Title Page
Contents
ABSTRACT 10
Chapter 1. Literature Review 12
Ⅰ. Literature Review 13
1-1. Accelerator Mass Spectrometry 13
1-2. ¹⁴C-Nanotracing technique 15
1-3. Mesenchymal stem cell 17
1-4. Human embryonic stem cells-ECs 19
Chapter 2. In vivo Tracking of ¹⁴C-Thymidine labeled Mesenchymal Stem Cells using Accelerator Mass Spectrometry 21
Ⅰ. Introduction 22
2-1. Introduction 22
Ⅱ. Materials and Methods 24
2-1. Cell culture and labeling of ¹⁴C-thymidine 24
2-2. Cell viability assay 24
2-3. DNA extraction 25
2-4. Analysis of liquid scintillation counter (LSC) 25
2-5. Characterization of ¹⁴C-labeled AD-MSCs 25
2-6. Assessment of multilineage differentiation 26
2-7. Calibration curves of LSC and AMS analysis 26
2-8. Confirm to distribution of ¹⁴C-labeled AD-MSCs in nude mice 27
2-9. Graphitization of samples and AMS measurement 27
2-10. Detection of Dil labeled AD-MSCs in nude mice 28
2-11. Statistical analysis 28
Ⅲ. Results 29
2-1. Selection of optimal ¹⁴C-thymidine concentration for AD-MSCs 29
2-2. Characterization of ¹⁴C-labeled AD-MSCs 36
2-3. Pre-treatment process and calibration the LSC and AMS method 39
2-4. Quantification of ¹⁴C-labeled AD-MSCs in nude mice 42
2-5. Detection of Dil-labeled AD-MSCs in nude mice 48
Ⅳ. Discussion 50
2-1. Discussion 50
Chapter 3. Verification of therapeutic effect through AMS based single cell level quantification of ECs distributed into an ischemic model 52
Ⅰ. Introduction 53
3-1. Introduction 53
Ⅱ. Materials and Methods 56
3-1. Cell culture and generation of hESC-ECs 56
3-2. Immunocytochemistry and ac-LDL uptake 57
3-3. Tube formation assay 57
3-4. Labeling of ¹⁴C-thymidine in CB-EPCs and hESC-ECs 57
3-5. Cell proliferation assay 58
3-6. Measurement of concentration of small molecule using LC-MS/MS 58
3-7. Genomic DNA (gDNA) extraction and qPCR 59
3-8. RNA extraction and cDNA synthesis for qPCR analysis 59
3-9. Liquid scintillation counter (LSC) measurement 59
3-10. Modeling of hindlimb ischemia in nude mice and injection of cells 60
3-11. Laser Doppler imaging for blood flow measurement 60
3-12. Histological analysis 61
3-13. Detection of engrafted Dil labeled CB-EPCs and hESC-ECs 61
3-14. Pretreatment of urine and feces for ¹⁴C signal measurement 61
3-15. Graphitization of samples and AMS measurement 62
3-16. Statistical analysis 63
Ⅲ. Results 64
3-1. Generation and characterization of hESC-ECs 64
3-2. Selection of optimal conditions for ¹⁴C-thymidine concentration 67
3-3. Improvement of ischemic region by hESC-ECs transplantation 76
3-4. Verification of neovascularization according to hESC-ECs 83
3-5. In vivo cell tracking and quantification of ECs 87
3-6. Tracking of organ according to migration of ECs 95
Ⅳ. Discussion 101
3-1. Discussion 101
References 107
Appendix 117
Abstract (in Korean) 119
Table 1. Comparison of cell number and ¹⁴C activity according to the dose of ¹⁴C-thymidine. 35
Table 2. Quantification of cell number per organ by LSC analysis. 46
Table 3. Quantification of cell number per organ by AMS analysis. 47
Table 4. Comparison of ¹⁴C activity and ¹⁴C uptake rate according to the dose of ¹⁴C-thymidine. 73
Table 5. Blood flow ratio (ischemic/non-ischemic) results according to times after injection of PBS, hESC-ECs, and CB-EPCs group. 80
Table 6. Quantitative results in ischemic limb after ¹⁴C-labeled hESC-ECs and CB-EPCs group injection using LSC analysis. 93
Table 7. Quantitative results in ischemic limb after ¹⁴C-labeled hESC-ECs and CB-EPCs group injection using AMS analysis. 94
Table 8. The result of concentration in organ after ¹⁴C-labeled hESC-ECs group injection using AMS analysis. 98
Table 9. The result of total amount in organ after ¹⁴C-labeled CB-EPCs group injection using AMS analysis. 99
Figure 1. The composition of KIST accelerator mass spectrometry. 14
Figure 2. The comparison of microdosing and ¹⁴C-nanotracing. 16
Figure 3. The differentiation potential of mesenchymal stem cells. 18
Figure 4. In vitro differentiation of human embryonic stem cells to endothelial cells and the molecular characteristics of human embryonic... 20
Figure 5. Method of labeling to prepare ¹⁴C-labeled AD-MSCs using ¹⁴C-thymidine. 31
Figure 6. Optimization of media conditions for AD-MSC labeling with ¹⁴C-thymidine. 32
Figure 7. Cell morphology, proliferation, and viability according to ¹⁴C-thymidine concentration. 33
Figure 8. Optimization of ¹⁴C-thymidine concentration for AD-MSCs. 34
Figure 9. Confirmation of MSC specific marker expression of unlabeled and ¹⁴C-thymidine labeled AD-MSCs by FACS analysis. 37
Figure 10. Analysis of biological characteristics of ¹⁴C-thymidine labeled AD-MSCs. 38
Figure 11. Pretreatment process of LSC and AMS analysis. (A) Flow chart of pretreatment process for LSC. (B) Flow chart of pretreatment process for AMS. 40
Figure 12. Calibration curves of LSC and AMS analysis. 41
Figure 13. Quantification result of infused ¹⁴C-labeled AD-MSCs in nude mice using LSC and AMS analysis. 45
Figure 14. Image of infused AD-MSCs with Dil staining. Distribution of Dil-labeled AD-MSCs were analyzed in lung, liver, spleen, kidney, brain,... 49
Figure 15. Generation of hESC-ECs. 65
Figure 16. Characteristics of hESC-ECs. 66
Figure 17. Optimal conditions of cell proliferation for labeling of ¹⁴C-thymidine in hESC-ECs and CB-EPCs. 70
Figure 18. Comparison of ¹⁴C signal for labeling of ¹⁴C-thymidine in hESC-ECs and CB-EPCs. 71
Figure 19. Expression of thymidine kinase1 and concentration of thymidine, dTMP, and dUMP in intracellular space. 72
Figure 20. Characteristics of ¹⁴C-thymidine in hESC-ECs and CB-EPCs. 74
Figure 21. Verification of angiogenic effects of hESC-ECs according to ¹⁴C-thymidine (2.5 nCi/ml) labeling. 75
Figure 22. Blood flow improvement effect in hindlimb ischemia model following ECs transplantation. 78
Figure 23. Measurement of blood flow between groups using laser Doppler. 79
Figure 24. Hematoxylin and eosin staining (H&E) of the lesion area at day 28 after transplantation of PBS, CB-EPC, and hESC-EC. Hematoxylin and... 81
Figure 25. Masson's trichrome staining (MT) of the lesion area at day 28 after transplantation of PBS, CB-EPC, and hESC-EC. Masson's... 82
Figure 26. Verification of capillaries formed by Dil-labeled ECs (CB-EPC and hESC-EC: Dil+CD31+) and mouse ECs (Only CD31+) through... 85
Figure 27. Verification of angiogenesis/neovascularization at the lesion site 28 days after transplantation of PBS, CB-EPC and hESC-EC. 86
Figure 28. Strategies to analyze the quantification of cells engraftment and migration after injection of ¹⁴C-labeled ECs into the hindlimb... 89
Figure 29. The quantification of hESC-EC (n=3) and CB-EPC (n=3) group after injection to the ischemic limb region using LSC 90
Figure 30. The quantification of ¹⁴C-labeled hESC-ECs after injection in ischemic limb. 91
Figure 31. The quantification of ¹⁴C-labeled hESC-ECs and CB-EPCs after injection in hindlimb ischemia model. 92
Figure 32. Quantification of ¹⁴C-labeled hESC-ECs and CB-EPCs in organs other than transplantation sites using AMS. 97
Figure 33. Amount of cell and ¹⁴C signal in urine and feces after administration of hESC-ECs. 100