and washes were repeated and decanted. Detection buffer (500 L) was placed on the membranes for 5 minutes, membranes were lifted with forceps to drain excess buffer, placed on Whatman paper, covered with Saran, exposed to x-ray film (1 to 5 minutes) and developed. 4.3.8 ELISA for Rat GRO/CINC-1 and MCP-1 in Ischemic Tissue Extracts Ischemic tissue extracts were prepared as described above. The striatum, hippocampus, and cortex areas in ischemic rats taken at 4, 6, 12, 24, 48, 72 hours and 1 week after MCAO from the ipsilateral and contralateral sides to the occlusion were used in both rat ELISAs. In the rat GRO/CINC-1 ELISA, 100 L of ischemic and control tissue extracts for each condition were incubated in 96 well micro-plate (unknown and standards were performed in triplicate) for 1 hour with immobilized polyclonal antibody to rat GRO/CINC-1. The bound cytokine was incubated with appropriate labeled antibody, substrate solutions, and standard curve performed according to manufacturers instructions (TiterZyme EIA, Assay Designs, Inc.) with a range of 0 300 pg/mL. In the rat Monocyte Chemoattractant Protein-1 (MCP-1) ELISA, 50 L of ischemic and controls tissue extracts were incubated in a 96-well plate (each unknown and standards were performed in triplicate) for 1 hour with immobilized antibody to rat MCP-1. Bound cytokines were incubated with appropriate labeled antibody, substrate solutions, and 109

PAGE 123

standard curve performed all in accordance to manufacturers instructions (Rat MCP-1 ELISA Kit, Pierce Endogen). The assay had a range of 0 1500 pg/mL. The amount of bound GRO/CINC-1 and MCP-1 were measured in a chemoluminescence reader (Synergy, BioTek, Winooske, VT ) set to absorbance at 450nm and 550nm, respectively. 4.3.9 Statistical Analysis Migration assay unknowns (the tissue extracts), standards, and controls were performed in triplicate and the migration assay was performed twice for a total of 6 data points per condition. A two-way analysis of variance was used to analyze data for the migration assay and significance was set at p<0.05. Further analyses of planned comparisons were performed using Dunns Bonferroni (corrected) test. The same analysis was used for the ELISAs, and Students t-Tests were used for the rat cytokine arrays after using Image Pro Plus software (Media Cybernetics, Inc Silver Spring, MD) to determine optical density. 4.4 Results 4.4.1 Migration of HUCB Cell to Ischemic Tissue Extracts When we examined the HUCB cells after migration to the ischemic tissue extracts from striatal and hippocampal conditions at 24, 48, and 72 hours, we observed the formation of cell aggregates which were progressively larger in the later times after stroke (Figure 4.1). This migration of the HUCB cells to the stroked tissue extract is typical of true chemotaxis (the attraction of cells in a positive gradient). Aggregates did not form in the non-stroke brain areas (contralateral sides). The positive control, SDF-1, 110

PAGE 124

showed migration of these cells in a random fashion with no cell aggregation or cluster formation upon reaching the bottom well, indicative of chemokinesis (migration in a random direction). Figure 4.1 Migrated HUCB Cells in Striatal Ischemic Tissue Extracts 111 Figure 4.1 Bright-field photomicrographs of HUCB cells that have migrated to MCAO tissues extracts and controls. Pictures were taken of the bottom well in the 96-well plate on an inverted microscope at 10x. (A,C,E) Shows the numerous HUCB cells that migrated to striatal and hippocampal extracts at both 24 hr and 72 hr after MCAO. Upon reaching the bottom plate, the cells began to form cell-clusters in these conditions. (B, D, F, H) Very few cells migrated to the control conditions and this was typical for all samples tested. (G) Cells also migrated to this chemoattractant SDF-1, however, the pattern of migration was more random with no defined cell clusters. Bar = 100 m applies to all photomicrographs

PAGE 125

This is the first study to report a time-dependent factor in which the mononuclear fraction of HUCB cells migrate to ischemic tissue extracts; the time at which the brains of stroked rats are harvested after MCAO directly effects the number of HUCB cells that migrate toward brain areas damaged by ischemia (Table 4.1). Significantly more HUCB cells migrated to the ipsilateral striatal extract at 24, 48 and 72 hours after MCAO compared to the same area on the contralateral side ( p <0.0001) and to media control (Figure 4.2.A). Significantly fewer cells migrated to striatal ipsilateral tissue extracts at 4, 6, and 12 hours compared to their contralateral control side ( p <0.05). However, by 1 week there were no significant differences between the tissue extracts of either side. The tissue extracts for the hippocampal area showed similar results as the striatum, at both 48 and 72 hours (Figure 4.2.B); significantly more HUCB cells migrated to the stroke side than to the non-stroke side, media control, and when compared to the stroke side at 4, 6, 12 hours and 1 week ( p <0.0001). A trend similar to that seen with striatal extracts were observed in the earlier time points with fewer HUCB cell migration at 4 and 6 hours to the hippocampal ipsilateral tissue extracts when compared to their contralateral side (p<0.005). When we examined the cortex, there were significant main effects for the side of the brain and time factors (p <0.0001, Table 4.1) and a side by time interaction ( p <0.0001). Upon further analysis, there were only significant differences between the stroke and non-stroke cortex extracts at 24 hours and 1 week (Figure 4.2.C). Fewer cells migrated to the cortex stroke brain tissue extract at 24 hours compared to the non-stroke side. Lastly, at 1 week more HUCB cells migrated to the cortex stroke brain tissue extract than the non-stroke side. 112

PAGE 126

Table 4.1. Analysis of Variance for HUCB Cells Migration to Striatum Source df F-Ratio Prob Striatum (A) 1 0.35482 0.5333 Time (B) 6 467.3022 <0.0001 A X B 6 137.6980 <0.0001 Within Cell 70 Total 83 Analysis of Variance for HUCB Cells Migration to Hippocampus Source df F-Ratio Prob Hippocampus (A) 1 14.9061 <0.0002 Time (B) 6 559.209 <0.0001 A X B 6 178.2521 <0.0001 Within Cell 70 Total 83 Analysis of Variance for HUCB Cells Migration to Cortex Source df F-Ratio Prob Cortex (A) 1 32.4057 <0.0001 Time (B) 6 99.1623 <0.0001 A X B 6 71.2945 <0.0001 Within Cell 70 Total 83 113

PAGE 127

114 Figure 4.2 Migration of HUCB Cells to Stroke Tissue Extracts 020406080100120140160180200220240 02040608010012014160180200220240 Non-Stroke Stroke A. B. ATP Luminescence (RLUs) * 0 # # # 4 hrs 6 hrs 12 hrs 24 hrs 48 hr 72 hrs 1 w k Striatal Extracts B. * ATP Luminescence (RLUs) # # 4 hrs 6 hrs 12 hrs 24 hrs 48 hr 72 hrs 1 w k Hippocampal Extracts

PAGE 128

115 020406080100120140160180200220240 020406080100120140160180200220240 Non-Stroke Stroke C. ATP Luminescence (RLUs) # # # 4 hrs 6 hrs 12 hrs 24 hrs 48 hr 72 hrs 1 wk Cortex Extracts D. ** ATP Luminescence (RLUs) (358) Figures 4.2 The relative number of HUCB cells that migrated to MCAO brain tissue extracts at 4, 6, 12, 24, 48, 72 hrs and 1 week after ischemia and to controls. (A) There was asignificant number of HUCB cells that migrated to the stroked striatal tissue extracts at 24 hours (*) and to the stroked striatal and (B) hippocampal tissue extracts at 48 and 72 hour conditions (*) when compared to tissue extracts from non-stroke side or plain media. (A) Interestingly, at 4, 6, and 12 hours significantly fewer cells (#) migrated to the striatal stroke tissue extracts compared to non-stroked control. Controls SDF-1 Media Figure 4.2 (continued) (B) And at 4 and 6 hours after stroke fewer cells (#) migrated to hippocampal stroke tissue extracts compared to non-stroke hippocampal extracts. (C) At 1 week (#) more cells migrated to cortex stroke extracts than from cortex non-stroke extracts while at 24 hr fewer cells migrated to the stroke side when compared to non-stroke side. (D) Significantly more HUCB cells (**) migrated to SDF-1 chemoattractant and fewercell migrated to the media control (#) compared to all other conditions.

PAGE 129

4.4.2 Rat Cytokine Array The rat cytokine array showed that the cytokine with the most intensity was Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) for striatal tissue extracts at 12, 48 hours and 1 week when compared to non-stroke tissue extracts and control media (Figure 4.3). Figure 4.3 Rat Ischemic Extracts Cytokine Array Figure 4.3 Photomicrographs of the rat cytokine membrane film. (A,B) Shows cytokines expressed at 12 hours after ischemia in striatal extracts. TIMP-1 was more intense at 12 and 48 hours and 1 week in striatal ipsilateral sides (B, D, and F). (C and D) At 48 hours MCP-1 andCINC-1 were also, elevated compared to control sides. (E and F) By 1 week CINC-1 and -NGF weremore intensely labeled on the stroked side than the non-strokedside. (G) DMEM (the media) used for tissue processing,shows no cytokine expression. (H) Normal striatal rat brain extract had no measurable cytokine expression. Students T-test was used to test significance ( p < 0.05). Optical density of stroke conditions (B,D,F) were compared to non-stroke conditions (A,C,E). In addition, the other cytokines visualized and found significant in order of intensity were MCP-1 and Cytokine-induced Neutrophil Chemoattractant-1 (CINC-1) at both 48 hours and 1 week, CINC-3 and Nerve Growth Factor-beta (-NGF) at 1 week 116

PAGE 130

compared to non-stroke tissue extracts and media controls. Based on these data we chose to investigate further Growth-Regulated Oncogene/CINC-1 (GRO-CINC-1) and MCP-1 using ELISA to determine expression over time after MCAO in rats. GRO-CINC-1 has been reported to be the rat equivalent to human Interleukin 8 (IL-8) (Ramos, Heluy-Neto, Ribeiro, Ferreira, & Cunha, 2003). Even though rats do not produce IL-8 they do respond to it. Mast cells in the HUCB cell population have also been reported to produce IL-8. 4.4.3 Rat ELISAs Unexpectedly, both GRO/CINC-1 and MCP-1 showed a time-dependent pattern that was similar to the time course of cell migration (Figure 4.4 and 4.5). The striatal ischemic tissue extracts had significantly more GRO/CINC-1 at 4, 6, 24, and 48 hours compared to non-ischemic striatal extracts at the same time points (Figure 4.4.A). Similar results were seen with hippocampal tissue extracts where GRO/CINC-1 concentrations significantly increased at 4, 12, 48 and 72 hours, while the cortex extracts had elevated levels at all time points compared to non-ischemic extracts (Figure 4.4.B). By 1 week for striatal and hippocampal ischemic extracts levels of GRO/CINC-1 had returned to normal, however the level remain elevated in the cortex extracts. In sham surgery and normal rat brain extracts GRO/CINC-1 were significantly lower than the ischemic striatal, hippocampal, and cortex concentrations at all times (Figure 4.4.C and D). 117

PAGE 131

Figure 4.4 Levels of GRO/CINC-1 in Ischemic Extracts 0.00050.000100.000150.000200.000250.000300.000350.0004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK Stroke Non-Stroke 0.00050.0000.0000.0000.0000.000300.000350.0004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK Stroke Non-StrokeGRO/CINC-1 pg/mL A. * 10152025 Striatal Extracts B. * GRO/CINC-1 pg/mL * Hippocampal Extracts 118

PAGE 132

Figure 4.4 Continued Cortex Extracts C. D. 350.00 Controls 0.00050.000100.000150.000ShamsLeft ShamsRightNormalLeft NormalRight 0.0050.00100.00150.00200.00250.00300.004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK Stroke Non-Stroke GRO/CINC-1 pg/mL * * Figure 4.4 ELISA graph of GRO/CINC-1 in rat ischemic and non-ischemic tissue extracts. (A, B, C) GRO/CINC-1 concentration in ischemic (stroke) rat tissue extracts from 4 hours to 1 week. (*) shows significant difference from controls (non-stroke) at corresponding time points. (D) GRO/CINC-1 concentration in striatal extracts from sham surgery and normal brain controls. Presented controls represents the most elevated levels from sham and normal brain. 119

PAGE 133

The MCP-1 in ischemic tissue extracts was expressed later then GRO/CINC-1. In striatal ischemic extracts, significant elevations of MCP-1 were shown at 6, 12, 48 and 72 hours (Figure 4.5.A), while hippocampal ischemic extracts showed increased levels at 12, 24, 48, and 72 hours (Figure 4.5.B) and the cortex only showed increases in MCP-1 at 6, 12 hours and 1 week (Figure 4.5.C) compared to non-ischemic tissues. MCP-1 for sham surgery and normal rat brains was extremely lower compared to concentrations observed in ischemic conditions (Figure 4.5.D). 120

PAGE 134

Figure 4.4 Levels of MCP-1 in Ischemic Extracts A. Non Stroke 0.000300.000600.000900.0001200.0004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK * 1500.000 MCP-1 Level in pg /mL Striatal Extract Striatal Extract B. * 0.000300.000600.000900.0001200.0001500.0004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK * MCP-1 Level in pg /mL Hippocampal Extract 121

PAGE 135

Figure 4.5 Continued C. Stroke Non Stroke 0.0000300.0000600.0000900.00001200.00001500.00004HRS 6HRS 12HRS 24HRS 48HRS 72HRS 1WK D. Cortex Extracts * * MCP-1 Level in pg /mL MCP-1 Level in pg /mL 122 0255075100ShamsLeft ShamsRightNormalLeft NormalRight Controls Figure 4.5 ELISA graph of MCP-1 in rat ischemic and non-ischemic tissue extracts. (A, B, C) MCP-1 concentration in ischemic rat tissue extracts from 4 hours to 1 week. (*) shows significant difference from control at corresponding time points. (D) MCP-1 concentration from sham surgery and normal brain controls. The controls present here represent the most elevated levels in all control conditions.

PAGE 136

4.5 Discussion In utilizing an in vitro model of migration, we were able to emulate the actions of HUCB cells in the microenvironment of stroke. The present findings are the first to show the aggregation and migration of HUCB cells to ischemic tissues extracts with a time-dependent factor. In addition, this is the first report of the levels of GRO/CINC-1 and MCP-1 in ischemic extracts at several periods after stroke has occurred. Once the HUCB cells migrated to the tissue extracts, cell clusters formed on the bottom well with smaller aggregates of cells in the striatal and hippocampal tissue extracts at 24 hours, and larger aggregates at 48 and 72 hours. Although some cells migrated to extracts of the contralateral sides, these cells upon reaching the bottom well did not form clusters (Figure 4.1) suggesting a random migration. Even with the SDF-1 chemoattractant, in which a large number of HUCB cells migrated, no clustered formations were observed which is consistent with our previous observation of this chemoattractant in vitro. The aggregation of these cells in the stroke tissue extracts suggest that in this microenvironment there is cell-to-cell interaction and signaling, which could be mimicking what occurs upon transplantation of these cells in this model of brain injury. We used the MCAO animal model of stroke in rats to establish the optimal time for delivery of these cells for use in transplantation. This model produces localized CNS infarction in which intervention strategies such as neural transplantation can easily be evaluated. The middle cerebral artery receives approximately 80% of the blood passing 123

PAGE 137

through the internal carotid artery. The MCAO model has been shown to produce marked cell damage and neurobehavioral deficits that parallel the ischemic strokes observed in humans. The occlusion of the MCA results in cell damage and/or loss of cells in the striatum, hippocampus, and cortex ipsilateral to the occlusion. Typically, our laboratory and others have transplanted HUCB cells 24 hours after induction of ischemia. The findings from this study suggest that a later time (48 hours) may be a more optimal time to deliver these cells, with more cells migrating to the ischemic extract (Figure 4.2) resulting in greater therapeutic benefits. This is especially important when cells are administered intravenously, after which the cells must migrate a rather far distance to the site of injury. However, research by this laboratory and others have established the proof of principle that intravenous administration of HUCB cells results in vast improvement in behavioral deficits in animal models of stroke (Chen et al., 2001; Taguchi et al., 2004, Willing et al., (2003); Vendrame et al., 2004; Willing, Vendrame et al., 2003). Also, the same has been shown in spinal cord injury (Saporta et al., 2003), TBI (Lu et al., 2002), and ALS (Chen & Ende, 2000; Garbuzova-Davis et al., 2003). The observed migration of HUCB cells to extracts from the injured brain is consistent with earlier reports (J. Chen et al., 2001; Lu et al., 2002). Previously, HUCB cells have been shown to migrate toward ischemic cerebral tissue extract taken at 24 hours after stroke while bone marrow cells migrated to cerebral extracts at 6, 24, and 48 hours after stroke compared to controls (Chen et al., 2001; Chen et al., 2001). However, this is the first study to examine systematically the migration of HUCB cells to selected areas affected by MCAO over time in rats. Together these studies suggest that 124

PAGE 138

endogenous signals generated by the pathology are capable of attracting HUCB cells to the area of insult, and migration is dependent on the area of the brain and the time the cells are administered after stroke. The results also suggest that at earlier time points the injured tissue may be sending out inhibitory cues that inform the cells of a hostile environment. After the initial insult, both cytokines and chemokines are expressed in a complex temporal cascade of events. The pro-inflammatory cytokines have been reported to be released early in response to the loss of blood flow followed by excitotoxicity of the cells within the core of the infarct (for review see (Leker & Shohami, 2002). There is an increase in IL-1, IL-6, TNF-, interferon(IFN-), and transforming growth factor (TGF1 & 2) mRNA expression as early as 1 hour after stroke (Leker & Shohami, 2002). Expression of these cytokines peaks between 12 and 24 hours in permanent focal ischemia as determined by mRNA (Hill et al., 1999). Resident reactive microglia are believed to be one of the sources of these cytokines (Kostulas, Pelidou, Kivisakk, Kostulas, & Link, 1999; Leker & Shohami, 2002). These pro-inflammatory cytokines may exacerbate neuronal damage (Barone et al., 1997; Tarkowski et al., 1995; Yang, Zhao, Davidson, & Betz, 1997) and are known to induce neutrophil infiltration possibly by the stimulation of CINC. Both IL-8 and CINC belong to CXC family of chemokines and are present in ischemic brain. In the cytokine array and ELISAs we showed that CINCs were present in ischemic tissue extracts (Figure 4.3 & 4.4). In the cytokine array, CINC-1 was observed at 48 hours and 1 week, while CINC-3 was weakly observed at 1 week. In the ELISAs although there were peaks at certain time points, GRO/CINC-1 was shown to decrease gradually from 4 hours to 1 week in all brain tissue extracts. CINC-1, GRO/CINC-1 and 125

PAGE 139

IL-8 all serve as proinflammatory agents, attract neutrophils, and serve as the first line of defense in inflammation, which may be why GRO/CINC-1 is highly elevated early in the ischemic striatal extracts (4hrs.). This is especially true if the progression of degenerating tissue is considered (from the core outwards), which may indicate that neutrophils within the HUCB cells are migrating to the core in a progressive manner and aid in reducing the infarct size. Indirect evidence of this has been reported in Vendrame et al. (2004) who showed that HUCB cell infusion reduced the volume of the infarct in MCAO rats. In humans, IL-8 indirectly regulates the migration of neutrophils by stimulating CINC-1 in mast cells (Ramos et al., 2003), directly attracts polymorphonuclear neutrophils (Kostulas et al., 1999), and participates in mediating the inflammatory response and the trafficking of leukocytes into the ischemic area. The infiltration of leukocytes to the ischemic area are believed to be aided by intercellular adhesion molecule (ICAM), endothelial leukocyte adhesion molecule (Pantoni, Sarti, & Inzitari, 1998) and tissue metalloproteinases (MMPs) which are zinc-dependent proteolytic enzymes that are known to weaken the blood brain barrier (Crocker, Pagenstecher, & Campbell, 2004). TIMP-1 was present on cytokine arrays for all ischemic extracts (12hrs, 48hrs, and 1wk) and has also been shown in ischemic rats using other methods (Hedrick, Cohen, Nielsen, & Davis, 1984; Romanic et al., 2002). This cytokine is believed to inhibit MMPs in diverse conditions such as wound healing, angiogenesis, cancer metastasis, and stroke (Crocker et al., 2004). One role of TIMP-1 is to rebuild or remodel the extracellular matrix by the inhibition of MMPs. We suggest here that TIMP-1 is performing a similar function in rebuilding the extracellular matrix that has been 126

PAGE 140

destroyed by MCAO and at a future point may serve as an adjunct therapy, especially because TIMP-1 is seen at 1 week, on both the ipsilateral and contralateral side to the occlusion (even though the intensity is weaker on the contralateral side of the brain). However, the exact function of TIMP-1 in the ischemic brain requires further investigation. In addition, to GRO/CINC-1 an elevation in MCP-1 was also observed in both the rat cytokine array and ELISA. MCP-1 has previously been shown to increase the migration of bone marrow stromal cells to rat cerebral ischemic tissue extracts (Wang, Li, Chen, Gautam et al., 2002; Wang, Li, Chen, Chen et al., 2002). Here MCP-1 concentration peaked at 48-hours in striatal extract on cytokine arrays, and in striatal and hippocampal tissue extracts at 48 and 72-hours in the ELISAs. This is similar to other reported findings for MCP-1 in ischemic tissue (Wang, Li, Chen, Chen et al., 2002). This chemokine strongly attracts monocytes and serves as a potent immunoregulator, which would explain why we see more HUCB cells migrating at the 48-hour period than at earlier or later times. IL-1, IL-6, and TNFparticipate in mediating the inflammatory response and are chemoattractants for leukocytes as well. Although the exact actions of these individual cytokines remains unknown; both TNFand IL-1 have been shown to increase the infarct size (Barone et al., 1997; Yamasaki et al., 1995), while IL-6 may act as both a pro and anti inflammatory agent (for review see Benveniste, 1998). Furthering our understanding of initial cytokines expression will aid in establishing whether they are responsible for inhibiting the migration of HUCB cells at the earlier times to ischemic tissue extracts observed in this study. 127

PAGE 141

SDF-1 was used for a positive control in this study as it has been shown to be a strong chemoattractant for hematopoietic progenitor cells (CD34 + ) from HUCB (Kim & Broxmeyer, 1998) and for monocytes and lymphocytes. In addition, CD34 + HUCB cells express a higher level of the CXCR4 (SDF-1 receptor) which is believed to aid in their ability to home to bone marrow when transplanted (Yong et al., 1999). In a mouse model of MCAO, SDF-1 was reportedly associated with reactive astrocytes, mostly localized within the penumbra, and transplanted green fluorescent protein expressing bone marrow cells were found in areas highly immunoreactive for SDF-1 thus suggesting a chemoattractant role for SDF-1 (Hill et al., 2004). There is evidence that SDF-1 has a broader role by stimulating release of other chemokines, regulating leukocyte trafficking, stimulating the renewal of stem cell, and the release of maturing cells from bone marrow for continued hematopoiesis (Broxmeyer et al., 1999; Lee et al., 2002; Onai et al., 2000). Two studies have reported that SDF-1 selectively induces the chemokine IL-8 from cord blood-derived human mast cells (Lin, Issekutz, & Marshall, 2000, 2001). Therefore, evidence suggests that multiple chemokines are involved in the migration of HUCB cells to ischemic tissue extracts and furthermore, it is likely that several factors, including blood brain barrier breakdown, inflammatory response, and trophic factor production may be involved in the trafficking of these mononuclear cells to the site of injury. The results from this study showed that 48-hours is the optimal time for the migration of HUCB cells to ischemic tissue and suggest that these cells are induced to migrate perhaps by MCP-1, IL-8 (GRO/CINC-1) or other chemokines, and once there the chemokine(s), or other factors, induce these cells to aggregate. Further studies of the 128

PAGE 142

intrinsic and extrinsic mechanisms are required to understand the signals involved in the migration of cells to the site of injury. Our research group is currently investigating the optimal time of 48-hours to deliver HUCB cells in our in vivo model of MCAO in rats, as well as examining the cytokines and chemokines produced by cord blood cells. In conclusion, these new results suggest that a wider therapeutic window (from 48 to 72 hours after stroke) may exist for the treatment of stroke victims using cord blood cells, compared to the existing 3-hour post stroke t-PA. This may also decrease mortality rate and improve long-term outcomes for patients. 129

PAGE 143

4.6 References America Heart Association (AHA). (2005). Heart Disease and Stroke Statistics.Unpublished manuscript, Dallas, Texas. Barone, F. C., Arvin, B., White, R. F., Miller, A., Webb, C. L., Willette, R. N., et al. (1997). Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke, 28(6), 1233-1244. Benveniste, E. N. (1998). Cytokine actions in the central nervous system. Cytokine Growth Factor Rev, 9(3-4), 259-275. Broxmeyer, H. E., Kim, C. H., Cooper, S. H., Hangoc, G., Hromas, R., & Pelus, L. M. (1999). Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors. Ann N Y Acad Sci, 872, 142-162; discussion 163. Butler, T. L., Kassed, C. A., Sanberg, P. R., Willing, A. E., & Pennypacker, K. R. (2002). Neurodegeneration in the rat hippocampus and striatum after middle cerebral artery occlusion. Brain Res, 929(2), 252-260. Chen, J., Li, Y., Wang, L., Lu, M., Zhang, X., & Chopp, M. (2001). Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. Journal of the Neurological Sciences, 189(1-2), 49-57. Chen, J., Sanberg, P. R., Li, Y., Wang, L., Lu, M., Willing, A. E., et al. (2001). Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 32(11), 2682-2688. Chen, R., & Ende, N. (2000). The potential for the use of mononuclear cells from human umbilical cord blood in the treatment of amyotrophic lateral sclerosis in SOD1 mice. J Med, 31(1-2), 21-30. Crocker, S. J., Pagenstecher, A., & Campbell, I. L. (2004). The TIMPs tango with MMPs and more in the central nervous system. J Neurosci Res, 75(1), 1-11. Garbuzova-Davis, S., Willing, A. E., Zigova, T., Saporta, S., Justen, E. B., Lane, J. C., et al. (2003). Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J Hematother Stem Cell Res, 12(3), 255-270. Hedrick, S. M., Cohen, D. I., Nielsen, E. A., & Davis, M. M. (1984). Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature, 308(5955), 149-153. 130

PAGE 144

Hill, J. K., Gunion-Rinker, L., Kulhanek, D., Lessov, N., Kim, S., Clark, W. M., et al. (1999). Temporal modulation of cytokine expression following focal cerebral ischemia in mice. Brain Res, 820(1-2), 45-54. Hill, W. D., Hess, D. C., Martin-Studdard, A., Carothers, J. J., Zheng, J., Hale, D., et al. (2004). SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol, 63(1), 84-96. Kim, C. H., & Broxmeyer, H. E. (1998). In vitro behavior of hematopoietic progenitor cells under the influence of chemoattractants: stromal cell-derived factor-1, steel factor, and the bone marrow environment. Blood, 91(1), 100-110. Kostulas, N., Pelidou, S. H., Kivisakk, P., Kostulas, V., & Link, H. (1999). Increased IL-1beta, IL-8, and IL-17 mRNA expression in blood mononuclear cells observed in a prospective ischemic stroke study. Stroke, 30(10), 2174-2179. Lee, Y., Gotoh, A., Kwon, H. J., You, M., Kohli, L., Mantel, C., et al. (2002). Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines. Blood, 99(12), 4307-4317. Leker, R. R., & Shohami, E. (2002). Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities. Brain Res Brain Res Rev, 39(1), 55-73. Lin, T. J., Issekutz, T. B., & Marshall, J. S. (2000). Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha. J Immunol, 165(1), 211-220. Lin, T. J., Issekutz, T. B., & Marshall, J. S. (2001). SDF-1 induces IL-8 production and transendothelial migration of human cord blood-derived mast cells. Int Arch Allergy Immunol, 124(1-3), 142-145. Liu, M. H., Jin, H. K., Floten, H. S., Yang, Q., Yim, A. P., Furnary, A., et al. (2001). Vascular endothelial growth factor-mediated endothelium-dependent relaxation is blunted in spontaneously hypertensive rats. J Pharmacol Exp Ther, 296(2), 473-477. Lu, D., Sanberg, P. R., Mahmood, A., Li, Y., Wang, L., Sanchez-Ramos, J., et al. (2002). Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant, 11(3), 275-281. 131

PAGE 145

Maxwell, W. L. (1999). Cellular response to ischaemic CNS injury. In M. Berry & A. Logan (Eds.), CNS Injuries: cellular responses and pharmacological strategies. (Vol. Chapter 2, pp. 19-42). New York: CRC Press. Newman, M. B., Davis, C. D., Borlongan, C. V., Emerich, D., & Sanberg, P. R. (2004). Transplantation of human umbilical cord blood cells in the repair of CNS diseases. Expert Opin Biol Ther, 4(2), 121-130. Newman, M. B., Davis, C. D., Kuzmin-Nichols, N., & Sanberg, P. R. (2003). Human umbilical cord blood (HUCB) cells for central nervous system repair. Neurotox Res, 5(5), 355-368. Newman, M. B., Emerich, D. F., Borlongan, C. V., Davis Sanberg, C., & Sanberg, P. R. (2004). Human umbilical cord blood (HUCB) cells to repair the damaged brain. Curr Neurovascl. Res., 1(3), 269-281. NINDS, T. N. I. o. N. D. a. S. r.-P. S. G. (1995). Tissue plasminogen activator for acute ischemic stroke. N Engl J Med, 333(24), 1581-1587. Onai, N., Zhang, Y., Yoneyama, H., Kitamura, T., Ishikawa, S., & Matsushima, K. (2000). Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine. Blood, 96(6), 2074-2080. Pantoni, L., Sarti, C., & Inzitari, D. (1998). Cytokines and cell adhesion molecules in cerebral ischemia: experimental bases and therapeutic perspectives. Arterioscler Thromb Vasc Biol, 18(4), 503-513. Ramos, C. D., Heluy-Neto, N. E., Ribeiro, R. A., Ferreira, S. H., & Cunha, F. Q. (2003). Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1. Cytokine, 21(5), 214-223. Romanic, A. M., Harrison, S. M., Bao, W., Burns-Kurtis, C. L., Pickering, S., Gu, J., et al. (2002). Myocardial protection from ischemia/reperfusion injury by targeted deletion of matrix metalloproteinase-9. Cardiovasc Res, 54(3), 549-558. Saporta, S., Kim, J. J., Willing, A. E., Fu, E. S., Davis, C. D., & Sanberg, P. R. (2003). Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior. J Hematother Stem Cell Res, 12(3), 271-278. Taguchi, A., Soma, T., Tanaka, H., Kanda, T., Nishimura, H., Yoshikawa, H., et al. (2004). Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. The Journal of Clinical Investigation, 114(3), 330-338. 132

PAGE 146

Tarkowski, E., Rosengren, L., Blomstrand, C., Wikkelso, C., Jensen, C., Ekholm, S., et al. (1995). Early intrathecal production of interleukin-6 predicts the size of brain lesion in stroke. Stroke, 26(8), 1393-1398. Vendrame, M., Cassady, C. J., Newcomb, J., Bulter, T., Pennypacker, K. R., Zigova, T., et al. (2004). Infusion of human unbilical cord blood cells in rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke, 35, 1-6. Vila, N., Filella, X., Deulofeu, R., Ascaso, C., Abellana, R., & Chamorro, A. (1999). Cytokine-induced inflammation and long-term stroke functional outcome. J Neurol Sci, 162(2), 185-188. Wang, L., Li, Y., Chen, J., Gautam, S. C., Zhang, Z., Lu, M., et al. (2002). Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture. Experimental Hematology, 30(7), 831-836. Wang, L., Li, Y., Chen, X., Chen, J., Gautam, S. C., Xu, Y., et al. (2002). MCP-1, MIP-1, IL-8 and Ischemic Cerebral Tissue Enhance Human Bone Marrow Stromal Cell Migration in Interface Culture. Hematology (Amsterdam, Netherlands), 7(2), 113-117. Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., et al. (2003). Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res, 73(3), 296-307. Willing, A. E., Vendrame, M., Mallery, J., Cassady, C. J., Davis, C. D., Sanchez-Ramos, J., et al. (2003). Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant, 12(4), 449-454. Yamasaki, Y., Matsuura, N., Shozuhara, H., Onodera, H., Itoyama, Y., & Kogure, K. (1995). Interleukin-1 as a pathogenetic mediator of ischemic brain damage in rats. Stroke, 26(4), 676-680; discussion 681. Yang, G. Y., Zhao, Y. J., Davidson, B. L., & Betz, A. L. (1997). Overexpression of interleukin-1 receptor antagonist in the mouse brain reduces ischemic brain injury. Brain Res, 751(2), 181-188. Yong, K., Fahey, A., Reeve, L., Nicholls, C., Thomas, N. S., Pizzey, A., et al. (1999). Cord blood progenitor cells have greater transendothelial migratory activity and increased responses to SDF-1 and MIP-3beta compared with mobilized adult progenitor cells. Br J Haematol, 107(2), 441-449. 133

PAGE 147

Chapter 5 5.0 Cytokines Produced by Cultured Human Umbilical Cord Blood Cells 5.1 Abstract The potential therapeutic benefits from Human Umbilical Cord Blood (HUCB) cells for the treatment of certain injuries, diseases, and neurodegeneration is becoming increasingly recognized. The utilization of cord blood cells in various animal models, such as ischemia/stroke, traumatic brain injury, myocardial infarction, Parkinsons Disease, and Amyotropic Lateral Scelerosis, has resulted in improvements of behavioral deficits and for some diseases, prolonged lifespan. Previously, we reported the migration of HUCB cells to ischemic brain tissue extracts is time-dependent, and that expression of certain cytokines/chemokines within the ischemic extracts corresponds to this migration pattern. However, the mechanism(s) responsible for the functional improvements in the animal models and for the migration of these cells to site of injury or tissue extracts are unknown. Here we investigated the expression of cytokines/chemokines produced by HUCB cells under various experimental conditions in vitro. Certain cytokines were consistently expressed in the majority of experimental conditions, such as interleukin-8, monocyte chemoattractant protein-1, and interleukin-1. This research provides insight as to the possible cytokines from cord blood cells involved in the functional improvements seen in the animal models of injury/disease. 134

PAGE 148

5.2 Introduction The field of cell replacement/repair for the treatment of diseases and injuries, especially those of the central nervous system (CNS), is a crucial area of research that could eventually benefit millions of people. In the last 20 years, although tremendous progress has been achieved in the cell transplantation field, negative outcomes reported from clinical trials along with ethical concerns involving the use of fetal cells or tissue and embryonic stem cells have delayed progress in the cell replacement/repair field. Our group, along with others, has turned to other potential cell sources in order to continue the progression of this field. We have been utilizing cells of hematopoietic origin. The most promising has been those from human umbilical cord blood (HUCB). Theses cells have been used extensively in the treatment of various nonmalignant and malignant hematopoietic diseases, especially in children, for the last 15 years (for reviews see (Lu, Shen, & Broxmeyer, 1996) (Cohen, Gluckman, Rubinstein, & Madrigal, 2000). Functional improvements in behavioral deficits have been confirmed in rats that were administered HUCB cells following middle cerebral artery occlusion (MCAO) (Chen et al., 2001; Vendrame et al., 2004; Willing, Lixian et al., 2003; Willing, Vendrame et al., 2003). Despite reports that HUCB cells express neural phenotypes both in vitro and in vivo (Garbuzova-Davis et al., 2003; Ha et al., 2001; J. R. Sanchez-Ramos, 2002; J R Sanchez-Ramos et al., 2001; Zigova et al., 2002), few cord blood cells are present in the ischemic region when compared to the number of cells injected (Vendrame et al., 2004), suggesting that cell replacement is not the responsible mechanism for the functional improvements seen. 135

PAGE 149

Previously, we have demonstrated a temporal pattern in the migration of HUCB cells to ischemic tissue extracts (Newman et al., In press; Newman et al., 2003). The time at which the rats brains were harvested after MCAO proved to be a critical factor in the migration of HUCB cells to ischemic brain tissue extracts. The optimal time after stroke proved to be 48 hours. In addition, the striatal ischemic tissue expressed cytokines and the chemoattractants, cytokine-induced neutrophil chemoattractant-1 (CINC-1) and monocyte chemoattractant protein-1 (MCP-1), at 48 hours after MCAO. Further analysis showed that both growth-regulated oncogene/CINC-1 [GRO/CINC-1, the rat equivalent of human interleukin-8 (IL-8)] and MCP-1 from ischemic tissue had time-dependent pattern similar to that observed in the migration assays. The mechanism by which HUCB cells induce functional improvements in behavioral deficits of MCAO rats remains unknown. We have suggested previously that HUCB cells, once at the infarct, are releasing certain cytokines, chemokines or trophic factors that are aiding in the repair and are involved in the functional recovery seen in the stroke animal. Also, the HUCB cells could be releasing signals that attract endogenous stem cells to the site of injury, which may further aid in the recovery of the injured animals. In this study we sought to identify properties of HUCB cells that may be responsible for the therapeutic benefit seen in the rat model of MCAO. Specifically, we have investigated cytokines and chemokines produced by HUCB cells under various culture conditions. 136

PAGE 150

5.3 Materials and Methods 5.3.1 Preparation of HUCB Cells Cryopreserved mononuclear fractions of HUCB were obtained from Saneron CCEL Therapeutics, Inc. (Tampa, FL.). Frozen samples were thawed either in 10 mL of clear DMEM with 5% FBS and Gentamicin (50 g/mL, Sigma) or with Ex Vivo 10 media (Cambrex) with Gentamicin (50 g/mL). Cells were centrifuged at 400 g for 15 minutes, the supernatant was removed, and the cells re-suspended in 1 mL of the same media. The viability of all samples ranged from 73 to 95 percent as determined by the trypan blue exclusion method. Cord blood cells were cultured in 5 mL of media in low adherence 6 well plates (Costa, Corning) in an incubator set at 37 0 C and with 5% CO 2.. Cells were treated in accordance with the protocols for each experiment as described below, after which Conditioned Media (CM) (supernatant) was transferred to sterile conical tubes by gentle pipetting, and centrifuged at 400 g for 15 minutes to remove any remaining cells and stored at -80 0 C. 5.3.2 Human Cytokine Array Protocol 137 Human cytokine antibody array kits (RayBiotech, Inc) simultaneously profiled either 23 (Array 1.0) or 43 (Array 3.0) active cytokine/chemokine proteins in CM. Array membranes were placed in the provided 6-well tray with 2 mL of blocking buffer and incubated for 30 minutes at room temperature. After washing [3 times with 2 mL of Wash Buffer I (provided), then 2 times with Wash Buffer II (provided) while on a shaker for 5 minutes] 1 mL of CM and control media were added to separate wells and incubated for 2 hours with the membrane to allow cytokine binding to immobilized antibodies on

PAGE 151

the arrays. Membranes were washed again as described above and 1 mL of diluted biotin-conjugated antibodies (4 L in 996 L of blocking buffer) was added, incubated for 2 hours, and washes were repeated. Diluted HRP-conjugated streptavidin (2 L in 1998 L of blocking buffer) was incubated on membranes for 60 minutes, washes were repeated and the supernatant decanted. Detection buffer (500 L) was placed on the membranes for 5 minutes, membranes were carefully lifted with forceps to drain excess buffer, placed on Whatman paper, covered with Saranwrap, exposed to x-ray film (1 to 5 minutes) and the film was developed. In addition to the positive and negative controls on each array, we employed cultured media that had not been used to culture the cells as a control array, which was used to compare with CM arrays. 5.3.2.1 Human Cytokine Array Time Response Cells from the mononuclear fraction of HUCB (10 million/5mL) were cultured in DMEM with 5% FBS and Gentamicin (50 g/mL) for 4 days. Media was removed and fresh DMEM with no FBS was incubated with the cells for an additional 6 days for a total of 10 days or for additional 18 days for a total of 28 days, with media changes occurring every 3 days. The CM and control media were then processed as described above and 1 mL of each used in the human cytokine array (Ver. 1.0). 5.3.2.2 Human Cytokine Array Seeding Density Cells from the mononuclear fraction of HUCB were cultured in serum free DMEM with Gentamicin (50 g/mL) for 3 days at concentrations of 5, 10, or 30 million cells/5mL of media. The CM and control media were then processed as described above and 1 mL of each used in the human cytokine array (Ver. 1.0). 138

PAGE 152

5.3.2.3 Human Cytokine Array Hematopoietic Media and Stimulants HUCB cells (10 million/5mL) were cultured for 1, 5, or 12 days in serum free Ex Vivo 10 media with Gentamicin (50 g/mL). In addition, cord blood cells (10 million/5mL) were cultured for 4 days in serum free Ex Vivo-10 media with Gentamicin and then media was changed and supplemented with either human Interleukin-3 [5 ng/mL, (IL-3); BioSource International, Camarillo, CA], thrombopoietin [25 ng/mL, (TPO); Chemicon International, Temecula, CA] or no supplementation for an additional 5 days after which media was changed to plain Ex Vivo-10 for additional 3 days for a total of 12 days in culture. The CM and control media were then processed as described above and 1 mL of each used in the human cytokine array (Ver. 3.0). 5.3.3 ELISA for Human IL-8 and MCP-1 in Culture HUCB Cells HUCB cells were cultured for 3 and 7 days in serum free DMEM or Ex Vivo-10 with Gentamicin (50 g/mL). CM was obtained according to the protocol described above. ELISA kits (Endogen, Pierce) were used to measure the amount of human IL-8 and MCP-1 in CM produced by HUCB cells according to the manufacture instructions. Briefly, 50 L of CM for each condition and controls were pipetted, in triplicate, along with serial diluted standards, into a 96-strip well plate, incubated for 1 hour with immobilized antibody at room temperature and wells were washed 3 times. Then biotinylated antibody (50 L) was added, incubated for 1 hour at room temperature, and washed 3 times. Next 100 L of streptavidin-HRP solution was added to wells incubated for 30 minutes and wells washed 3 times. To allow enzymatic color reaction TMB Substrate Solution was added to each well (100 L) incubated for 30 minutes in the dark, stop solution (100 L) was added incubated for 30 minutes, and ELISAs plates were 139

PAGE 153

measured in a chemoluminescence reader (Synergy, BioTek, Winooske, VT ) set to absorbance at 450nm and 550nm. 5.3.4 Migration Assays HUCB cells were prepared as described above and cultured for 24 hours before use. The 96-Chemotx Chambers (Neuro Probe, Gaithersburg) were used for the migration assays. The chamber is a 96-well plate consisting of bottom wells that hold the unknowns or chemoattractant and a top plate, which is a polycarbonate membrane with 5 m pore size. The chemoattractants used were human recombinants MCP-1 (5, 10, 20, and 30 ng/mL; Endogen, Woburn, MA), IL-3 (1, 5, and 10 ng/mL) TPO (10, 25, and 40 ng/mL), and Stromal cell-derived factor 1 [100 ng/mL, positive control (SDF-1); Serologicals, Norcross, GA], along with the serum free DMEM and Ex Vivo-10 (300 L/well) were pipetted directly into the bottom wells, in triplicate, after which the top plate was securely attached. The HUCB cells were pipetted directly into the top well at a concentration of 62,000 cells per 25 L. The migration chambers were then placed in a cell culture incubator at 37C with 5% CO 2 for 4 hours. The top plates were removed and the bottom plates were then centrifuged (300 g for 10 minutes) so that all migrating cells would be forced to the bottom. Half the media (150 L) was then removed and a cell viability assay (CellTiter-Glo Kit, Promega) based on the incorporation of adenosine 5 triphosphate (ATP) into live cells was used to determine the number of cell that had migrated. The migration plate was read in an automated plate reader (BioTek) set to luminescence. 140

PAGE 154

5.3.5 Statistical Analysis The membranes used in the cytokines arrays were developed onto x-ray film. The x-ray film was then scanned and the image analysis program, ImagePro-Plus (Version 5.0, Media Cybernetics, San Diego, CA) was used to measure optical density. Higher concentrations of cytokines were denser (darker) on the membrane and had a lower numeric value, which equated to the optical density. The measurements were performed three times by individual investigators blind to the treatment conditions and interrelator reliability was between 85 and 98%. The three measurements were used in the statistical analysis, which was performed using Students t-Test with significant set at p < 0.05 and compare to the control array(s), which was cultured media (no cells). ELISAs also employed Students t-Test to determine significance (p < 0.05), and each condition was compared to control (DMEM or Ex Vivo-10 media). 5.4 Results This is the first study to report that the mononuclear fractions of HUCB cells produce various cytokines and chemokines in response to culture conditions. Interestingly UCB cells in all culture conditions, except for TPO, consistently produced two chemokines, IL-8 and MCP-1. These results support our working hypothesis that transplanted HUCB cells release factors that contribute to the recovery of behavioral deficits in stroke rats and other injury/disease models, and produce chemokines that may be attracting endogenous cells to the area of insult, which are thought to aid in repair and recovery. The most important factors that contributed to the chemokine production were 141

PAGE 155

the amount of time the HUCB cells were in culture and the media and stimulants used. The concentration of cells did not contribute to additional cytokine production. The stronger concentration of a cytokine the denser (darker) it appears on the membrane, which equates to smaller numeric values on the graphs. 5.4.1 Human Cytokine Array Time Response: Cord blood cells were cultured for a total of either 10 or 28 days in DMEM. The CM from cells that were cultured for the 28 days showed more cytokines compared to the 10 days condition (Figure 5.1). All cytokines/chemokines presented in Figure 5.1 were significantly different from control. IL-8 and MCP-1 were at both times and showed an increase in intensity by 28 days. At 10 days in vitro (DIV) the CM from HUCB cells contained epithelial cell-derived neutrophil activating protein (ENA-78), macrophage derived chemokine (MDC), onocostation (OSM), and vascular endothelial growth factor (VEGF) all of which were not present by 28 days in culture. However, by day 28 several additional cytokines and chemokines were present: IL-6, tumor necrosis factor(TNF-), IL-1, IL-10, TNF-, granulocytes-macrophage colony stimulating factor (GM-CSF), interferon(IFN-), MCP-2, and MCP-3. 142

PAGE 156

Figure 5.1 Cytokines Produced by HUCB Cells 10 and 28 DIV 020406080100120140160180200220IL-8MCP-1ENA-78MDCOSMGROVEGFPositiveNegativeBackground A. Optical Density 020406080100120140160180200220IL-8IL-6MCP-1TNF-aIL-1aIL-10TNF-bGROGMCSFIFN-gMCP-2MCP-3PositiveNegativeBackground B. Optical Density 143

PAGE 157

Figure 5.1. Human cytokines produced by HUCB cells at 10 and 28 days in vitro (DIV). Graphs represent the optical density of cytokines in the conditioned media (CM) from cultured HUCB cells that were bound to the membrane array. The higher the concentration of bound cytokines to the membrane the denser (darker) it appears, which when electronically measured for optical density equates to: the smaller the numeric value the greater the concentration of the cytokine. (A) Cytokines in CM from HUCB cells that were culture in DMEM for total of 10 days. (B) Cytokines in CM from HUCB cells that were culture in DMEM for total of 28 days. (A,B) All cytokines present were significantly different from control array (DMEM) and from negative control. The background, negative control and control array were between 90 to 100% identical for optical density measures. All cytokines presented in the graph were significantly different from control array (Students T-test, p < 0.05). 5.4.2 Human Cytokine Array Seeding Density To determine whether the number of cells influenced cytokine production, cord blood cells were cultured at increasing concentrations (5, 10, and 30 million cells). HUCB cells were cultured for 3 days and the CM from all concentrations expressed the same 5 cytokines; IL-8, MCP-1, IL-1, IL-3, and RANTES, which were all significantly denser than control (Figure 5.2.). There was a significant increase in intensity of these cytokine that corresponded to the increase in HUCB cell concentration. 144

PAGE 158

Figure 5.2 Effect of Seeding Density on Cytokine Secretion 020406080100120140160180200220IL-8MCP-1IL-1aIL-3RANTESPositiveNegativeBackground 5 Million Cells 10 Million Cells 30 Million Cells Optical Density Figure 5.2 Human cytokines produced by HUCB cells at increasing concentration. Graph represents the effect of seeding density on cytokine production in cultured HUCB cells. Cells were plated at seeding densities of 5, 10, and 30 million cells/5mL of media (DMEM) for 3 days in vitro (DIV). Cytokines showed a progressive increase in optical density that corresponded to the concentration of HUCB cells plated. All cytokines present were significantly different from control array (DMEM) and from negative control. The background, negative control and control array were between 98 to 100% identical for optical density measures. All cytokines presented in the graph were significantly different from control array (Students T-test, p < 0.05). 145

PAGE 159

5.4.3 Human Cytokine Array Hematopoietic Media and Stimulants To determine whether a serum free media designed to support hematopoietic cells or the addition of proliferative agents would induce or inhibit the release of chemokines in cord blood cells, they were cultured in hematopoietic media with or without the addition of IL-3 or TPO (Figure 5.3 & 5.4). In Ex Vivo-10 (a serum free media designed to support long-term cultures of hematopoietic cells), cord blood cells were induced to release the chemoattractant SDF-1, which was not seen in the DMEM conditions. Again IL-8 was the most prominent chemokine released in all Ex Vivo-10 conditions. Macrophage colony-stimulating factor (MCSF) was present in the 5 and 12 DIV conditions; it was not previously seen in the CM from cells cultured in DMEM (Figure 5.3 B & C). In addition, only in the 5 DIV condition was the cytokine Leptin present in the CM (Figure 5.3 B). Figure 5.3 Effects Hematopoietic Media has on Cytokine Secretion A. IL-8 IL-6 MCP-1 TNF-a IL-10 IL-1a 0 20 40 60 80 100 120 140 160 Optical Density 146

PAGE 160

B. IL-8 MCP-1 OSM IL-1a SDF-1 Leptin TNF-a IL-10 MCSF 0 20 40 60 80 100 120 Optical Density C. IL-8 IL-6 MCP-1 OSM SDF-1 RANTES MDC MCSF Positive Negative Background 0 50 100 150 200 250 Optical Density Figure 5.3 Effects that HUCB cells cultured with hematopoietic media has on cytokine production. Cord blood cells were cultured with serum free Ex Vivo-10 media at a seeding density of 10 million cells/5 mL for either, (A) 1 DIV, (B) 5 DIV, or (C) 12 DIV. (A) Most cytokines in CM from HUCB cells at 1 DIV were not as intense compared to same cytokines at 5 DIV (B) or 12 DIV (C). (B,C) At 5 and 12 DIV is first time SDF-1 was shown to be present in CM from HUCB cells. All cytokines presented in the graph were significantly different from control array (Students T-test, p < 0.05). 147

PAGE 161

When IL-3 was added to the Ex Vivo-10 media a down regulation of several cytokines occurred (Figure 5.4 A). While TPO inhibited MCP-1, it induced the HUCB cells to produce transforming growth factor(TGF-) and macrophage inflammatory protein-1 (MIP-1), which were not seen in any of the other conditions (Figure 5.4 B). Figure 5.4 Effects on Cytokines Secretion by IL-3 and TPO A. IL-8 SDF-1 TNF-a MCP-1 OSM TNF-b 0 20 40 60 80 100 120 140 180 160 Optical Density B. IL-8 SDF-1 TGF-b OSM IL-10 IL-1a RANTES MIP-1d 0 20 40 60 80 100 120 140 160 180 200 Optical Density 148

PAGE 162

Figure 5.4 Cytokine release after IL-3 and TPO added to hematopoietic media. HUCB cells were cultured for 4 day in serum free Ex Vivo-10, after which cells were cultured with fresh media and either (A) IL-3 (5 ng/mL) or (B) TPO (25 ng/mL) for 5 days (with media refreshed at 2.5 days). Cells were then cultured in Ex Vivo-10 for additional 3 days for a total of 12 DIV. (A,B) Overall fewer cytokines were present in CM from these conditions. However, the most prevalent cytokine remained IL-8 and surprisingly followed by SDF-1 in both conditions. (B) Although the additions of TPO down regulated MCP-1, this condition was the only one to show TGFand MIP-1 in the CM. All cytokines presented in the graph were significantly different from control array (Students T-test, p < 0.05). 5.4.4 ELISA results for Human IL-8 and MCP-1 in CM from HUCB Cells To investigate further IL-8 and MCP-1 enzyme link immunoassays were performed (Figure 5.5 A & B). In agreement with the cytokine arrays, IL-8 was present in CM from both Ex Vivo-10 and DMEM conditions at significantly higher levels than controls, however, there were no significant differences seen between 3 and 7 DIV conditions or between media conditions (Figure 5.5 A). Consistent with the cytokine arrays, there was significantly more MCP-1 in the CM from cord blood cells cultured for 3 days with Ex Vivo-10 than in CM from day 7 Ex Vivo-10 and day 3 DMEM conditions (Figure 5.5 B). 149

PAGE 163

Figure 5.5 ELISA of IL-8 and MCP-1 from CM of HUCB Cells A. 00000000018002000 0200400160018002000 20040060080010121416 Human IL-8 Levels in pg/mL 3 DIV 7 DIV Media 3 DIV 7 DIV Media PBS EX VIVO 10 DMEM B. 600800100012001400Human MCP-1 Levels in pg/mL 3 DIV 7 DIV Media 3 DIV 7 DIV Media PBS 150

PAGE 164

Figure 5.5 ELISA confirmation of human IL-8 and MCP-1 in CM from HUCB cells.HUCB cells at 10 million cells/5 mL were culture in serum free Ex Vivo-10 or DMEM for 3 or 7 DIV. (A) IL-8 (pg/mL) was found in all conditions, except controls (media and PBS), and there were no significant differences. (B) MCP-1 (pg/mL) was also shown in all conditions, except controls (media and PBS), and the CM from 3 DIV Ex Vivo-10 condition contained significantly more MPC-1 then at 7 DIV or in the DMEM 3 DIV condition. (Students T-test, p < 0.05). 5.4.5 Migration Assays To examine whether MCP-1, IL-3 or TPO were chemoattractants for HUCB cells, migration assays were performed. Previously we have shown that striatal ischemic tissue extracts from rats contain MCP-1 and here we show that HUCB cells strongly migrate to this extract (Figure 5.). IL-3 and TPO are often used to maintain long-term cultures and for progenitor proliferation. Therefore, we wanted to determine whether either had chemoattractant properties, as well. Significantly more HUCB cells migrate to MCP-1, IL-3 and TPO compared to controls (DMEM and PBS). However, there were no significant differences between the doses used for each protein, which was likely due to the closeness in range of the doses employed. 151

PAGE 165

Figure 5.6 Migration Assay Luminescence Expression of ATP (RLUs) 0200400600 * 5 10 20 30 1 5 10 10 25 40 MCP-1 ng/mL IL-3 ng/mL TPO ng/mL SDF-1 C1 C2 Figure 5.6 Migration of HUCB cells to MCP-1, IL-3 and TPO. HUCB cells show a strong chemoattraction to all three cytokines/chemokines MCP-1, IL-3 and TPO. Although a dose range for each chemokine was performed there were no significant differences between the doses. A standard curve was also performed with the HUCB cells from 0 to 80,000 cell which corresponded to ATP expression in relative light units (RLUs) 0 to 2,500. (Students T-test, p < 0.05). C1 = DMEM, C2 = PBS 152

PAGE 166

5.5 Discussion Cytokine arrays, in which multiple cytokines are simultaneously identified (23 to 43 per membrane), allowed us to obtain cytokine profiles of what HUCB cells are producing in various culture conditions and at various times. This study demonstrates the significant signal interaction that must be occurring between HUCB cells in order to produce this variety cytokines and chemokines and whose profiles change as the culture conditions change. IL-8 and MCP-1 are more extensively produced than any other chemokines in the human body and have been implicated as the first line of defense in the inflammatory reaction. In this study, both IL-8 and MCP-1 were significantly elevated in the CM of HUCB cells under varying culture conditions. IL-8 was detected in the CM from HUCB cells in all culture condition. Additionally, the intensity of IL-8 was the highest in every condition. IL-8 belongs to the super family of cytokines, under the category of chemokines, and more specifically is considered an -chemokine (or chemotactic cytokine). This chemokine is a strong chemoattractant for neutrophils and lymphocytes (T-cells), and induces a full pattern of response from these cells, with activation of the motile apparatus and directional migration, increase in surface adhesion molecules, lysosomal enzyme release, and reactive oxygen intermediates production (Wang, Xu, Murphy, Taub, & Chertov, 1996). We have previously shown an elevated presence of IL-8 (Growth-Regulated Oncogene/ Cytokine-induced Neutrophil Chemoattractant-1 GRO/CINC-1 rat equivalent of human IL-8) in striatal, hippocampal, and frontal cortex of ischemic rat tissue extracts, which was elevated from 24 hours to 72 hours after stroke when compare to non-stroked tissues (Newman et al., 2003; Newman, Willing, Manresa, 153

PAGE 167

Davis Sanberg, & Sanberg, 2004). IL-8 is also elevated in the sera from patients in a number of human injuries and diseases, such as, Multiple Sclerosis (Lund et al., 2004), coronary heart disease (Qi et al., 2003), traumatic brain injury (Kushi, Saito, Makino, & Hayashi, 2003), and ischemic stroke patients {Kostulas, 1999 #6608;Kostulas, 1999 #6611}. In addition, IL-8 from cord blood alone or together with other cytokines is being used as a determinant for neonatal sepsis (Berner, Tuxen, Clad, Forster, & Brandis, 2000; Dollner et al., 2001; Krueger et al., 2001). TNFand IL-1 have been implicated as the cytokines responsible for stimulating the release of IL-8 and MCP-1 (Kasahara, 1991; Kim, 1996; Yang, Hu, Chang, Tai, & Leu, 2004) However, recently neutrophil chemoattraction to IL-8 was shown to be dependent on CINC-1 produced from mast cells (Ramos, Heluy-Neto, Ribeiro, Ferreira, & Cunha, 2003). This discovery may be of importance in the migration of HUCB cells to ischemic tissue. Both neutrophils and mast cells are within the heterogeneous population of HUCB cells and depending on the culturing conditions may be maintained for long periods (Dvorak, Estrella, Mitsui, & Ishizaka, 1993; Dvorak, Mitsui, & Ishizaka, 1993). In addition, ischemic tissue extracts were previously shown to contain significant levels of CINC-1 and CINC-3 (Newman et al., In press) and this study has shown the presence of IL-8 in every HUCB cell culture condition. We believe that these converging lines of evidence suggest that HUCB cells are partially attracted to ischemic tissue as a result of its content of CINC-1, the variety of and interaction of cells within the cord blood (including neutrophils and mast cells), and the production of IL-8 from these cells. MCP-1 a -chemokine, was the second most prevalent cytokine present in every condition except TPO. As the name implies MCP-1 is a strong chemoattractant for 154

PAGE 168

monocytes and has a role in the accumulation of monocytes over a 48 hour period after interaction of antigen and sensitized lymphocytes (Yoshimura & Ueda, 1996). However, in the Ex Vivo-10 conditions for 1 and 12 DIV, there was a higher level of IL-6 than MCP-1, and in IL-3 stimulated condition, both SDF-1 and TNFlevels were higher than MCP-1. In the brain, MCP-1 is well documented as a major chemoattractant for monocytes especially after disease state onset or after injury as part of the inflammatory response. IL-1, TNF-, IFN, and several other stimuli are known to induce the expression of MCP-1 from many cell types (for review see Yoshimura & Ueda, 1996). More recently, MCP-1 has been reported to induce the migration of rat-derived neural stem cells in vitro (Widera et al., 2004). Previously, we have shown that the peak expression of MCP-1 in ischemic rat brain corresponds to the time when the greatest number of HUCB cells migrate to ischemic striatal and hippocampal extracts. The down regulation of MCP-1 seen in the IL-3 condition and more so in the TPO condition may be a function of the time at which the CM was taken, which was 12 DIV. It is possible that MCP-1 was expressed at an earlier time in these conditions. Although IL-1 was present in the TPO condition the level of expression was not very strong and TNFwas not present at all in this CM, which suggest cells were not stimulated enough to induce MCP-1 secretion. In addition, both IL-3 and TPO are used as a culturing tool to stimulate cell proliferation and maturation, and the exposed HUCB cells may have been reacting to these cytokines and their microenvironment by down-regulating unnecessary proteins to conserve energy for proliferation. Interestingly, when HUCB cells were cultured in the hematopoietic media (Ex Vivo-10) at 5 and 12 DIV and with IL-3 or TPO, SDF-1 was secreted from the cells, 155

PAGE 169

which was not seen in any conditions using DMEM. SDF-1 has been shown to induce the homing and mobilization of hematopoietic stem cells (HSC), especially to bone marrow (Broxmeyer et al., 1999). Enhanced cell survival (Lee et al., 2002) and the potentiation of angiogenesis (Mirshahi et al., 2000; Salvucci, Basik, Yao, Bianchi, & Tosato, 2004) has also been shown to be induced by SDF-1. The chemoattraction of cord blood HSC to SDF-1 has been reported as well (Yong et al., 1999). This chemoattractant has been shown to induce IL-8 production in cord blood-derived human mast cells (Lin, Issekutz, & Marshall, 2000, 2001). However, in this study, HUCB cells in cultured conditions without SDF-1 also induced IL-8 secretion, which indicates that the presence of SDF-1 is not necessary for IL-8 production in cord blood cells. Another unique finding was the presence of Leptin, which was induced when HUCB cells were cultured in hematopoietic media at 5 DIV. Leptin is a protein predominantly produced by adipocytes and has a role in regulating long-term body weight. Although Leptin has been found in the serum of cord blood and in cells of the placenta (Senaris et al., 1997), its function or importance in cultured HUCB cells is unknown. The amount of time in culture and the media plus the supplements used in culture were the major influences in the production of cytokines by the HUCB cells. The cytokine profiles demonstrate the direct reaction of the cord blood cells to their culture environment, with cytokines being produced or inhibited in concert with changes in their environment. While these findings give us insight into the ability of HUCB cells to produce both cytokines and chemokines, there are remaining questions that need to be addressed. Are selected cells producing only certain cytokines? If cells are selected 156

PAGE 170

based on a phenotype of interest, will they work independently or is the heterogeneous population needed for the correct cell-to-cell interaction? Lastly, could it be that this heterogeneous microenvironment of cells is necessary for the benefits seen in the previous transplant studies? 157

PAGE 171

5.6 Reference Berner, R., Tuxen, B., Clad, A., Forster, J., & Brandis, M. (2000). Elevated gene expression of interleukin-8 in cord blood is a sensitive marker for neonatal infection. Eur J Pediatr, 159(3), 205-210. Broxmeyer, H. E., Kim, C. H., Cooper, S. H., Hangoc, G., Hromas, R., & Pelus, L. M. (1999). Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors. Ann N Y Acad Sci, 872, 142-162; discussion 163. Chen, J., Sanberg, P. R., Li, Y., Wang, L., Lu, M., Willing, A. E., et al. (2001). Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke, 32(11), 2682-2688. Cohen, S. B., Gluckman, E., Rubinstein, P., & Madrigal, J. A. (Eds.). (2000). Cord blood characteristics: Role in stem cell transplantation. London: Martin Dunitz Ltd. Dollner, H., Vatten, L., Linnebo, I., Zanussi, G. F., Laerdal, A., & Austgulen, R. (2001). Inflammatory mediators in umbilical plasma from neonates who develop early-onset sepsis. Biol Neonate, 80(1), 41-47. Dvorak, A. M., Estrella, P., Mitsui, H., & Ishizaka, T. (1993). C-kit ligand induction of immature neutrophils in cultures of human umbilical cord blood. Eur J Cell Biol, 62(2), 422-431. Dvorak, A. M., Mitsui, H., & Ishizaka, T. (1993). Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures. J Leukoc Biol, 54(5), 465-485. Garbuzova-Davis, S., Willing, A. E., Zigova, T., Saporta, S., Justen, E. B., Lane, J. C., et al. (2003). Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation. J Hematother Stem Cell Res, 12(3), 255-270. Ha, Y., Choi, J. U., Yoon, D. H., Yeon, D. S., Lee, J. J., Kim, H. O., et al. (2001). Neural phenotype expression of cultured human cord blood cells in vitro. Neuroreport, 12(16), 3523-3527. Kasahara, T. (1991). Immunology, 74, 60-67. Kim, J. S. (1996). Cytokines and adhesion molecules in stroke and related diseases. J Neurol Sci, 137(2), 69-78. 158

PAGE 172

Krueger, M., Nauck, M. S., Sang, S., Hentschel, R., Wieland, H., & Berner, R. (2001). Cord blood levels of interleukin-6 and interleukin-8 for the immediate diagnosis of early-onset infection in premature infants. Biol Neonate, 80(2), 118-123. Kushi, H., Saito, T., Makino, K., & Hayashi, N. (2003). IL-8 is a key mediator of neuroinflammation in severe traumatic brain injuries. Acta Neurochir Suppl, 86, 347-350. Lee, Y., Gotoh, A., Kwon, H. J., You, M., Kohli, L., Mantel, C., et al. (2002). Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines. Blood, 99(12), 4307-4317. Lin, T. J., Issekutz, T. B., & Marshall, J. S. (2000). Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha. J Immunol, 165(1), 211-220. Lin, T. J., Issekutz, T. B., & Marshall, J. S. (2001). SDF-1 induces IL-8 production and transendothelial migration of human cord blood-derived mast cells. Int Arch Allergy Immunol, 124(1-3), 142-145. Lu, L., Shen, R. N., & Broxmeyer, H. E. (1996). Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: current status and future application. Crit Rev Oncol Hematol, 22(2), 61-78. Lund, B. T., Ashikian, N., Ta, H. Q., Chakryan, Y., Manoukian, K., Groshen, S., et al. (2004). Increased CXCL8 (IL-8) expression in Multiple Sclerosis. J Neuroimmunol, 155(1-2), 161-171. Mirshahi, F., Pourtau, J., Li, H., Muraine, M., Trochon, V., Legrand, E., et al. (2000). SDF-1 activity on microvascular endothelial cells: consequences on angiogenesis in in vitro and in vivo models. Thromb Res, 99(6), 587-594. Newman, M. B., Willing, A. E., Cassady, C. J., Manresa, J. J., Davis Sanberg, C., Saporta, S., et al. (2003). In vitro migration and phenotype identification of human umbilical cord blood (HUCB) cells to stroke brain. Exp Neurol, 181, 101-102. Newman, M. B., Willing, A. E., Manresa, J. J., Davis Sanberg, C., & Sanberg, P. R. (2004). Identification of cytokine in ischemic tissue extracts of the CNS and those released by human umbilical cord blood (HUCB) cells. Exp Neurol, 187, 216. Qi, X., Li, J., Gu, J., Li, S., Dang, Y., & Wang, T. (2003). Plasma levels of IL-8 predict early complications in patients with coronary heart disease after percutaneous coronary intervention. Jpn Heart J, 44(4), 451-461. 159

PAGE 173

Ramos, C. D., Heluy-Neto, N. E., Ribeiro, R. A., Ferreira, S. H., & Cunha, F. Q. (2003). Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1. Cytokine, 21(5), 214-223. Salvucci, O., Basik, M., Yao, L., Bianchi, R., & Tosato, G. (2004). Evidence for the involvement of SDF-1 and CXCR4 in the disruption of endothelial cell-branching morphogenesis and angiogenesis by TNF-alpha and IFN-gamma. J Leukoc Biol, 76(1), 217-226. Sanchez-Ramos, J. R. (2002). Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res, 69(6), 880-893. Sanchez-Ramos, J. R., Song, S., Kamath, S. G., Zigova, T., Willing, A., Cardozo-Pelaez, F., et al. (2001). Expression of neural markers in human umbilical cord blood. Experimental Neurology, 171(1), 109-115. Senaris, R., Garcia-Caballero, T., Casabiell, X., Gallego, R., Castro, R., Considine, R. V., Dieguez, C., & Casanueva, F. F. (1997). Synthesis of leptin in human placenta. Endocrinology, 138(10), 4501-4504. Sivan, E., Lin, W. M., Homko, C. J., Reece, E. A., & Boden, G. (1997). Leptin is present in human cord blood. Diabetes, 46(5), 917-919. Vendrame, M., Cassady, C. J., Newcomb, J., Bulter, T., Pennypacker, K. R., Zigova, T., et al. (2004). Infusion of human unbilical cord blood cells in rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke, 35, 1-6. Wang, J. M., Xu, L., Murphy, W. J., Taub, D. D., & Chertov, O. (1996). IL-8-Induced T-Lymphocyte Migration: Direct as Well as Indirect Mechanisms. Methods, 10(1), 135-144. Widera, D., Holtkamp, W., Entschladen, F., Niggemann, B., Zanker, K., Kaltschmidt, B., et al. (2004). MCP-1 induces migration of adult neural stem cells. Eur J Cell Biol, 83(8), 381-387. Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., et al. (2003). Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res, 73(3), 296-307. Willing, A. E., Vendrame, M., Mallery, J., Cassady, C. J., Davis, C. D., Sanchez-Ramos, J., et al. (2003). Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant, 12(4), 449-454. 160 Yang, Y. Y., Hu, C. J., Chang, S. M., Tai, T. Y., & Leu, S. J. (2004). Aspirin inhibits monocyte chemoattractant protein-1 and interleukin-8 expression in TNF-alpha

PAGE 174

stimulated human umbilical vein endothelial cells. Atherosclerosis, 174(2), 207-213. Yong, K., Fahey, A., Reeve, L., Nicholls, C., Thomas, N. S., Pizzey, A., et al. (1999). Cord blood progenitor cells have greater transendothelial migratory activity and increased responses to SDF-1 and MIP-3beta compared with mobilized adult progenitor cells. Br J Haematol, 107(2), 441-449. Yoshimura, T., & Ueda, A. (1996). Monocyte chemoattractant protein-1. In B. B. Aggarwal & J. U. Gutterman (Eds.), Human cytokines:Handbook for basic and clinical research. (2 ed., pp. 198-221). Cambridge: Blackwell Science. Zigova, T., Song, S., Willing, A. E., Hudson, J. E., Newman, M. B., Saporta, S., et al. (2002). Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant, 11(3), 265-274. 161

PAGE 175

Chapter 6 6.0 Relevance of the Dissertation Studies, Conclusions, Limitations, and Future Directions The main objective of this study was to determine whether HUCB cells had the ability to migrate to the injured brain. The previous transplantation studies in the ischemic rat model, which have used these cells, all reported significant functional improvements in behavioral deficits (see Chapter 1). However, these studies did not report a larger number of transplanted cells at the site of injury. Therefore, we needed to determine whether the cord blood cells were attracted to or inhibited from the area of injury. The secondary objectives were to determine, whether there was an optimal time, after stroke, in which more cells migrated to ischemic extracts and to identify factors that were involved with their migration. An in vitro model of cell migration and tissue extracts from MCAO rat model of stroke were used in this study. 162 The results from our preliminary data, which was used to characterize the properties of the HUCB cells (in vitro), showed that these cells will survive for an extended period and proliferate in culture. Additionally, these data showed five distinct cell types based on morphological visualization, and that the mononuclear fraction of cord blood cells are heterogeneous and contains both stem and progenitor cells, which was shown by immunophenotyping analysis and their exocytosis of certain nuclear dyes.

PAGE 176

These results are important especially if the cells are approved to be used in human transplantation to repair the injured brain. The expansion of cord blood cells might be necessary if a certain fraction contains unique properties particular for an individual(s) (such as their HLA antigens) or if a subpopulation of cells, within the cord blood, are determined to have properties exclusive for the repair of a certain brain injury. Although these cells have been used to treat nonmalignant and malignant hematopoietic diseases (see Chapter 1) and more recently cord blood cells have been successfully transplanted into infants with Krabbes disease (Escolar et al., 2005) and in children with Hurlers syndrome (Staba et al., 2004), they have not been approved for other treatments or for use in brain repair. In the migration experiments, the HUCB cells were shown to migrate to the chemoattractant SDF-1, in a dose dependent manner, 100 ng/mL was the optimal dose and 4 hours was the optimal time for migration of the cells. In the neonatal brain assays, HUCB cells migrated to the 2 and 7 day brain extracts and the hippocampal extracts in the 7 day condition. The PD animal model that was studied used the toxin 6-OHDA to create a lesion in the rat brain. The toxin is not the cause of human PD, but does result in pathology and symptoms similar to those seen in PD patients. In the migration assay, HUCB cells significantly migrated to tissue extracts from the 6-OHDA lesioned substantia nigra and striatum. In addition, this toxin had an inhibitory effect on the cord blood cells, in that the cells did not migrate to the toxin when in solution with media only. The 6-OHDA rat brains were harvested 3 weeks post-lesion, which corresponds to the time when lesioned rats have the symptoms and pathology (significant decrease of DA neurons and levels) of PD seen in humans. In addition, 3 weeks post-lesion should 163

PAGE 177

be sufficient time for the toxin to clear or at least appreciably decrease so that it would not interfere, to a great extent, with the cell migration. More importantly and the primary focus of this study was the MCAO rat model of stroke. HUCB cells were attracted to ischemic tissue extracts in a time dependent manner. More cells migrated to the ischemic extracts that were taken and processed between 24 and 72 hours after induction of MCAO in the rat, with the peak migration occurring at 48 hours after inducing the stroke. These results suggested a window of opportunity in which to deliver the cells. A typical protocol for transplantation studies in the MCAO model is to administer the cells 24 hours after inducing the stroke. These results showed that 48 hours after stroke might be more optimal for cell transplantation studies; we are now examining this in vivo. Dr. Willings group, from our laboratory, has been investigating this phenomenon. Adult male rats received either HUCB cells 48 hours after MACO or no cells. The results showed almost full amelioration of behavioral and neurophysiological deficits produced by the stroke, and a reduction of the infarct volume in MCAO rats. The results also suggested that the neurons in the core of the infarct may not die immediately, and that it is more probable that these cells die slowly through apoptotic cell death (Newcomb et al., 2005). Together the results suggest that additional therapeutic strategies, such as anti-inflammatory agents in addition to the administration of HUCB cells would increase survival rate and overall recovery. Most importantly, these studies provide strong evidence that the therapeutic window could be extended from the current 3 hour treatment and thus allow more stroke victims to be treated. 164

PAGE 178

The results from the MCAO study also showed an inhibitory effect in the migration of HUCB cells to the earlier time points in the ischemic tissues. Both the 4 and 6 hours ischemic striatal and hippocampal extracts attracted fewer cells than the corresponding contralateral sides, signifying an inhibitory effect. This effect is possibly the result of an increased invasion of pro-inflammatory cytokines (e.g. TNF-, IL-1), which are known to be released soon after ischemia occurs and peak about 12 hours post-stroke. In studying this model, we also determined some of the cytokine and chemokines present in the striatal ischemic tissue extract (see Chapter 4). TIMP-1 was the strongest expressed cytokine and present in all striatal ischemic extracts assayed and in the contralateral side extracts as well. The TIMPs family has a significant role in rebuilding the extracellular matrix that has been damaged during an insult to the brain (Crocker, Pagenstecher, & Campbell, 2004). The strong presence of TIMP-1, in the tissue extracts, suggest that in the ischemic brain this protein is performing a similar function probably by inhibiting the tissue metalloproteinases (MMPs), which are known to participate in the breakdown of extracellular matrix and in weakening the blood brain barrier. Therefore, it is reasonable to assume that TIMP-1 is produced and released by surviving cells around the infarct in a response to protect the break down of the BBB extracellular matrix. Two known chemoattractants were also strongly present in the ischemic extracts. Both MCP-1 and CINC-1 were expressed on the cytokine arrays from the striatal ischemic extracts at 48 hours and 1 week. To help determine whether they had a role in the migration of HUCB cells we further investigated their expression in ischemic and non-ischemic tissue extracts overtime, by ELISA. Interestingly, the presence of 165

PAGE 179

GRO/CINC-1 (IL-8) and MCP-1 in the ischemic extracts, over the same time periods as the migration studies, showed a pattern that was similar to the results of the migration of HUCB cells to the ischemic extracts. In that both IL-8 and MCP-1, levels were lower in the earlier time periods and peaked between 24 to 72 hours, which is when cell migration to ischemic extracts was at its peak. Our findings are in agreement with the findings of other researchers. The expression of MCP-1 in ischemic brain peaks 2 to 3 days after stroke (Yamagami et al., 1999); (Che, Ye, Panga, Wu, & Yang, 2001). MCP-1 may be stimulated by several factors, such as, TNF-, IL-1, IFN, LPS, and platelet derived growth factor (PDGF). TNF-, IL-1 and IFN are present in ischemic tissues as early as 3 to 6 hours after stroke (Kim, 1996) and are key responders to inflammation (Sharma & Kumar, 1998). In vitro studies have shown that TNFand IL-1 may induce the production of IL-8 and MCP-1 from astrocytes (Kim, 1996). This suggests that these two pro-inflammatory cytokines may have a role in chemotaxis. Both IL-8 and MCP-1 are chemoattractants for monocytes, and both were present in the ischemic tissue and in the conditioned media from the cord blood cells in culture without any inflammatory agents present. Although, the exact chemokines that induce the cells to migrate remain to be identified, we now have insight as to which chemokine families are involved and we believe that more than one chemokine is responsible for the attraction of HUCB cells to ischemic tissue. A second primary objective of this study was to identify the cytokines and chemokines that may be produced by HUCB cells in culture (see Chapter 5). The amount of time the cells were cultured and the culturing conditions are the two principal factors that influenced the cord blood cells secretion of cytokines and chemokines. The 166

PAGE 180

most prevalent chemokine was IL-8, which was present in every culture condition. This was followed by the presence of MCP-1. Several other cytokines and chemokines were also produced from the cultured HUCB cells. We postulate that significant cell-to-cell signaling (interactions) occurred with the cultured HUCB cells in order for the production of such diversity in the cytokines and chemokines. Further evidence of cell to cell signaling is observed in the cytokine profiles, which change as the culturing conditions change. However, within the population of cord blood cells the cells that are producing and secreting certain cytokines or chemokines remain to be determined. In conclusion, the results suggest that a wider therapeutic window (from 48 to 72 hours after stroke) may exist for the treatment of stroke victims using cord blood cells, when compared to the existing 3-hour post-stroke use of t-PA. The chemokines that may be partially responsible for the migration of HUCB cells were identified (IL-8 and MCP-1). The cytokine profiles demonstrated the direct reaction of the cells to their culturing environment, with cytokines being produced or inhibited in concert with changes to their environment. This suggests that when cells are administered in the ischemic rat they would have the ability to produce cytokines or chemokines that may aid in the repair of the injured brain. An intriguing possibility is that the two most prevalent chemokines produces by HUCB cells (IL-8 and MCP-1) may induce the attraction of endogenous cells (stem cells) to the site of injury, thus further aiding in the repair of the damaged tissue. Converging lines of evidence, from the transplanted post-stroke animals, the migration assays showing an extended therapeutic window (from 3 hours to now 48 hours), and the production of cytokines and chemokines by HUCB cells, suggest their use 167

PAGE 181

as a treatment for stroke, which may improve the mortality rate and long-term outcomes for patients. 6.1 Limitations In these studies, as with any study, there are the existences of intrinsic limitations. Using in vitro cultures and assays is the major limitation in this set of studies. One major problem with in vitro model systems is that they do not necessarily reflect the activity that takes places in vivo. This is especially true with the migration assays that employ a chamber, which is an artificial condition that does not contain all the components of the in situ environment. Although this type of design affords the opportunity to examine the migration functions in an isolated environment, which has its advantages, it does not allow for the complex interactions that undoubtedly occur during the endogenous migration of HUCB cells with the surrounding cells, matrix, and the numerous cytokines, chemokines or trophic factors that are produced in reaction to ischemia. The in vitro assays allows for isolating the experiment from extraneous variables and may lead to significant findings, it is also possible, and must be considered, that false leads could be generated. This problem can be circumvented, to a certain extent, by verifying the in vitro results with animal studies, which is currently underway in our laboratory. Nonetheless, it must still be appreciated that both the migration assays and the cytokines/chemokines produced by cultured HUCB cell systems are an artificial construct that can only suggest possible mechanisms that must be explored and the results compared to intact animal experiments. 168

PAGE 182

6.2 Future Directions Cell transplantation for CNS diseases has always held tremendous medical potential. Chapter 1 discussed the problems associated with fetal or embryonic cells and the selection of HUCB cells as an alternative source for cell therapy. HUCB cells have been found to exert pronounced effects when administered systemically in the treatment of animals with CNS diseases and injuries as reviewed. If the functional benefits of HUCB cells endure scientific scrutiny and with appropriate standard pre-clinical safety and toxicological, testing initial Phase I clinical evaluation is conceivable in the short term. The FDA has already approved clinical trials using HUCB cells for hematologic malignancies, aplastic anemia, immunodeficiency diseases, and metabolic disorders (Thomson et al., 2000; Wagner, Kernan, Steinbuch, Broxmeyer, & Gluckman, 1995). While these clinical trials are not for neurological disorders, the level of comfort that regulatory agencies already have with this general approach together with the data collected from ongoing trials (related to dosage, GVHD, cell survival, safety of the HUCB cells, and their efficacy) will facilitate entry into clinical trials for cellular transplantation therapies. With over 1500 cord blood transplants performed worldwide, a strong safety and efficacy database is being compiled (Gluckman, 2001; Laughlin et al., 2001)(Gluckman et al., 2001; Laughlin et al., 2001). Before HUCB cells, are ready for the clinic, fundamental issues need to be addressed. One of the most obvious issues is the cell type(s) underlying the behavioral effects of HUCB injections, which needs to be identified. HUCB cells are a heterogeneous population of cells and have been shown to differentiate into neural cells 169

PAGE 183

both in vitro and in vivo. While it is tempting to speculate that neural cells are one of the essential ingredients for the improved deficits in behavior, this remains an open question. The animal transplant studies utilizing cord blood cells thus far are not indicating cell replacement as the mechanism for the improvements in the behavioral deficits. The possibility exist that these cells, upon migrating to the injured tissue, are releasing cytokines and chemokines that are either directly or indirectly responsible for these effects. Alternative factors, such as these, are being addressed by our laboratory and others. Future studies should systematically isolate the various cell components in HUCB and evaluate their therapeutic role in animal models. These studies should also identify effective and non-effective clonal stem cell lines as well as identify methods of expanding those lines prior to transplantation. Serious attention should be paid to identifying culturing conditions, media, serum, and other factors to enhance or maintain expansion as well as the stage of differentiation of the cells for specific disease indications. Another issue is the role of the recipients immune system after the transplantation of HUCB cells. Grafted HUCB cells (which are considered foreign to the host) were shown to have a low rejection rate (see Chapter 1), but they may also induce a low level of chronic inflammatory response that contributes to or minimizes their therapeutic potential. Transplant studies across discordant hosts would provide insight into this issue. Perhaps the observed effect in animals (where a xenograft is being used) underestimates the potential in humans. This is an important point when considering the optimization of this or any transplant approach that borrows heavily from pre-clinical animal data. In addition to the host immune response, the role of the host stem cells 170

PAGE 184

should be evaluated. It is possible that HUCB cells are providing benefits through these host cells. Studies should begin to determine whether grafted HUCB cells stimulate or augment the proliferation, migration, and differentiation of endogenous stem cells. Refinements in cell labeling coupled with cross-species grafts should be used to determine the proportion of host versus donor stem cells within the region of interest. Lastly, long-term safety studies along with duration of the behavioral effects and survival of the grafted cells should be addressed. The current studies are relatively short term and do not permit determination of any continued improvements, diminishments in efficacy over time or overall safety. These studies should incorporate detailed anatomical analysis of cell types within the injured region with particular emphasis on the appearance of specific cell types prior to the onset of behavioral improvements and from any anatomical abnormalities. Certainly, this is only a partial list and we are in the process of fully evaluating the effectiveness of HUCB cells in a number of different injury and disease models. Despite the need to evaluate further, the issues discussed above, the pre-clinical transplantation studies utilizing cord blood cells and the results of theses studies, it is reasonable to propose that HUCB cells are a formidable cell source for cellular therapies in CNS disorders. 171

PAGE 185

6.3 References Che, X., Ye, W., Panga, L., Wu, D. C., & Yang, G. Y. (2001). Monocyte chemoattractant protein-1 expressed in neurons and astrocytes during focal ischemia in mice. Brain Res, 902(2), 171-177. Crocker, S. J., Pagenstecher, A., & Campbell, I. L. (2004). The TIMPs tango with MMPs and more in the central nervous system. J Neurosci Res, 75(1), 1-11. Escolar, M. L., Poe, M. D., Provenzale, J. M., Richards, K. C., Allison, J., Wood, S., et al. (2005). Transplantation of umbilical-cord blood in babies with infantile Krabbe's disease. N Engl J Med, 352(20), 2069-2081. Gluckman, E. (2001). Hematopoietic stem-cell transplants using umbilical-cord blood. The New England Journal of Medicine, 344(24), 1860-1861. Kim, J. S. (1996). Cytokines and adhesion molecules in stroke and related diseases. J Neurol Sci, 137(2), 69-78. Laughlin, M. J., Barker, J., Bambach, B., Koc, O. N., Rizzieri, D. A., Wagner, J. E., et al. (2001). Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. The New England Journal of Medicine, 344(24), 1815-1822. Newcomb, J., Ajmo, C. T., Collier, L., Davis Sanberg, C., Pennypacker, K. R., Sanberg, P. R., et al. (2005). Infarct core resuced by intravenous treatment of human umbilibcal cord blood cells. Exp Neurol, 193(1), 255. Sharma, B. K., & Kumar, K. (1998). Role of proinflammatory cytokines in cerebral ischemia: a review. Metab Brain Dis, 13(1), 1-8. Staba, S. L., Escolar, M. L., Poe, M., Kim, Y., Martin, P. L., Szabolcs, P., et al. (2004). Cord-blood transplants from unrelated donors in patients with Hurler's syndrome. N Engl J Med, 350(19), 1960-1969. Thomson, B. G., Robertson, K. A., Gowan, D., Heilman, D., Broxmeyer, H. E., Emanuel, D., et al. (2000). Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation. Blood, 96(8), 2703-2711. 172

PAGE 186

Wagner, J. E., Kernan, N. A., Steinbuch, M., Broxmeyer, H. E., & Gluckman, E. (1995). Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet, 346(8969), 214-219. Yamagami, S., Tamura, M., Hayashi, M., Endo, N., Tanabe, H., Katsuura, Y., et al. (1999). Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats. J Leukoc Biol, 65(6), 744-749. 173

PAGE 187

174 About the Author Mary B. Newman received her Bachelors Degree ( cum laude) in Psychology (1998) and her Masters in Experimental Psychology (2000) from the University of South Florida. She initially volunteered in Dr. Douglas Shytles laboratory, which focus was on the treatment of Attention Deficit Disorder a nd Tourette s Syndrome with nicotine. She was hired as a research assist ant and accepted into graduate sc hool with Dr. Shytle as her Major Professor. After working with Dr. Shy tle, she became very intrigued by the work of Dr. Paul R. Sanberg, Director of Research for Neurosurgery. Dr. Sanberg accepted her into his laboratory as a graduate student in the area of Cognitive & Neural Sciences and hired her as a research assist ant. After receiving her Mast ers Degree, she began teaching Lifespan Development, Neurobiology, and Experimental Psychology at Pasco Hernando Community College. She continued teaching at the Community College and USF until she earned her Ph.D. While earning her Ph.D. in Dr. Sanbergs la boratory at The Center for Excellence in Aging and Brain Repair, she was first author on 17 published scie ntific articles, co-author on several more, wrote 4 book chapters, was invited to speak at scientif ic conferences (both national and international), presented her research at several conferen ces, and peer reviewed articles for established scientific journals. In addition, she was an inventor on three U.S. patents, received a grant for Parkinsons Dis ease research, was elected twice to the position of Student Council Member for America Societ y of Neural Transpla ntation and Repair Society, and she also completed the Kauffman Entrepreneur Internship Program. Upon completion of her training with Dr. Sanber g, she accepted a postdoctoral position at Rush University Medical Center, in Chic ago, with Dr. Paul M. Carvey.