博碩士論文 106324023 詳細資訊



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姓名 蘇煥喬(Huan-Chiao Su)  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 人類多能性幹細胞分化心肌細胞培養於熱敏性高分子塗佈細胞外間質
(Differentiation of Human Pluripotent Stem Cells into Cardiomyocytes Cultured on Thermo-Responsive Polymer Coated with Extracellular Matrix)
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摘要(中) 人類多能性幹細胞對於組織再生醫學是具有吸引力的來源。人體多能性幹細胞包括人體胚胎幹細胞與人體誘導多能性幹細胞已被使用在心血管研究包括 (a) 人類心臟發育 (b) 藥物發現 (c) 心臟毒性篩查和 (d) 疾病模型。此外，人體胚胎幹細胞分化的心肌前驅細胞已被用於臨床實驗中，用於治療心肌梗塞的患者。近年來，一直迫切需要在無異種條件下使用化學成分確定的培養基，發展再現性高且高效率的人體多能性幹細胞分化成心肌細胞的方法。特別是，一直以來都難以從培養皿上的未分化細胞中分離已分化或前驅的細胞。在這項研究中，熱敏性聚合物，如聚N-異丙基丙烯酰胺（polyNIPAAm），聚（N-異丙基丙烯酰胺-丙烯酸丁酯）（polyNIPAAm-co-BA）其在20 ℃ 時具有低臨界溶液溫度(LCST)，作為與幾種細胞外間質（ECM）綴合的細胞培養皿上的表面塗層聚合物。使用這個【智能生物材料】，開發出具有細胞分選功能的培養皿，可以分離或純化人體多能性幹細胞分化的心肌細胞在這個具有熱敏性的表面上。比起用其他細胞外間質(LN-511（層粘連蛋白-511）、纖連蛋白、第一型膠原蛋白或重組玻連蛋白)塗層於聚N-異丙基丙烯酰胺表面上，在塗有LN-521（層粘連蛋白-521）的聚N-異丙基丙烯酰胺表面上發現人體多能性幹細胞分化的心肌細胞具有較強的貼附能力。在低臨界溶液溫度(LCST)下，從熱敏感性表面收集的分離細胞其表面標誌物cTnT（心肌肌鈣蛋白T，心肌細胞標記物）表達高於未分離細胞。此外，發現人體多能性幹細胞分化的心肌細胞再次利用熱敏性表面分選後其cTnT的表現量可以高於90%。此外，人體多能性幹細胞分化的心肌細胞在塗覆有細胞外間質的熱敏性聚合物皿顯示出比直接塗覆有細胞外間質的細胞培養皿有著更好的細胞附著力。還證明了人體多能性幹細胞分化成心肌細胞的過程中，在用重組玻連蛋白（rVN）塗覆的聚N-異丙基丙烯酰胺共聚物表面上有更高的存活率比起直接塗覆重組玻連蛋白（rVN）的表面。可以得出結論，塗有LN-521的熱敏性聚合物表面可以用作細胞分選培養皿，並且可以在無異種細胞培養條件下分離具有高純度的人體多能性幹細胞分化的心肌細胞。
摘要(英) The human pluripotent stem cells (hPSCs) are attractive source for tissue regeneration medicine. hPSCs including hiPSCs (human induced pluripotent stem cells) and hESCs (human embryonic stem cells) have been used in the cardiovascular investigation of (a) human cardiac development, (b) drug discoveries, (c) cardiotoxicity screening, and (d) disease modeling. Furthermore, hESC-derived cardiac progenitors have started to be used in clinical trials for the treatment of myocardiac infarction patients. The development of reproducible and highly efficient protocols for hPSC differentiation into cardiomyocytes under xeno-free conditions using chemically defined medium has been in great demand in recent years. Especially, it is difficult to isolate differentiated/progenitor cells from undifferentiated cells on culture dishes. In this study, thermo-responsive polymers such as poly-N-Isopropylacrylamide (polyNIPAAm) and Poly(N-isopropylacrylamide-co-butylacrylate) (polyNIPAAm-co-BA), which have lower critical solution temperature (LCST) around 20 degrees, are selected as surface coating polymers on the cell culture dishes coated with several extracellular matrices (ECMs). Using these smart biomaterials, the cell sorting dishes were developed to isolate/purify hPSCs derived cardiomyocytes (hPSCs-derived-CMs) on the thermoresponsive surface in this study. It was found that the polyNIPPAm surface coated with LN-521 (laminin-521) had stronger affinity to hPSCs-derived-CMs than the surface coated with other ECMs (LN-511 (laminin-511), fibronectin, collagen type I, or recombinant vitronectin (rVN)). The cTnT (cardiac Troponin T, cardiomyocyte marker) was expressed higher on the detached cells from the thermoresponsive surface than non-detached cells, which were collected below LCST. Moreover, cTnT expression of hPSCs-derived-CMs were found to be higher than 90% after resorting on the thermoresponsive surface. The thermoresponsive polymeric dishes coated with ECMs showed better cell attachment of hPSCs-derived-CMs than tissue culture polystyrene (TCPS) dishes coated with ECMs directly. It was also demonstrated that hPSC-derived-CMs had higher survival rate on polyNIPAAm copolymer surface coated with rVN compared to dishes directly coated rVN during differentiation stage into CMs. It is concluded that the thermoresponsive polymer surface coated with LN-521 can serve as the cell sorting dishes and can isolate hPSCs-derived-CMs with high purity in xeno-free cell culture conditions.
關鍵字(中) ★ 熱敏性聚合物
★ 人類多能性幹細胞衍生心肌細胞
★ 細胞外基質		 關鍵字(英) ★ embryonic stem cell
★ induced pluripotent stem cell
★ differentiation
★ thermo-responsive polymer
★ cardiomyocyte
★ extracellular matrix
論文目次 Abstract i
摘要 vi
目錄 vii
Index of Figures x
Index of Tables xvii
Chapter 1. Introduction 1
1-1 Human Pluripotent Stem Cells (hPSCs) 1
1-1.1 Human embryonic stem cells (hESCs) 3
1-1.2 Human induced pluripotent stem cells (hiPSCs) 5
1-1.3 Regenerative medicine 7
1-2 Biomaterials for hPSCs Cultivation 9
1-2.1 Human Pluripotent Stem Cells Cultivation (2D system) 9
1-2.2 Integrin-binding site of ECMs for hPSCs culture 12
1-3 hPSCs Culture on Thermoresponsive Surface 13
1-3.1 Characteristics of Poly(N-isopropylacrylamide) 13
1-3.2 New approach for tissue engineering by using thermoresponsive surface 15
1-4 hPSCs-derived Cardiomyocyes Differentiation 16
1-4.1 Functional protocols for cardiac differentiation 16
1-4.2 Cardiomyocytes differentiated from Embryoid Body (EB) 18
1-4.3 Cardiomyocytes differentiation by co-culture system 20
1-4.4 Cardiomyocytes differentiated on monolayer substrates 22
1-4.5 Cardiomyocytes differentiation in suspension culture 24
1-4.6 Effect for biomaterials during cardiomyocytes differentiation 27
1-5 Characterization of Cardiomyocytes 28
1-5.1 Morphologies of hPSCs-derived cardiomyocytes 28
1-5.2 Flow cytometry analysis of cardiomyocytes 29
1-5.3 Immunofluorescence staining analysis of cardiomyocytes 31
1-5.4 Goal of This Research 33
Chapter 2. Material and Methods 34
2-1 Materials 34
2-1.1 Cell lines 34
2-1.2 Materials for thermo-responsive surface 34
2-1.3 Stem cells culture medium and others 34
2-1.4 Cardiomyocytes differentiation medium and chemicals 35
2-1.5 Immunostaining and flow cytometry analysis 35
2-2 Methods 37
2-2.1 Preparation of thermoresponsive surface dishes (2D) 37
2-2.2 Culture and subculture of human pluripotent stem cells 38
2-2.3 Seeding Density Measurements of hPSCs 39
2-3 Cardiomyocytes Differentiation 40
2-3.1 Differentiation medium preparation 40
2-3.2 Cardiomyocyte differentiation protocol 41
2-4 Characterization of hPSC-derived CMs 44
2-4.1 Immunofluorescence staining protocol of cardiomyocytes analysis 44
2-4.2 Flow cytometry measurement protocol for hPSC-derived cardiomyocytes analysis 45
Chapter 3. Result and Discussion 47
3-1 Thermoresponsive Polymers Selection for Thermoresponsive Coated Surface 47
3-1.1 hPSCs culture on thermoresponsive coated surface conjugated with rhVN: hPSCs morphologies for different concentration of thermoresponsive polymers 47
3-1.2 Expansion fold and attachment ratio of hESCs on polyNIPAAm and polyNIPAAm-co-BA coated surface 50
3-1.3 hESCs-derived-CMs culture on polyNIPAAm and polyNIPAAm-co-BA coated surface immobolized with rhVN by Jove protocol 53
3-1.4 hESCs-derived-CMs culture on polyNIPAAm-coated and polyNIPAAm-co-BA-coated surface immobilized with rhVN by the revised protocol (protocol 4) 55
3-2 Characterization of polyNIPAAm Immobilized with ECMs 57
3-2.1 X-ray photoelectron spectroscopy (XPS) analysis 57
3-2.2 Water contact angle (CA) analysis 60
3-3 Optimal Thermoresponsive Surface Immobilized with Laminin-521 for Cardiomyocytes Differentiation 62
3-3.1 hESCs-derived CMs culture on polyNIPAAm-coated and polyNIPAAm-co-BA-coated surfaces immobilized with laminin-521 by Jove protocol 62
3-3.2 Beating Colonies Number and Beating Frequency of hESC-derived CMs on Thermoresponsive Surface 64
3-4 Sorting High Purified Cardiomyocytes on Thermoresponsive Surface 66
3-4.1 Thermoresponsive sorting method 67
3-4.2 Immunostaining analysis of hESC-derived CMs on thermoresponsive surface 68
3-4.3 Length determination of sarcomere structure 72
3-4.4 low cytometry analysis of cardiomyocytes differentiated from hPSCs on smart sorting surface 74
3-4.5 Sorting hPSC-deruved CMs on comparative surfaces 75
Chapter 4. Conclusion 78
Reference 80
Supplementary Data 89
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2019-8-19
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