無滋養層培養人類胚胎幹細胞及誘導型多能性幹細胞於 不同軟硬度之奈米片段接枝表面

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高士軒(Shih-hsuan Kao) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

無滋養層培養人類胚胎幹細胞及誘導型多能性幹細胞於不同軟硬度之奈米片段接枝表面
(Feeder-free Culture of Human ESCs & iPSCs on Dishes Grafted with Cell Adhesion Peptides and Having Different Elasticity)

相關論文

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★ 可見光對羊水間葉幹細胞成骨分化之影響	★ 可見光調控神經細胞之基因表現及突觸生長
★ 膜純化法及免疫抗體磁珠法用於分離及體外增殖血液幹細胞之研究	★ 人類表皮成長因子的結構穩定性及生物活性測定
★ 微環境對羊水間葉幹細胞多功能性基因表現及分化之影響	★ 奈米片段與細胞外基質之改質膜用於臍帶血中造血幹細胞之純化與培養
★ 小鼠脂肪幹細胞之膜純化法及細胞外間質對人類脂肪幹細胞影響之研究	★ 利用具有奈米片段與細胞外間質蛋白質的表面改殖材質進行臍帶血造血幹細胞體外培養
★ 在不同培養條件下針對大腸癌細胞及組織中癌細胞進行純化、剔除及鑑定之研究	★ 羊水間葉幹細胞培養於細胞外間質改質表面其分化能力及多能性之研究
★ 人類脂肪幹細胞的膜純化法與分化能力研究	★ 具有抗藥性之大腸癌細胞株能提高癌胚抗原的表現，但並非是癌症起始細胞
★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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摘要(中)

人類多能性幹細胞擁有特殊的潛在治療應用，可以助於損壞的器官修復及疾病的治療。然而，臨床實驗的測試受限於細胞培養於老鼠纖維母細胞培養層。在未來，必須開發一個能培養人類多能性幹細胞之無外源汙染的材料。無滋養層培養是利用合成生醫材料接枝奈米片段作為幹細胞的培養盤，他可以幫助人類多能性幹細胞 (人類胚胎幹細胞及人類誘導型多能性幹細胞) 繁殖且同時在無滋養層及外源汙染的培養下維持其多能性。
本人研究在無老鼠纖維母細胞之滋養層下培養人類多能性幹細胞在聚乙烯醇-共-衣康酸 (PVA-IA) 表面接枝奈米片段 (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN]) 能夠生長且維持其多能性。利用PVA-IA薄膜表面接枝高濃度之寡肽纖粘蛋白且具有最適合軟硬度的培養盤培養人類多能性幹細胞，比較於市面世上販售合成生醫材料 (Synthemax II)，細胞表現較高的貼覆率及成長倍率且較低的分化比率。
更者，經10代培養人類胚胎幹細胞 (WA09) 及人類誘導型多能性幹細胞 (HPS0077) 於PVA-IA塗層接枝1000µg/mL的寡肽纖粘蛋白且具有理想軟硬度25.3kPa的表面，其擁有極好的多能性蛋白及分化蛋白表現。此結果指出人類多能性幹細胞可以培養於PVA-IA培養系統且維持多能性。在未來，PVA-IA培養系統可以被用於在無外源汙染及無滋養層的環境下利用人類原始的組織來製造人類誘導型多能性幹細胞。

摘要(英)

Human pluripotent stem cells (hPSCs) have significant potential in therapeutic applications for damaged organs or diseases. However, the tentative clinical potential of hPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. It is necessary to develop a suitable culture system without using xeno-contaminated materials for hPSCs culture in future. The feeder-free cultures using synthetic biomaterials having nanosegments as stem cell culture materials can support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures.
I investigated that hPSCs could proliferate and keep pluripotenty without usage of a feeder layer of MEFs where hPSCs were cultured on polyvinylalcohol-co-itaconic acid (PVA-IA) grafted with nanosegment (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN]). The hPSCs on PVA-IA film grafted with high concentration of oligoVN having optimal elasticity showed higher colony attachment ratio, higher colony expansion fold, and lower differentiation ratio compared to those on commercially availiable synthetic biomaterial (Synethemax II).
Moreover, hESCs (WA09) and hiPSCs (HPS0077) cultured on PVA-IA films grafted with 1000µg/ml of oligo-VN with 25.3kPa elasticity after 10 passage showed excellent pluripotent protein and differentiation protein expression in embryoid boid. This result indicates that hPSCs can be cultured and maintain their pluripotency on PVA-IA culture system. In future, the PVA-IA culture system could be used to generate hiPSCs from primary human tissue cells on xeno-free and feeder-free conditions.

關鍵字(中)

★ 多能性幹細胞

關鍵字(英)

論文目次

Chapter 1: Introduction XIII
1-1 Stem Cells 1
1-1-1 Stem Cell Therapy and Lineage Plasticity 2
1-1-2 Totipotency and Nuclear Transfer 4
1-1-3 Pluripotent Stem Cells 6
1-1-4 Adult Stem Cells 10
1-2 Microenvironment Effect on Human Pluripotent Stem Cell 11
1-2-1 Physical Cues 11
1-2-2 Chemical Effect 13
1-3 Characterization of Pluripotent Stem Cells 44
1-3-1 Colony Formation 46
1-3-2 Alkali Phosphatase Activity 46
1-3-3 Pluripotent Gene Expression 46
1-3-4 Pluripotent Protein Expression 46
1-3-5 Differentiation Ability 47
1-4 Immunofluorescence 49
Chapter 2: Materials and Methods 51
2-1 Material 51
2-1-1 Cell line 51
2-1-2 Chemical 51
2-2 Cell Culture 53
2-2-1 Culture and passage of human pluripotent stem cells 53
2-2-2 Storage of human pluripotent stem cell 54
2-2-3 Thawing frozen stock of human pluripotent stem cell 55
2-3 Immunofluorescence 55
2-4 Colony attachment ratio 57
2-5 Colony differentiation ratio 57
2-6 Alkaline phosphatase live staining assay 58
2-7 Embryoid body formation 59
2-8 Teratoma formation 60
2-9 Preparation of PVA-IA film 61
2-10 Preparation of PVA-IA coating dish grafted with oligopeptide 62
2-11 XPS analysis of dish surface 63
Chapter 3: Result and Discussion 64
3-1 Characterization of PVA-IA film grafted with oligovitronectin by X-ray photoelectron spectroscopy 64
3-2 Cultivation of hPSCs on PVA-IA films grafted with oligo-VN 68
3-2-1 Transfer of hESCs from feeder-layer culture system to feeder-layer free culture system using Matrigel coated dish 68
3-2-2 Characterization of hESCs before cultivation on PVA-IA culture system 70
3-2-3 Cultivation of hESCs on PVA-IA films having different stiffness grafted with oligo-VN with different concentration 72
3-2-4 Characterization of hESCs after cultivation on PVA-IA culture system 84
3-2-5 Cultivation of hiPSCs on PVA-IA films having different stiffness grafted with different concentration of oligo-VN 88
3-2-6 Characterization of hiPSCs after cultivation on PVA-IA culture system 100
3-3 Cultivation of hPSCs on PVA-IA films grafted with different concentration of Oligo-VN having optimal elasticity (25.3kPa) for 20 passages 104
Chapter 4: Conclusion 108
Supplement Data 110
Supplement tables 110
Reference 121

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2015-7-30

推文