臺灣博碩士論文加值系統

光遺傳學是一項技術，可通過非侵入性光刺激對生物系統進行有針對性和快速的控制。Channelrhodopsin-2（ChR2）是一種膜蛋白，能夠透過光激發對膜離子通道進行細胞調節。傳統轉基因技術可能限制光遺傳學在誘導多能性幹細胞（iPSC）中的臨床應用。本研究測試了ChR2可通過病毒轉染至神經前驅細胞（NPC）而在iPSC衍生的神經元中表達。結果顯示ChR2通過病毒轉染至NPC而在iPSC分化為運動神經元中長時間穩定表達。藉由藍光激發的神經元能夠調節膜電壓，並且使用全細胞膜片鉗技術觀察到的神經元具有去極化現象，光學激活 的神經元也顯示出與運動神經元一致的自發放電模式。我們的研究結果表明攜帶ChR2基因的運動神經元可以在長時間內維持ChR2基因表達，藉由光學調控該特定細胞群體中的作用。本論文提供光基因病毒轉染方法可能有益於iPSC研究和未來臨床應用。

Optogenetics is a technology that allows targeted and fast control of biological systems with noninvasive optical stimulation. Channelrhodopsin-2 (ChR2) is a membrane protein that enables cellular regulation of transmembrane ion conductance through light-gated pores. Previous methods of transgenic approach may limit the clinical application of optogenetics in human induced pluripotent stem cells (iPSC). This study tested the hypothesis that ChR2 can be expressed in iPSC-derived neurons through AAV transfection to the neuronal progenitor cells (NPC). The cultured NPCs were transfected with ChR2 optogene using AAV with a vector concentrations of 3.25×10^13 vg/mL. The experimental procedure included cloning of vectors, virus purification, transfection of cultured neurons cells and patch-clamp electrophysiological measurement of the derived neurons. The results showed that ChR2 was stably expressed for at least 20 days in human iPSC-derived motor neurons through AAV transfection to the NPC. The neurons exposed to 430 nm blue light stimulation enabled regulation of membrane voltage and depolarized the cells from −55 mV to −15 mV observed with the whole-cell patch-clamp technique. The optically activated neurons also showed a spontaneous firing pattern consistent with that of the motor neurons. Our results demonstrated that iPSC-derived NPC carrying ChR2 optogene differentiated into motor neurons can maintain ChR2 gene expression in an extended period of time, thus allowing optical manipulation of activity in this specific cell population. The AAV optogene transfection method may benefit iPSC research and future clinical applications.

摘 要 i
ABSTRACT ii
誌 謝 iii
Contents iv
Figure Captions vi
Table Captions viii
Chapter 1 Introduction 1
1.1 Motor Neuron Disease 1
1.2 Amyotrophic lateral scleerosis (ALS) 2
1.2.1 Description and clinical symptoms 2
1.3 Induced pluripotent stem cells (iPSC) 3
1.3.1 Historical overview 3
1.3.2 Generation of iPSC 5
1.3.3 iPSC reprogramming 5
1.4 Optogenetics 7
1.4.1 Mechanism 10
1.4.2 Application in medical neuroscience 11
1.5 Motivation 12
1.6 Objectives 13
Chapter 2 Cell Culture 14
2.1 Materials and Methods 14
2.1.1 Culture Conditions 15
2.1.2 Differentiation of neural progenitor cells 17
2.1.3 Characterization of motor neurons 19
2.2 Design Goals 21
2.3 Characterization of iPSC 22
2.3.1 Detection of expression of exogenous reprogramming factors 22
2.3.2 Flow cytometry of pluripotent markers 23
2.3.3 Expression of pluripotency-related markers were analyzed 24
2.3.4 Differentiate into the three germ layers in vitro demonstrated 25
Chapter 3 Development of Recombinant AAV Vectors 27
3.1 Adeno-associated virus (AAV) History 27
3.2 Materials and Methods 27
3.2.1 Plasmid Construction 28
3.2.2 Viral Purification 29
3.3 AAV Transfection of neural progenitor cells 31
3.4 Optogenetics in neural progenitor cells 31
3.4.1 Statistics 32
3.4.2 Analyzed trans-differentiation efficiency 33
Chapter 4 Experimental Results 34
4.1 Electrophysiological recording 34
4.1.1 Membrane potential 35
4.1.2 Whole-cell patch-clamp technique 36
Chapter 5 Establishment of a iPSC-derived MNs co-culture with muscle cell 39
5.1 Assembly of co-culture 39
5.1.1 Neuromuscular junction with myotube 41
5.1.2 Muscle contraction 43
Chapter 6 Conclusions and Future Work 44
6.1 Conclusions 44
6.2 Future works 44
References 45
Appendix 51

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