Title Page
Contents
ABSTRACT 13
Ⅰ. Introduction 16
1.1. Mesenchymal stem cells (MSCs) 16
1.1.1. Definition of MSCs 16
1.1.2. Resource of MSCs and special feature 17
1.2. Characteristics of MSCs 18
1.2.1. Multipotency of MSCs 18
1.2.2. Immunomodulatory function and anti-inflammatory action of MSCs 20
1.2.3. Regenerative effect of MSCs 23
1.2.4. Anti-fibrotic effect 24
1.2.5. Anti-oxidative stress effect 26
1.2.6. Anti-apoptotic effect 28
1.2.7. Anti-tumor effect 28
1.2.8. Anti-microbial effect 29
1.2.9. Homing effect 31
1.3. Application of MSCs 34
1.3.1. First clinical applications of the MSCs 34
1.3.2. MSCs in anti-inflammatory therapy 35
1.3.3. MSCs in regenerative medicine 39
Ⅱ. Anti-inflammatory effects of MMSCs in DNCB-induced atopic dermatitis mice 64
2.1. Introduction 64
2.2. Material and methods 67
2.2.1. Chemicals and reagents 67
2.2.2. hPSC culture 67
2.2.3. Isolation of MMSCs and culture 68
2.2.4. MMSC cultured and conditioned media concentration 68
2.2.5. Antibody-based protein microarray 69
2.2.6. Animals 70
2.2.7. Induction and treatment of AD-like skin lesions in mice 71
2.2.8. Histopathological analysis 72
2.2.9. Quantitative real-time PCR (qPCR) 72
2.2.10. Serum enzyme-linked immunosorbent assay (ELISA) 75
2.2.11. Statistical analysis 75
2.2.12. Ethics statement 75
2.3. Results 76
2.3.1. Protein antibody array of MMSCs 76
2.3.2. Induction of AD-like lesions on mouse skin and histopathological remission 82
2.3.3. RT-qPCR analysis of genes associated with inflammation and IgE levels in serum by ELISA analysis 85
2.4. Discussion 87
Ⅲ. Immunomodulation of pluripotent stem cell derived mesenchymal stem cells in rotator cuff tears model 90
3.1. Introduction 90
3.2. Material and methods 93
3.2.1. Isolation and culture of MMSCs 93
3.2.2. RNA-sequencing for MMSCs 94
3.2.3. Transcriptomic analysis for MMSCs compared to adult MSCs 95
3.2.4. Mixed lymphocyte reaction (MLR) for MMSCs, BM-MSCs, and AD-MSCs 97
3.2.5. Rotator cuff tear (RCT) model of rats 97
3.2.6. Long-term in vivo cell tracking using Dil 98
3.2.7. Grip strength measurement in rats 99
3.2.8. RT-qPCR analysis to monitor physiological status of in vivo muscles 100
3.2.9. Antibody-based protein microarray 102
3.2.10. Histopathological analysis of in vivo muscles 103
3.2.11. Statistical analysis 104
3.2.12. Ethics statement 105
3.3. Results 106
3.3.1. Transcriptomic characteristics of MMSCs and mixed lymphocyte reaction 106
3.3.2. Grip strength test for quantification of rotator muscular function retrieval 114
3.3.3. Morphological evaluation of muscles in vivo based on H&E and MT staining 117
3.3.4. Inflammatory cell counting based on TB staining and IHC for CD68 121
3.3.5. RT-qPCR analysis of genes associated with inflammation, adipogenicity, and myogenicity 125
3.3.6. Long-term in vivo MSC tracking and apoptosis 128
3.4. Discussion 131
References 137
Appendix 168
Abstract (in Korean) 171
Table 1. Primer sequences. (Mouse) 74
Table 2. Proteins including each node in Fig. 4C and Fig. 4D. 81
Table 3. Primer sequences. (Rat) 101
Figure 1. Applications of MSCs with multiple differentiation potential for repair of various tissues. 19
Figure 2. MSCs-Immune cell interactions. 21
Figure 3. MSCs in the context of a "galaxy" of intercellular signals. 33
Figure 4. Schematic mechanisms of MSCs for cardiac regeneration. 49
Figure 5. Examples of potential applications of MSCs. 63
Figure 6. Characterization of MMSCs via comparison with BM-MSCs. 78
Figure 7. Schematic diagram of the study protocol, Photographs of the dermal skin lesions. 83
Figure 8. Histological analysis. Effects of MCMC on the histology of lesions in DNCB-treated mice. 84
Figure 9. MCMC inhibits cytokine expression. Effects of MCMC on cytokine expression. 86
Figure 10. Characterization of MMSCs compared to adult MSCs. 109
Figure 11. Transcriptomic analysis of MMSCs compared to adult MSCs. 111
Figure 12. The percentages of genes per GOs for each DEG. 112
Figure 13. Antibody-Based Protein Microarray. 113
Figure 14. Schematic diagram of the study, effects of MSCs injection on grip strength recovery after RCT induction. 115
Figure 15. Morphological evaluation of H&E stained slides. 119
Figure 16. Morphological evaluation of MT-stained slides. 120
Figure 17. Confirmation of mast cells. 122
Figure 18. Confirmation of CD68+ macrophages. 123
Figure 19. Relative expressions of genes related to inflammation, adipogenicity, and myogenicity in supraspinatus. 126
Figure 20. Relative expressions of genes related to inflammation, adipogenicity, and myogenicity in infraspinatus. 127
Figure 21. Long-term in vivo MSC tracking and apoptosis. 129