Title Page
Contents
ABSTRACT 12
Chapter 1. Literature Review 14
Ⅰ. Literature Review 15
1-1. Induced pluripotent stem cells (iPSCs) 15
1-2. hiPSC derived cardiomyocyte (hiPSC-CMs) 17
1-3. Organoids 19
Chapter 2. Dual-Cardiotoxicity Evaluation Methods based on human iPSC-derived Cardiomyocyte and Multi-Electrode Array 21
Ⅰ. Introduction 22
2-1. Introduction 22
Ⅱ. Materials and Methods 24
2-1. Generation of hiPSC-CMs 24
2-2. Analysis of cardiac structure immunocytochemistry 24
2-3. Confirmation purity using flow cytometry analysis 25
2-4. Measurement of FP and contractility using MEA 25
Ⅲ. Results 27
2-1. Generation of hiPSC-CMs for cardiotoxicity test 27
2-2. Parameters for measuring MEA-based FP and contractility 29
2-3. Assessment of FP following treatment with TdP drugs 32
2-4. Assessment of contractility following treatment with TdP drugs 35
2-5. Dual-cardiotoxicity evaluation by nifedipine treatment 37
Ⅳ. Discussion 39
2-1. Discussion 39
Chapter 3. Cardiotoxicity Evaluation Methods based on Human iPSC-derived Heart Organoids 41
Ⅰ. Introduction 42
3-1. Introduction 42
Ⅱ. Materials and Methods 45
3-1. Generation of hiPSCs derived heart organoids 45
3-2. PCR and mRNA seq 46
3-3. Single-cell RNA seq (scRNA seq) 47
3-4. Immunofluorescent staining and histological analysis 47
3-5. Live/dead assay 48
3-6. Sprouting assay of CF-hHO 48
3-7. In vivo vascularization of CF-hHOs in nude mice 49
3-8. Chemical reagents 50
3-9. Microscopy video analysis of beating rate and contraction distance 50
3-10. Intrinsic optical signal (IOS) 50
3-11. Measurement of calcium flux 51
3-12. Multi-electrode assay (MEA) 51
3-13. Statistical analysis 52
Ⅲ. Results 53
3-1. Generation of CF-hHOs 53
3-2. Gene profiling in CF-hHOs 60
3-3. Self-organization of CMs in CF-hHOs 70
3-4. Morphological and functional maturation of CF-hHOs 80
3-5. Epicardium and cardiac specific cells in CF-hHOs 88
3-6. Validation of in vitro and in vivo vascularized CF-hHOs 98
3-7. Mechanical analysis related to contractility of CF-hHOs 106
3-8. Electrophysiological function of CF-hHOs 113
Ⅳ. Discussion 120
3-1. Discussion 120
References 125
Appendix 140
Abstract (in Korean) 141
Table 1. ISO analysis to measure changes in contraction magnitude with drug treatment. 109
Table 2. Calcium imaging to measure changes in calcium flux with drug treatment. 115
Table 3. MEA analysis to measure changes in main parameters of field potential with drug treatment. 117
Figure 1. Human iPSC (hiSPCs) derivation, differentiation, and applications. 16
Figure 2. The most common tools for evaluation of drug effects in hiPSC-CM-based in vitro models. 18
Figure 3. Process for the establishment of human PSC-derived and adult stem cell (AdSC)-derived organoids. 20
Figure 4. Characterization of hiPSC-CMs. 28
Figure 5. FP and contractility analysis of hiPSC-CMs using MEA. 30
Figure 6. Main parameters for MEA-based FP and contractility. 31
Figure 7. Changes in 4 main parameters of FP activities during treatment of clinical TdP drugs. 34
Figure 8. Changes in beat amplitude, which is the main parameter of contractile force during treatment of clinical TdP drugs. 36
Figure 9. Monitoring of changes in FP and contractility according to nifedipine dosing in hiPSC-CMs. 38
Figure 10. Generation of hiPSC-derived CF-hHOs. 56
Figure 11. Selection of CF-hHOs. 57
Figure 12. The morphology of hHOs with chambers fabricated for different experiments. 58
Figure 13. Live-dead assay to determine the viability of CF-hHOs. 59
Figure 14. Electrophoresis for cardiac constituent cell analysis by hHOs differentiation step. 63
Figure 15. Total mRNA sequencing analysis of hHOs. 64
Figure 16. mRNA sequencing analysis of EB and hHOs. 65
Figure 17. PCA clustering of hHOs and GEO dataset. 66
Figure 18. GO analysis of day 20 hHOs. 67
Figure 19. GO analysis of day 20 hHOs. 68
Figure 20. scRNA seq of hHOs. 69
Figure 21. Immunofluorescence staining for cTnT to confirm the distribution of CMs of CF-hHOs. 73
Figure 22. Identification of CMs in CF-hHOs. 74
Figure 23. Comparison of CMs distribution between non-chambered and chambered hHOs in day 20. 75
Figure 24. Morphological identification of atrium/ventricle-like areas in CF-hHOs. 76
Figure 25. Functional identification of atrium/ventricle-like areas in CF-hHOs. 77
Figure 26. Analysis of atrium/ventricle-similar areas in CF-hHOs. 78
Figure 27. Analysis of atrium/ventricle-similar areas in CF-hHOs on day 10 and day 20. 79
Figure 28. Verification of maturation of CMs in CF-hHOs using MEA. 82
Figure 29. Comparison of electrophysiological maturation of day 10-13 hHOs and day 19-21 CF-hHOs using MEA. 83
Figure 30. IOS for measuring the change of contraction magnitude according to maturation of CF-hHOs. 84
Figure 31. Verification of maturation of CMs in CF-Hho using mRNA seq. 85
Figure 32. Verification of maturation of CMs in CF-hHOs using IF. 86
Figure 33. IF image for sarcomere structure analysis of CMs following maturation of CF-hHOs. 87
Figure 34. Distribution validation of cells expressing αSMA or/and VIM expression in CF-hHOs at day 20. 91
Figure 35. Analysis of epicardium naturally formed in CF-hHOs. 92
Figure 36. Changes in the distribution of WT1+ cells according to the differentiation and maturation of CF-hHOs. 93
Figure 37. Analysis of epicardium and cardiac fibroblasts naturally formed in CF-hHOs. 94
Figure 38. Analysis of the epicardium naturally formed in CF-hHOs. 95
Figure 39. Comparison of distribution of WT1+ cells and VIM+ cells between non-chambered and chambered hHOs. 96
Figure 40. Major layers naturally formed in CF-hHOs. 97
Figure 41. Identification of ECs in CF-hHOs. 101
Figure 42. Verification of in vitro vascularized CF-hHOs. 102
Figure 43. Verification of in vivo vascularized CF-hHOs. 103
Figure 44. Analysis of blood vessels in tissues of mice in Matrigel transplanted into nude mice. 104
Figure 45. Morphology of GFP-tagged hPSC derived CF-hHO implanted subcutaneously in nude mice for 10 days. 105
Figure 46. Mechanical function analysis of CF-hHO through microscopy video and optical contractility analysis. 110
Figure 47. Comparison of drug differential effects on E-4031 between atrium-similar and ventricle-similar areas using IOS. 112
Figure 48. Analysis of electrophysiological function through calcium flux imaging. 116
Figure 49. Analysis of electrophysiological function through MEA 118
Figure 50. FPD analysis of CF-hHOs following nifedipine and isoproterenol treatment using MEA. 119