Isolation and Characterization of Mesenchymal Stem Cells from dogs, horses and deers

다국어 초록 (Multilingual Abstract)

Recent studies have shown that diverse tissues can be a source of stem cells in humans. There are common sources of stem cells such as bone marrow, umbilical cord blood, amniotic fluid, amniotic membrane, peripheral blood and fatty tissue. Stem cells have self-renewal and multipotent differentiation abilities. Especially, mesenchymal stem cells (MSCs) are one of the most useful and valuable cells in the therapeutic aspect of stem cells. Since MSCs show that the multilineage differentiation, being immune modulation effects, being able to secrete many cytokines, robust proliferation ability and not being a carcinogenic stem cells. Hence, it is necessary that the isolation and characterization of MSCs from various different species such as dog, horse and deer in veterinary medicine.
The several tissues are conducted for isolation and characterization of MSCs from canine derived tissues, including that wharton’s jelly, amniotic membrane and umbilical cord blood. Therefore, canine MSCs are successfully isolated and identified which are 3 different stem cell lines from canine wharton’s jelly-derived MSCs (cWJ-MSCs), canine amniotic membranine-derived MSCs (cAM-MSCs) and canine umbilical cord blood-derived MSCs (cUCB-MSCs). The isolated canine MSCs have typical stem cell characteristics such as a fibroblast-like shape and adhere to plastic culture dish. The immunophenotypes of canine MSCs are analyzed by FACS and the cell proliferation ability is measured by the cumulative population doubling level (CPDL). The differentiation ability of canine MSCs is investigated by a multipotent differentiation assay under various differentiation conditions. The isolated canine MSCs show chondrogenic, osteogenic and adipogenic differentiation abilities in vitro.
In equine derived tissues, there are 3 different stem cell lines from equine amniotic membrane-derived MSCs (eAM-MSCs), equine amniotic fluid-derived MSCs (eAM-MSCs) and equine umbilical cord blood MSCs (eUCB-MSCs). Also, the isolated equine MSCs show the typical stem cell characteristics in the cell morphology, FACS analysis, CPDL and multipotent differentiation assay.
In deer derived tissue, the deer antler is harvested and used for stem cell identification. Antler-derived MSCs have stem cells characteristics which are showed by conducting various assays such as FACS assay, CPDL and multipotent differentiation assay.
In conclusion, total 7 different stem cell lines are established from various tissues and small, large and wild animal. In aspect of veterinary medicine, these results can be useful and advantageous for clinical approach and various drugs screening assay.

목차 (Table of Contents)

• TABLE OF CONTENTS
• ABSTRACT i
• TABLE OF CONTENTS iv
• LIST OF ABBREVIATION x
• LITERATURE REVIEW xi
• CHAPTERⅠ １
• Isolation and Characterizatio of canine tissues-Derived Mesenchymal Stem Cells
• 1.1 INTRODUCTION 2
• 1.2 MATERIALS AND METHODS 5
• 1.2.1 Animals 5
• 1.2.2 Tissue collection & Culture 6
• 1.2.3 Cumulative Population Doubling Level Analysis 8
• 1.2.4 Flow Cytometry 9
• 1.2.5 Adipogenesis 10
• 1.2.6 Osteogenesis 11
• 1.2.7 Neurogenesis 12
• 1.2.8 Chondrogenesis 13
• 1.2.9 Immunostaining 14
• 1.2.10 RNA extraction and RT-PCR 15
• 1.3 RESULTS 17
• 1.3.1 Isolation and Cell cultured of cAM-MSCs 17
• 1.3.2 Immunophenotype of cAM-MSCs 18
• 1.3.3 Multipotent differentiation assay of cAM-MSCs 19
• 1.3.4 Isolation and Cell cultured of cWJ-MSCs 22
• 1.3.5 Immunophenotype of cWJ-MSCs 23
• 1.3.6 Multipotent differentiation assay of cWJ-MSCs 24
• 1.3.7 Isolation and Cell cultured of cUCB-MSCs 28
• 1.3.8 Immunophenotype of cUCB-MSCs 29
• 1.3.9 Multipotent differentiation assay of cUCB-MSCs 29
• 1.4 DISCUSSION 68
• CHAPTERⅡ 74
• Isolation and Characterization of equine tissues-Derived Mesenchymal Stem Cells
• 2.1 INTRODUCTION 75
• 2.2 MATERIALS AND METHODS 78
• 2.2.1 Tissue collection 78
• 2.2.2 Cell isolation 79
• 2.2.3 Cumulative Population Doubling Level assay 82
• 2.2.4 Karyotype analysis 83
• 2.2.5 Flow cytometry 83
• 2.2.6 Osteogenesis 84
• 2.2.7 Adipogenesis 86
• 2.2.8 Chondrogenesis 87
• 2.2.9 Statistical analysis 88
• 2.3 RESULTS 89
• 2.3.1 Isolation and cell culture of eAM-MSCs 89
• 2.3.2 Immunophenotype of eAM-MSCs 90
• 2.3.3 Multipotent differentiation assay of eAM-MSCs 90
• 2.3.4 Isolation and cell culture of eAF-MSCs 93
• 2.3.5 Immunophenotype of eAF-MSCs 95
• 2.3.6 Multipotent differentiation assay of eAF-MSCs 96
• 2.3.7 Isolation and cell culture of eUCB-MSCs 99
• 2.3.8 Immunophenotype of eUCB-MSCs 99
• 2.3.9 Multipotent differentiation assay of eUCB-MSCs 101
• 2.4 DISCUSSION 125
• CHAPTERⅢ 131
• Isolation and Characterization of Deer Antler-Derived Mesenchymal Stem Cells
• 3.1 INTRODUCTION 132
• 3.2 MATERIALS AND METHODS 135
• 3.2.1 Tissue collection from antler 135
• 3.2.2 Cell isolation and culture 136
• 3.2.3 Cumulative population Doubling Level assay 136
• 3.2.4 Karyotype Analysis 138
• 3.2.5 Flow cytometry analysis of surface antigen expression 139
• 3.2.6 Immunocytochemistry 139
• 3.2.7 Osteogenic differentiation 141
• 3.2.8 Adipogenic differentiation 142
• 3.2.9 Chondrogenic differentiation 143
• 3.2.10 Statistical analysis 144
• 3.3 RESULTS 145
• 3.3.1 Isolation and cell culture of Antler MSCs 145
• 3.3.2 Proliferation assay with serum and growth factors 146
• 3.3.3 Analysis of Immunophenotype and Karytype 148
• 3.3.4 Immunostaining of Antler MSCs with stem cell markers 149
• 3.3.5 Differentiation assay 150
• 3.4 DISCUSSION 166
• GENERAL CONCLUSION 173
• REFERENCES 176
• ABSTRACT IN KOREAN 198