臺灣博碩士論文加值系統

本論文的主題為: 「人體胚胎幹細胞在無滋養層及無異種條件的培養下於帶有生長因子的表面進行培養」。研究內容著重於利用已知成分之生醫材料對人體多能性幹細胞做培養。人體多能性幹細胞分為人體胚胎幹細胞以及人體誘導型多能性幹細胞兩大類，由於此等細胞於適當環境下具有優越的繁殖力以及多能性，近年來於再生醫療領域已成為相當有潛力的研究來源。然而，人體多能性幹細胞的培養成本之高，也一直是研究端及應用端面臨的困難；原因歸咎於人體多能性幹細胞生長必要之生長因子(纖維母細胞生長因子及轉化生長因子-β1)，單價相當昂貴且易在溶液中受溫度干擾或變質而降解(需低溫保存)，培養液之頻繁更換致使整體的培養成本居高不下。有鑑於此，本實驗的主軸除了著重於人體多能性幹細胞重要特性之維持，亦致力於培養成本之降低。基於此理念，本實驗發展出一生醫材料，試圖將生長因子吸附於培養皿表面，使生長因子的結構能被固定且穩定於培養皿表面，同時能減低培養液中生長因子的使用量，藉此降低整體培養的成本。實驗中的生醫材料設計運用了知名的蛋白質穩定物質—肝素，肝素藉由化學合成的方式固著於表面，作為生長因子吸附於材料上的媒介。人體胚胎幹細胞(細胞株型號WA09)貼附於帶有肝素及生長因子的培養皿上後，後續使用生長因子減量的培養液對WA09細胞做長期培養。本研究涵蓋了帶有生長因子的培養皿之設計、人體胚胎幹細胞於少量生長因子供應下培養之觀察及長期培養後人體胚胎幹細胞多能特性之鑑定，也於最終結果獲得最適於人體胚胎幹細胞之生醫材料的組成。

In this master thesis, “Human Embryonic Stem Cells Cultivation on Growth Factor-immobilized Surface under Feeder-free and Xeno-free Conditions”, a method was developed for culturing the hPSCs with chemically defined condition. Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have emerged as prospective resources in regenerative medicine due to their infinite proliferation ability and pluripotency. However, general hPSCs medium containing crucial growth factors, FGF-2 and TGF-β1, which are easily in degradation in solution but are highly contributing to the proliferation and pluripotency of hPSCs, are considerably expensive, which reflects on the high expense of hPSC cultivation. Therefore, a surface immobilized with growth factors was developed not only to stabilize the growth factors on the surface but also to reduce the usage of growth factor in culture medium. In this study, a well-known protein stabilizer, heparin, was chemically immobilized on the surface to serve as binding sites of growth factors for hESC (WA09) cultivation. After absorption of the growth factors on the heparin-immobilized surface, the hESCs were cultured on the modified surface with reduced usage of growth factor in medium for long-term cultivation. It is concluded that the expansion of hESCs (WA09) with decreasing usage of growth factors system containing heparin was successfully developed and a series of pluripotency examinations for hESCs were conducted after long-term cultivation.

ABSTRACT I
摘要 II
INDEX OF CONTENT III
INDEX OF FIGURES V
INDEX OF TABLES IX
CHAPTER 1 Introduction 1
1-1 From regenerative medicine to stem cell therapy 1
1-2 Stem cell therapy 3
1-3 Stem cells and stem cell research 4
1-3-1 Potency of stem cells 5
1-4 Pluripotent stem cells (PSCs) 7
1-4-1 Embryonic stem cells (ESCs) 7
1-4-2 Induced pluripotent stem cells (iPSCs) 9
1-5 Cultivation of hPSCs 10
1-6 Niches of hPSCs 11
1-6-1 Physical parameters of hPSCs niches 12
1-6-2 Cell-cell interactions of hPSCs 13
1-6-3 Cell-biomaterial (matrix) interactions on hPSCs 14
1-6-4 Soluble factors for hPSCs cultivation 20
CHAPTER 2 Materials and Methods 30
2-1 Materials 30
2-1-1 Cell cultivation 30
2-1-2 Immunofluorescence for pluripotency expression and differentiation ability of hESCs 33
2-2 Method 35
2-2-1 Preparation of heparin-immobilized surface 35
2-2-2 hESCs culture and passage procedure 37
2-2-3 Characterization of heparin-grafted surfaces 38
2-2-4 Characterization of cells growth, pluripotency expression, and differentiation ability in vitro and in vivo 39
CHAPTER 3 Results and discussion 45
3-1 Physical method of heparin immobilization 45
3-1-1 Surface preparation and screening 45
3-1-2 hESC cultivation on the PVA-H-OVN surfaces 48
3-2 Chemical method of heparin immobilization 54
3-2-1 Surface preparation and screening of heparin grafting 55
3-2-2 hESCs culture on the heparin-immobilized surfaces under reduced usage of FGF-2 and TGF-β1 66
3-2-3 Differentiation ability of hESCs cultured on CMC-HX-rVN: EB formation in vitro analyzed by immunostaining method 75
3-2-4 Differentiation ability of hESCs cultured on CMC-HX-rVN: Terotoma formation in vivo analyzed by H&E staining 81
CHAPTER 4 Conclusion 82
REFERENCE 83
Supplemental Data - Figure 97
Supplemental Data - Table 116

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