Title Page
Abstract
Contents
List of abbreviations 15
Chapter 1. Research background and objectives 18
Chapter 2. Literature review 22
2.1. Human stem cells 22
2.1.1. Human mesenchymal stem cells 22
2.1.2. Human embryonic stem cells 22
2.2. Stem cell research utilizing graphene oxide 23
Chapter 3. Experimental procedures 27
3.1. Preparation of GO 27
3.1.1. Ball-milling of graphite 27
3.1.2. Preparation of GO by ball-milled graphite 27
3.2. Characterization of GO 27
3.3. Preparation of magnetic nanoparticles 28
3.4. Cultivation and differentiation of hMSCs 28
3.5. Cultivation of hESCs 29
3.6. Generation of hEBs and neural differentiation 30
3.7. Cell adhesion assay of GO 30
3.8. Cell viability assay 31
3.8.1. CCK8 assay 31
3.8.2. Fluorescence-based live and dead assay 31
3.9. qRT-PCR analysis 31
3.10. Alkaline phosphatase staining and Alizarin Red S staining 34
3.11. Immunocytochemistry 34
3.12. Western blotting 34
3.13. Statistical analysis 35
Chapter 4. Material characteristics and cellular interactions of size-controlled graphene oxide flakes 37
4.1. Introduction 37
4.2. Characterization of GO processed by ball-milling 37
4.3. Morphology and cytotoxic effect of GO attached to hMSCs 43
4.4. Conclusions 46
Chapter 5. Enhanced osteogenic differentiation of bone marrow-derived human mesenchymal stem cells using size-controlled graphene oxide flakes 47
5.1. Introduction 48
5.2. Enhancing effect of GO on osteogenic differentiation of hMSCs 51
5.3. Promotion of early cell spreading and focal adhesion complex formation of hMSCs by GO-1.7 55
5.4. Expression and localization of osteogenic marker proteins by GO-1.7 59
5.5. Proposed mechanism of osteogenic differentiation enhanced by GO 63
5.6. Conclusions 68
Chapter 6. Enhancing effect of graphene oxide flakes on stem cell viability in single-cell detachment and shear stress-caused apoptotic circumstances 69
6.1. Introduction 70
6.2. Viability of hMSCs treated with GO-1.7 in non-adhesive condition and shear stress 72
6.3. Viability of hESCs treated with size-controlled GO flakes in non-adhesive condition 76
6.4. Conclusions 78
Chapter 7. Enhanced neural differentiation of adipose-derived human mesenchymal stem cells using size-controlled graphene oxide flakes 80
7.1. Introduction 81
7.2. 3D culture of ADSCs 83
7.3. Gene expression of neural induction markers in ADSCs 85
7.4. Expression of neural induction markers in ADSCs 88
7.5. Conclusions 91
Chapter 8. Overall discussion and further suggestions 93
Appendix. Enhanced neural differentiation of 3D human embryonic stem cells via magnetic nanoparticle-based physical stimuli 96
A.1. Introduction 97
A.2. Improved neural induction of MNP-incorporated hEBs, manufactured through a concentrated magnetic force system 100
A.3. Morphological analysis of neurally induced hESCs 103
A.4. Genetical analysis of neural induction marker genes 107
A.5. Related mechanisms to accelerated neural induction of hEBs 110
A.6. Conclusions 112
Bibliography 113
국문초록 125
Table 1. List of primers used in qRT-PCR 33
Figure 4.1. Synthesis and characterization of physical properties of GO. 41
Figure 4.2. Detailed characterization of graphene oxide (GO). 42
Figure 4.3. Morphology of GO on the surface of hMSCs and cytotoxicity of GO. 45
Figure 5.1. Osteogenic differentiation of hMSCs using GO flakes fabricated by ball-milling and oxidized by modified Hummers' method. 50
Figure 5.2. Enhancing effect of GO on hMSCs during osteogenic differentiation. 53
Figure 5.3. Promotion effect of GO on early cell spreading and focal adhesion complex formation of hMSCs. 57
Figure 5.4. Effect of GO on the expression and localization of osteogenic marker proteins. 62
Figure 5.5. Signaling pathway of osteogenic differentiation enhanced by GO. 66
Figure 5.6. Amount of relative oxygen species (ROS) of hMSCs treated with GO. 67
Figure 6.1. Research objective and schemes. 71
Figure 6.2. Viability and cell area of hMSCs treated GO-1.7 during 24 hours in non-adhesive condition (condition 1). 73
Figure 6.3. Viability and cell area of hMSCs treated GO-1.7 and GO-4.6 during 24 hours in non-adhesive condition with shear stress (condition 2). 75
Figure 6.4. Viability of hESCs treated 3 GO groups during 24 hours in non-adhesive condition (condition 1). 77
Figure 6.5. Decreased deterioration of cells on non-adhesive condition through the effect of size-controlled GO. 79
Figure 7.1. Schematic for experimental groups. 82
Figure 7.2. Morphology and dose-dependent viability of ADSCs in response of GO and MNPs. 84
Figure 7.3. Relative mRNA expression level of neural induction markers for 5 days of neural differentiation on ADSCs. 87
Figure 7.4. Immunocytochemistry of neural induction marker protein and Nissl staining. 90
Figure 7.5. Enhanced neural induction of ADSCs spheroid using size-controlled graphene oxide and magnetic nanoparticles. 92
Figure A1. Illustrations for neural induction of 2D and 3D hESCs. 99
Figure A2. Schematics of MNP-based hEB generation method and designation of experimental groups. 101
Figure A3. Morphological analysis of neurally induced 2D and 3D hESCs. 106
Figure A4. Genetical analysis of a pluripotency marker gene and neural induction marker genes. 109
Figure A5. Analysis of signaling pathways of the neurally induced 2D and 3D hESCs. 111