Title Page
ABSTRACT
국문 초록
Contents
INTRODUCTION 14
MATERIALS AND METHODS 16
PDMS Bending Cell Culture System 16
Human Cell Culture Conditions and Neuroepithelial Differentiation 18
Chemically Induced Inhibition of Cytoskeletal Components 19
Immunocytochemistry/Immunofluorescence (ICC/IF) 19
Confocal Microscopy, Light Microscopy and Live Imaging 21
Semi-Automated Division Angle and Cell Cycle Phase Measurement 22
Visual Representation of Data and Statistical Analysis 24
Computational Simulation of Division Angle 25
RESULTS 26
Human Pluripotent Stem Cells Cultured on PDMS Substrates and Differentiated with Dual SMAD Inhibition Protocol Show the Features of the Early Neuroepithelium 26
Live Imaging of Human Induced Pluripotent Stem Cell Line H2B-GFP Reveals Mitotic Division Time and the Semi-Automated Measurement of Cell Division Angle is Equivalent to the Conventional Division Angle Measurement 30
Cells of Neuroepithelium-like Colonies Undergo Oriented Division upon External Stress, Dose-Dependently to the Magnitude of the Force in a Direction with Respect to the Types of Stress. 34
Actin Cytoskeleton Takes Part in Oriented Division upon External Compression While Both ROCK-Mediated and Non-Muscle Myosin-Mediate Actomyosin Contraction is Dispensable 39
Temporal Profile of Oriented Division Reveals Strain is Increased and Relieved over Time in Stretching, But Strain Persists in Prolonged Compression 43
External Compression Has a Tissue Sculpting Capability, But It Does Not Derive from the Oriented Cell Division 47
DISCUSSION 51
REFERENCES 54
APPENDICES 56
Appendix A. A Python Script for Semi-Automated Cell Division Measurement 60
Appendix B. Cell Division Simulator Based on Vector Addition 62
Table 1. Chemical Agents Used to Inhibit Actomyosin Components 19
Table 2. The List of Primary Antibodies, Secondary Antibodies, and Fluorescence-Conjugated Small Molecules Used in this Work. 20
Figure 1. The Schematics and Outline for PDMS Bending System and Early Neuroepithelial Differentiation with Dual SMAD Inhibition (dSMADi) 29
Figure 2. Semi-Automated Measurement of Cell Division Angle and Cell Cycle Phase 33
Figure 3. The Cells of Neuroepithelium-like Colonies Undergo Oriented Division in Response to External Stress 38
Figure 4. Perturbation of Actin Cytoskeleton and Myosin Contractile Machineries During Stretching and Compression 42
Figure 5. Temporal Profile of Oriented Division upon External Stress 46
Figure 6. 48 Hours Culture under External Stress and Colony Tissue Morphology 50