人類脂肪幹細胞培養在具有細胞外基質接枝的水凝膠上之多能性與分化能力研究;Pluripotency and Differentiation of Human Adipose-derived Stem Cells Cultured on ECM-grafted Hydrogels Having Different Elasticity

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Title:	人類脂肪幹細胞培養在具有細胞外基質接枝的水凝膠上之多能性與分化能力研究;Pluripotency and Differentiation of Human Adipose-derived Stem Cells Cultured on ECM-grafted Hydrogels Having Different Elasticity
Authors:	吳孟學;Wu,Meng-hsueh
Contributors:	化學工程與材料工程學系
Keywords:	人類脂肪衍生幹細胞;彈性;間葉幹細胞;human adipose derived stem cell;elasticity
Date:	2014-06-16
Issue Date:	2014-08-11 18:13:48 (UTC+8)
Publisher:	國立中央大學
Abstract:	人類脂肪衍生幹細胞可以經由細胞培養皿來純化,並且顯示具有分化多支鏈的能力。在過去幾十年裡，某些細胞外基質常被塗佈於二維或是三維的幹細胞培養材料上,例如: 膠原蛋白、層黏蛋白質、纖維連接蛋白、玻璃連黏蛋白質以及幹細胞的分化及生長都是常被研究的。然而這些蛋白大多數是由動物衍生出來的所以如果用這些單白質來培養幹細胞，接下來如果要應用於臨床試驗會有異種污染的問題，加上這些蛋白質價格並不便宜。為了實現我們的最終負擔得起的，個性化的再生醫學的目的，臨床上安全和經濟的方式增殖的幹細胞是絕對必要的，同時保持其多能性，以及引導幹細胞譜系規格，而無需使用化學誘導。這裡我研究多能性和人類脂肪衍生幹細胞的分化能力培養於聚（乙烯醇共衣康酸），PVA-IA，水凝膠接枝某些細胞外基質（膠原蛋白第一型，纖連蛋白和寡玻連蛋白）。PVA-IA水凝膠的軟硬度可以透過6小時到48小時的交聯時間加以控制，藉由戊二醛溶液，生成的彈性從2.2千帕至3.7千帕的儲能模量，從E計算對應軟硬度為6.6千帕至11.1千帕的彈性模量= 3G′[生物材料29（2008）2757]。我們發現人類脂肪衍生幹細胞培養於PVA-IA接枝纖連蛋白及寡玻連蛋白於相對較軟的基材可以維持幹細胞的多能性但是於細胞培養聚苯乙烯的培養皿上卻沒有這個表現，從螢光染色的結果可以發現於我們改質過後的培養皿上有很好的表現但是於細胞培養聚苯乙烯的培養皿卻沒有什麼表現，在成骨分化的分析上細胞培養聚苯乙烯的培養皿比分化表現優於我們改質過後的培養皿。 ;Stem cells purified from human adipose tissue (hADSCs) via serial culture of stromal vascular fraction (SVF) on tissue culture plates show multilineage differentiation ability in vitro. In the past decades, extracellular matrices (ECMs) such as collagen, fibronectin, vitronectin, and laminin were used as coating or self-standing materials on 2D culture and 3D culture of stem cells, and the differentiation and proliferation ability of stem cells (e.g., ADSCs or bone marrow-derived mesenchymal stem cells) were investigated. However, the origin of these ECMs are mainly animal-derived or human recombinant, and, therefore, it is not suitable to use them for clinical applications due to xeno-origin contamination and high cost of ECMs. On the other hand, it has been shown that physical environmental factors, such as matrix elasticity, and small functional groups including oligopeptides can have significant effect in determining stem cell fate. To achieve our ultimate goal of affordable, personalized regenerative medicine, clinically safe and economical ways to proliferate stem cells are absolutely essential while maintaining their pluripotency as well as directing stem cell lineage specification without the use of induction chemicals. Here I investigated pluripotency and differentiation ability of hADSCs cultured on poly(vinylalcohol-co-itaconic acid), PVA-IA, hydrogels grafted with several ECMs (collagen type I, Fibronectin, and oligo-vitronectin [VN]). The stiffness of PVA-IA hydrogels can be controlled by the crosslinking time from 6 hr to 48 hr in glutaraldehyde solution, which generates the elasticity from a 2.2 kPa to 3.7 kPa storage modulus corresponding 6.6 kPa to 11.1 kPa elastic modulus from calculation of E=3G’ [Biomaterials 29 (2008) 2757]. Primary hADSCs were found to maintain their pluripotency on relatively soft hydrogels grafted with Fibronectin and oligopeptide derived from Vitronectin (KGGPQVTRGDVFTMP) from pluripotent gene expression (Nanog, Sox2, Oct4, and klf4) but not on TCPS. From the immunostaining data showed that hADSCs on ECM-grafted PVA-IA films have higher expression of pluripotent proteins than those on TCPS in expansion medium. In the osteogenic differentiation analysis, hADSCs on TCPS have higher differentiation ratio than those on ECM-grafted PVA-IA films after 21 days culture in ostogenic medium.
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