Title page
Contents
ABBREVIATIONS 11
ABSTRACT 12
1. GENERAL BACKGROUND 14
1.1. Identification and Characterization of Embryonic Stem Cells 14
1.2. Basic Techniques for In Vitro ESCs Differentiation 15
1.2.1. EBs-Based Systems 15
1.2.2. Stromal Feeder-Mediated System 16
1.2.3. Differentiation of ESCs by Default 16
1.3. Vasculogenesis : The Initial Formation of Blood Vessels 18
1.4. Defining Intermediate Stages during Endothelial Cell Differentiation 19
1.5. Human Embryonic Stem Cells as a Source for Vascular Progenitors 21
2. INTRODUCTION 23
3. MATERIALS AND METHODS 26
3.1. hESCs Culture and Endothelial Cells Differentiation 26
3.2. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 27
3.3. Cell Staining 29
3.4. Flow Cytometry 30
3.5. DiI-labeled ac-LDL Uptake and Matrigel Assay 30
3.6. Mouse Limb Ischemia 31
3.7. Cell Transplantation 31
3.8. Laser Doppler Imaging Analysis 32
3.9. Histological and Immunohistochemical Analysis 32
3.10. Fluorescence In Situ Hybridization (FISH) Staining 33
3.11. Estimation of Capillary and Arteriole Density 33
3.12. Statistical Analysis 34
4. RESULTS 35
4.1. Part I : In Vitro Differentiaion and Isolation of ECs Derived from hESCs 35
4.1.1. Overall Strategy to Purify hESC-ECs and Their Functional Test 35
4.1.2. Increased PECAM Expression during EBs Formation 36
4.1.3. Localization and Isolation of PECAM-Positive Cells in Attached EBs 39
4.1.4. Quantification of PECAM and vWF Expression by FACS Analysis 42
4.1.5. Formation of Vascular Tube-like Structure on Matrigel 43
4.2. Part II : In Vitro Characterization of ECs Derived from hESCs 45
4.2.1. Changes in The Marker Expressions during hESCs Differentiation, and Purification of hESC-ECs 45
4.2.2. Characterization of hESC-ECs 46
4.2.3. Endothelial-Specific Functional Characterization of hESC-ECs 47
4.3. Part III : In Vivo Test of hESC-ECs 50
4.3.1. Improvement of Ischemic Limb Salvage by hESC-ECs Transplantation 50
4.3.2. Improvement of The Blood Perfusion in Ischemic Limbs by hESC-ECs Transplantation 52
4.3.3. Enhancement of Neovascularization by hESC-ECs Transplantation 55
4.3.4. Expression of Human Angiogenic Growth Factors in Ischemic Limb Tissues by hESC-ECs Transplantation 57
4.3.5. Engraftment of Transplanted hESC-ECs into Vascular Structures in The Ischemic Limbs 59
5. DISCUSSION 62
6. GRAND CONCLUSIONS 66
7. REFERENCES 67
8. ACKNOWLEDGEMENTS 77
Table 1. Human-specific primers for each gene 28
Table 2. Mouse-specific primers for each gene 29
Figure 1.1-1. Basic techniques for differentiation of ESCs in vitro. (Elsevier, vol 1, 2004, Handbook of Stem cells) 17
Figure 1.1-2. The origin of endothelial cells. (Liao and Zon, 1999, Dev Bio) 18
Figure 1.1-3. Intermediate stages during differentiation of EC from ESCs. (Elsevier, vol 1, 2004, Handbook of Stem cells) 20
Figure 4.1-1. Overall strategy to isolate and culture hESC-ECs and to transplant them into a hindlimb ischemia mouse model. 36
Figure 4.1-2. EBs formation and PECAM expression and location during EBs differentiation. (A) EBs morphologies expanding continuously during EBsdifferentiation (40X magnification), (B) Expression of PECAM and Oct-4 during EBs differentiation (GAPDH was used as the internal... 38
Figure 4.1-3. PECAM localization in attached EBs and characterization of the cells isolated from center of attached EBs. (A) EBs were cultured on gelatin-coated plates for 7 days and the morphology of attached EBs were observed by phase contrast microscope (40X magnification). (B) A... 41
Figure 4.1-4. FACS analysis PECAM and vWF expression during the differentiation of hESCs and EBs. (A) undifferentiated hESCs, (B) day 9 EBs(PECAM and vWF expression increased as the hESCs differentiated gradually), (C) non-mechanical isolation and (D) mechanical isolation... 43
Figure 4.1-5. Vascular tube-like structure on Matrigel. (A) Non-mechanical isolation, (B) mechanical isolation (40X magnification). The cells isolated from center of attached EBs successfully formed vascular tube-like structures within 12 hr, whereas the cells not isolated failed to form... 44
Figure 4.2-1. Changes in the marker expressions during hESCs differentiation and purification of hESC-ECs. A, RT-PCR for examination of EC-specific marker expression in EBs during hESCs differentiation. B, FACS analysis for vWF of cells isolated mechanically from EBs. C, The... 46
Figure 4.2-2. Characterization of hESC-ECs ESC-ECs. Sorted cells were expanded up to 5 passages for in vivo injection.A, RT-PCR for various endothelial markers of hESC-ECs. HUVECs and undifferentiated hESCs were used as a positive and negative control, respectively. B, Immuno... 47
Figure 4.2-3. In vitro functional test of hESC-ECs. A, ac-LDL uptake by hESC-ECs. Red color in cytoplasm represents DiI-labeled ac-LDL. HUVECs and STO fibroblasts were used as a positive and negative control, respectively. B, Vascular tube-like networks formed by... 48
Figure 4.3-1. Improvement of ischemic limb salvage by hESC-ECs transplantation. A, Representative photographs of medium-(left) and hESC-ECs-(right) treated ischemic hindlimbs on days 0, 14, and 28 after treatment. B, Physiological status of ischemic limbs on 4 weeks... 51
Figure 4.3-2. Improvement of blood flow in ischemic hindlimbs after hESC-ECs transplantation. A, Serial analysis of laser Doppler perfusion imaging at 0, 14, and 28 days after treatment. A greater increase of limb blood perfusion was observed in the ischemic limbs of mice receiving... 53
Figure 4.3-3. Enhancement of neovascularization in ischemic limb tissues 4 weeks after hESC-ECs transplantation. A-C, Immuno histochemical staining with vWF (A), SMα-actin (B), and PECAM (C). D and E, Quantification of capillary density (D) and of arteriole density (E) in... 56
Figure 4.3-4. Expression of human angiogenic growth factors in ischemic limb tissues by hESC-ECs transplantation. A and B, Immuno histochemical staining for VEGF (A) and bFGF (B) with human-specific antibodies. Significant expression of these human factors was observed in hESC-ECs... 58
Figure 4.3-5. Engraftment of transplanted hESC-ECs into vascular structures in the ischemic limb tissues. A, RT-PCR for human β-actin to show the presence of human cells in mouse ischemic tissues. B, FISH staining showing human cells with Y chromosomes (arrows) in the... 60
List of Supplement Figure
Supplement Figure 3.1-1. In vitro and in vivo characterization of CHA3-hESCs. 27
Supplement Figure 4.3-1. Doppler imaging analysis to measure blood perfusion in ischemic hindlimbs after cell transplantation. A, Serial analysis of laser Doppler perfusion imaging at 5, 14, and 28 days after treatment. Blood perfusion increased in the ischemic limbs of mice... 54