Title Page
ABSTRACT
Contents
INTRODUCTION 12
1. Parkinson's disease 12
2. Viral gene delivery 16
3. Necessity of cerebral organoid-based disease model development 18
4. Proposal of Parkinson's disease model development using viral gene delivery in cerebral organoids 20
METHODS AND MATERIALS 21
Human Embryonic Stem Cells (hESCs) maintenance 21
Generation of hESCs-derived cerebral organoids 21
AAV production 22
Lentivirus production 22
Immunofluorescence staining 23
RT-PCR analysis of gene expression 23
Statistical analysis 24
Single-cell RNA sequencing 24
Ethical statements 25
RESULTS 26
1) Construction of various viral vectors 26
2) Viral gene delivery approaches in hESCs and cerebral organoids 28
3) Generation of cerebral organoids from hESCs 31
4) Exploring various virus-mediated gene deliveries in hESCs 35
5) Analysis of the characteristics of cerebral organoids with lentivirus-mediated fluorescence gene delivery 38
6) Validation of cerebral organoids with Parkinson's disease related genes as a Parkinson's disease model 41
DISCUSSION 49
REFERENCES 53
국문초록 66
Figure 1. Construction of various viral vectors for virus-mediated gene delivery in different cell types. 27
Figure 2. Gene delivery in hESCs and cerebral organoids with virus transduction method. 30
Figure 3. Establishment of hESC-derived cerebral organoids and analysis. 34
Figure 4. Comparison of transduction efficiency between viral vectors in hESCs. 37
Figure 5. Characterization of viral gene delivery in cerebral organoids using fluorescence microscopy and immunofluorescence staining. 40
Figure 6. Validation of cerebral oragnoids with α-syn gene delivered by lentivirus. 44
Figure 7. Validation of cerebral oragnoids with PD related genes delivered by lentivirus. 48