博碩士論文 104324067 詳細資訊



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姓名 劉政輝(Cheng-Hui Liu)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 人類多功能幹細胞於固定奈米片段 生醫材料的心肌細胞分化
(Human Embryonic Stem Cell Differentiation into Cardiomyocytes on Biomaterials Immobilized Nanosegments)
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摘要(中) 人類胚胎幹細胞中的多能幹細胞和誘導多能幹細胞原自於三種胚層擁有能夠分化成多種形式的潛力:內胚層、中胚層、外胚層。例如提供給阿茲海默症治療的多巴胺分泌細胞和提供給糖尿病治療的胰島素分泌細胞。然而由於多種的分化特性，如何引導人類胚胎幹細胞分化成特定型態是一個具有挑戰的議題。幹細胞的分化結果取決於不同的人類胚胎幹細胞培養微環境因素:生物觸發分子、細胞間的作用力、物理因素、生物和材料之間的作用力。透過生化材料來模擬人類胚胎幹細胞分化成特定細胞的培養環境通常是一種較合理的方法，且目前並沒有研究是針對細胞外基質或從細胞外基質培育出的奈米片段來分化出心肌細胞。因此我們發展具有不同黏彈性的奈米片段接枝生化材料來提供給人類胚胎幹細胞分化出心肌細胞。
我們準備塗佈有不同細胞外基質的培養皿 : 基質膠 、合成胜肽-丙烯聚酯、纖維連結蛋白、層黏連蛋白-521、層黏連蛋白-511、人造玻蛋白、玻蛋白。第零天，我們利用含有GSK3B抑制劑的心肌細胞分化培養液來取代原培養液。第一到第二天，我們發現30%~40%的細胞死亡並且從表面剝落。然而，存活下來的細胞聚落中間變得更厚更結實。在第五到第六天，細胞分化成心肌細胞。第八到第十天，我們連續在表面發現到跳動中的聚落。
我們分析和評估塗佈有細胞外基質用來分化人類胚胎幹細胞成心肌細胞的培養皿，這些人類胚胎幹細胞的心肌細胞誘導結果將能用於臨床實驗和細胞的專一性機制和心肌細胞的培養等研究。
摘要(英) Human pluripotent stem cells (hPSC) of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential ability to differentiate into many kind of cell types originated from three germ layers: endoderm, mesoderm and ectoderm cells such as dopamine-secreting cells for Alzheimer disease treatment and insulin-secreting cells for diabetes treatment.1 However, it is a challenging issue to guide hPSCs to differentiate into our desired lineages of cells due to their variety of differentiation ability. The fate of differentiation of stem cells is determined by different factors existed in the microenvironment of hPSCs: bioactive molecules, cell-cell interactions, physical factors and cell-biomaterial interaction. It is a reasonable strategy to mimic the stem cell microenvironment for the differentiation of hPSCs into specific lineages of cells using optimal biomaterials for hPSC culture. Currently, it has not yet investigated which extracellular matrices (ECMs) or nanosegments derived from ECMs promote hPSCs differentiation into cardiomyocytes. We developed nanosegment-grafted biomaterials having different elasticity for hPSCs differentiation into cardiomyocytes.
We developed several biomaterials for hPSCs differentiation into cardiomyocytes. We prepared (1) ECM-coated dishes where ECMs are Matrigel, Synthemax II, fibronectin (CellStartTM), laminin-521, laminin-511, recombinant vitronectin, vitronectin, and fibronectin. On day 0, we replaced the expansion medium into cardiomyocytes differentiation medium containing the GSK3B inhibitor. On days 1-2, we observed that 30%~40% of the cells were died and detached from the surface. However, the center of the colony of living cells were getting thicker and became compact. The cells were differentiated into cardiomyocytes between days 5-6. On day 8-10, we successively observed the contracting colonies on the surface.
We evaluated the optimal ECM-coating dishes for differentiation of hPSCs into cardiomyocytes. The results for cardiomyocyte induction from hPSCs will be used in clinical application and in the investigation of molecular mechanism of specification and maturation of cardiomyocytes.
關鍵字(中) ★ 人類多功能幹細胞
★ 生醫材料
★ 心肌細胞
★ 分化
★ 固定奈米片段		 關鍵字(英) ★ human pluripotent stem cell
★ biomaterial
★ cardiomyocytes
★ differentiation
★ Immobilized Nanosegments
論文目次
Chapter 1. Introduction 1
1.1 Human Pluripotent Stem Cells (hPSCs) 1
1.1.1 Human Embryonic Stem Cells (hESCs) 1
1.1.2 Human Induced Pluripotent Stem Cells (hiPSCs) 2
1.1.3 Mesenchymal Stem Cells (MSCs) 3
1.1.4 Stem Cell Therapy 5
1.2 Biomaterials for hPSCs Culture 6
1.2.1Extracelluar Matrices for hPSCs Expansion 6
1.2.2 Feeder Layer Substrates 6
1.2.3 Feeder Free Layer Substrates 6
1.2.4 Integrin-Binding Site of ECMs for hPSCs Culture 9
1.3 Cardiomyocytes Differentiation 10
1.3.1 Functional Methods for Cardiac Differentiation 10
1.3.2 Effect of Biomaterials on Cardiomyocytes Differentiation 21
1.4 Characteriation of Cardiomyocytes 26
1.4.1 Morphologies of Cardiomyocytes Derived from hESCs 26
1.4.2 Flow Cytometry Analysis of Cardiomyocytes 27
1.4.3 Immunofluorescence Staining Analysis of Cardiomyocytes 29
Chapter 2. Material and Methods 30
2.1 Materials 30
2.1.1 Cell Line 30
2.1.2 Commercial Coated Materials 31
2.1.3 Stem Cell Culture Medium 31
2.1.3 Cardiomyocytes Differentiation Medium and Chemicals 31
2.1.4 Immunostaining and Flow cytometry 31
2.1.5 Others 32
2.2 Stem Cell Culture 32
2.2.1 Human Pluripotent Stem Cell Culture and Passage 32
2.2.2 Cell density measurement 33
2.3 Cardiomyocytes Differentiation 34
2.3.1 Differentiation Medium Preparation 34
2.3.2 Cardiomyocyte differentiation protocol 35
2.4 Characterization of Cardiomyocytes 38
2.4.1 Immunofluorescence 38
2.4.2 Buffer preparation for flow cytometry 39
2.4.3 Protocol of cardiomyocytes analysis by flow cytometry 39
Chapter 3. Results and Discussion 40
3.1 Selection of Differentiation Protocol of hESCs into Cardiomyocytes 40
3.1.1 ECMs Used Differentiation Protocol Selection of hESCs into Cardiomyocytes 41
3.1.2 Cardiomyocytes Differentiation of hESCs Using Cardiomyocytes Differentiation Kit 41
3.1.3 Cardiomyocytes Differentiation of hESCs Using Jove Journal 43
3.1.4 Cardiomyocytes Differentiation of hESCs Using by Old and New Protocols of Nature Methods Journal 44
3.1.5 Analysis of Cells differentiated into Cardiomyocytes 46
3.1.6 Protocol Selection for Cardiomyocyte Differentiation 52
3.2 Cell Culture Substrates for Cardiomyocytes Differentiation of hESCs 52
3.2.1 Cardiomyocytes Differentiation of hESCs on Several ECMs 53
3.2.2 Survival Rate Analyses of Cardiomyocytes on Several ECM-Coating Dishes 56
3.2.3 Beating Colony Growth Curve on Several ECM-coating Dishes 58
3.2.4 Flow Cytometry Analysis of Cardiomyocytes on Several ECM-coating Dishes 60
3.2.5 Immunofluorescene Analyses of Cardiomyocytes 62
3.2.6 Analysis of Beating Frequency on Cardiomyocytes 68
Chapter 4. Conclusion 70
Reference 73
Supplemental Data - Figure 80
Supplemental Data - Table 104
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指導教授 樋口亜紺(Akon Higuchi) 審核日期 2017-8-22
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