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

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Title:	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
Authors:	高士軒;Kao,Shih-hsuan
Contributors:	化學工程與材料工程學系
Keywords:	多能性幹細胞
Date:	2015-07-30
Issue Date:	2015-09-23 10:52:59 (UTC+8)
Publisher:	國立中央大學
Abstract:	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。 ;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.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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