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研究生:蘇怡甄
研究生(外文):Yi-Chen Su
論文名稱:人類特定疾病模型之誘導式多能性幹細胞株之建立
論文名稱(外文):Establishment of Disease-Specific Human Induced Pluripotent Stem Cells
指導教授:李光申李光申引用關係
指導教授(外文):Oscar K. Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:77
中文關鍵詞:誘導式多能性幹細胞疾病模式
外文關鍵詞:iPSCdisease model
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人類誘導式多能性幹細胞(Induced pluripotent stem cells, iPSCs)具備自我更新(Self-renewal)與分化(Differentiation)的潛在能力。在先前的報告中也提到可透過此人類誘導型幹細胞建立出與特定性之疾病模式。誘導型幹細胞可結合臨床資源建立出具獨特基因背景之細胞疾病模式。除此之外,以病人自身細胞作為來源所建立出之誘導型幹細胞,對於解決現行臨床疾病問題是非常有用的一種創新方式 (例如:找出治病原因、建立藥物開發平台或進行細胞的移植)。為了能夠建立此系統以利於未來實驗室進行此類的相關研究工作,我們透過非基因嵌合型的基因轉送方法,將OCT3/4, SOX2, KLF4, L-MYC and LIN28同時送入細胞中進行轉型。為了分析纖維母細胞是否正確轉型,本研究透過觀察轉型處理之細胞是否具備胚胎幹細胞的型態(ES-like cells),並進一步以鹼性磷酸酶染色(Alkaline phosphatase live stain, ALP live stain)、免疫螢光染色法(Immunofluorescence, IF)並透過定量即時聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction, qPCR)了解相關基因表現,判斷纖維母細胞是否成功轉型為誘導性多功能幹細胞。本研究希望以建立神經表現細胞為例,建立可用於探討人類神經退化症的疾病模式,由本研究的數據顯示轉型後的細胞具備專一性的螢光表現,並且具備誘導型幹細胞的基因表現,並且結合神經誘導物建立出神經前趨細胞。由此研究中可知纖維母細胞可透過合適的誘導方法建立出特定疾病細胞之模式,未來我們更希望能夠將此技術可協助建立各式特定疾病模式,除了可提供更接近人類疾病的疾病模式,更能在短時間內提供一個良好的疾病研究平台,並進一步建立提供藥物篩選平台,更能有效加速藥物開發進程。
Human induced pluripotent stem cells (iPSCs) possess the characteristic of self-renewal and differentiation. They also have been reported to serve as a patient-specific disease model. iPSCs can be derived with the unique genetic background from clinical resource. These patients-derived iPSCs are useful for investigating medical problems (e.g. the cause of disease, drug development and cell transplantation). In order to establish this system in our lab, several pluripotency-related genes, including OCT3/4, SOX2, KLF4, L-MYC and LIN28 were used to reprogram somatic cells into iPSCs by integration-free method. The pluripotency of these fibroblast-derived iPSCs were analyzed by alkaline phosphatase (ALP) live stain, immunofluorescence (IF) staining and endogenous gene expression level. Then, these fibroblast-derived iPSCs were also analyzed for the ability of neural differentiation, and the immunofluorescence data indicated that they can differentiate into neural progenitor cells (NPCs). From these data, it was demonstrated that these fibroblast-derived iPSCs not only have the properties of pluripotency but also possess the ability of neural differentiation. In the future, these patient-specific iPSCs can provide a platform for exploring some neural degeneration diseases like spinocerebellar ataxia type 3 (SCA3), and measuring the effect of cell transplantation.
Table of Contents
Table of Contents I
Abstract VII
中文摘要 VIII
List of Abbreviations IX
1. Introduction 1
1.1 Definition of stem cells and their application 1
1.2 The properties of Induced pluripotent stem cells (iPSCs) and application 2
1.3 What is “disease in a dish” 3
1.4 The methods of reprogramming for generating patient- specific iPSCs 4
1.5 Take the spinocerebellar ataxia type 3 (SCA3) for example 5
2. Materials and Methods 7
2.1 Plasmids obtainment and amplification 7
2.2 Maintenance and expansion of human ES、iPS cell line and skin fibroblast-derived iPSCs 8
2.3 Reprogramming methods for human skin fibroblast-derived iPSCs 9
2.3.1 Electroporation 9
2.3.2 Transfection reagents 10
2.4 Institutional Review Board (IRB) of human subject 10
2.5 Reprogramming of CG28 and primary human skin fibroblasts into iPSCs 11
2.5.1 Maintenance and expansion of CG28 11
2.5.2 Isolation of primary human skin fibroblasts 11
2.5.3 Maintenance and expansion of primary human skin fibroblast 12
2.5.4 Reprogramming procedure 12
2.6 Characterization of fibroblast-derived iPSCs 13
2.6.1 Alkaline phosphatase (ALP) live staining 14
2.6.2 Immunofluorescence (IF) staining 14
2.6.3 RNA extraction and quantification 15
2.6.4 Reverse-transcriptase polymerase chain reaction (RT-PCR) 16
2.6.5 Quantitative real time polymerase chain reaction (qPCR) 16
2.7 Neural differentiation of fibroblast-derived iPSCs 17
2.7.1 Neural differentiation procedure 17
2.7.2 Immunofluorescence (IF) staining 17
3. Results 20
3.1 Sequencing of pluriopotency plasmids 20
3.2 The conditions of reprogramming methods by fibroblast cell line 20
3.3 Schematic of reprogramming induction procedure for CG28-derived iPSCs at different time points 21
3.4 Comparison the characteristics of pluripotency in human ES, iPS cell line and CG28-derived iPSCs 22
3.4.1 Alkaline phosphatase (ALP) live stain of CG28-derived iPSCs 22
3.4.2 Immunofluorescence (IF) staining of CG28-derived iPSCs 23
3.4.3 Quantitative real time polymerase chain reaction (qPCR) of CG28-derived iPSCs 23
3.5 Comparison the potential of neural differentiation in human ES, iPS cell line and CG28-derived iPSCs 24
3.5.1 Neural differentiation procedure and cell morphology of CG28 iPSC-derived neural progenitor cells (NPCs) 24
3.5.2 Immunofluorescence (IF) staining of CG28 iPSC-derived neural progenitor cells (NPCs) 24
3.6 Schematic of reprogramming induction procedure for healthy donor-derived iPSCs at different time points 25
3.7 Comparison the characteristics of pluripotency in human ES, iPS cell line and healthy donor-derived iPSCs 26
3.7.1 Alkaline phosphatase (ALP) live stain of healthy donor-derived iPSCs 26
3.7.2 Immunofluorescence (IF) staining of healthy donor-derived iPSCs 26
3.7.3 Quantitative real time polymerase chain reaction (qPCR) of healthy donor-derived iPSCs 27
3.8 Comparison the potential of neural differentiation in human ES, iPS cell line and healthy donor-derived iPSCs 28
3.8.1 Neural differentiation procedure and cell morphology of healthy donor iPSC-derived neural progenitor cells (NPCs) 28
3.8.2 Immunofluorescence (IF) staining of healthy donor iPSC-derived neural progenitor cells (NPCs) 28
4. Discussion 29
5. Conclusion 34
6. References 35
Tables 43
Table 1-1. Construction of pCXLE-HOCT3/4-shp53-F and pCXLE-hSK 43
Table 1-2. Construction of pCXLE-hUL and pCXLE-eGFP 44
Table 2. Sequence of qPCR primers for four pluripotent plasmids 45
Table 3. Sequence of qPCR primers for fibroblast derived-iPSCs 45
Figures 48
Figure 1. Sequencing of pluripotent plasmids from addgene 47
Figure 2. Tic-Tac cut method for human ES, iPS cell line and fibroblast-derived iPSCs passage 48
Figure 3. First delivery method: The efficiency of electroporation for human skin fibroblast cell line 49
Figure 4. Second delivery method: The efficiency of commercial transfection reagents for human skin fibroblast cell line 50
Figure 5. The best situation of cell morphology and maintenance density of CG28 for reprogramming 51
Figure 6. Schematic of the CG28-derived iPSCs induction procedure 52
Figure 7. The situation of cell maintenance in CG28-derived iPSCs 53
Figure 8. Comparison the signal of alkaline phosphatase live stain in CG28-derived iPSCs with that in human ES, iPS cell line 54
Figure 9-1. Comparison the signal of immuno-fluorescence staining in CG28-derived iPSCs with that in human ES, iPS cell line (SSEA-4 and Nanog) 55
Figure 9-2. Comparison the signal of immuno-fluorescence staining in CG28-derived iPSCs with that in human ES, iPS cell line (TRA-1-60 and OCT-4) 56
Figure 9-3. Comparison the signal of immuno-fluorescence staining in CG28-derived iPSCs with that in human ES, iPS cell line (TRA-1-81 and SOX-2) 57
Figure 9-4. Comparison the signal of immuno-fluorescence staining in CG28-derived iPSCs with that in human ES, iPS cell line (SSEA-3) 58
Figure 10. Comparison the endogenous gene expression level of pluripotency in CG28-derived iPSCs with that in human ES, iPS cell line 60
Figure 11. Comparison the situation of neural induction in CG28-derived iPSCs with that in human ES, iPS cell line 62
Figure 12. Comparison the signal of immuno-fluorescence staining in CG28 iPSC-derived NPCs with that in human ES, iPS-derived NPCs 63
Figure 13. Schematic of primary human skin fibroblasts isolation from healthy donor 64
Figure 14. The best situation of cell morphology and maintenance density of healthy donor-derived primary human skin fibroblasts for reprogramming 65
Figure 15. Schematic of the healthy donor-derived iPSCs induction procedure 66
Figure 16. The situation of cell maintenance in healthy donor-derived iPSCs 67
Figure 17. Comparison the signal of alkaline phosphatase live stain in healthy donor-derived iPSCs with that in human ES, iPS cell line 68
Figure 18-1. Comparison the signal of immuno-fluorescence staining in healthy donor-derived iPSCs with that in human ES, iPS cell line (SSEA-4 and Nanog) 69
Figure 18-2. Comparison the signal of immuno-fluorescence staining in healthy donor-derived iPSCs with that in human ES, iPS cell line (TRA-1-60 and OCT-4) 70
Figure 18-3. Comparison the signal of immuno-fluorescence staining in healthy donor-derived iPSCs with that in human ES, iPS cell line (TRA-1-81 and SOX-2) 71
Figure 18-4. Comparison the signal of immuno-fluorescence staining in healthy donor-derived iPSCs with that in human ES, iPS cell line (SSEA-3) 72
Figure 19. Comparison the endogenous gene expression level of pluripotency in healthy donor-derived iPSCs with that in human ES, iPS cell line 74
Figure 20. Comparison the situation of neural induction in healthy donor-derived iPSCs with that in human ES, iPS cell line 76
Figure 21. Comparison the signal of immuno-fluorescence staining in healthy donor iPSC-derived NPCs with that in human ES, iPS-derived NPCs 77


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