羊水間葉幹細胞培養於細胞外間質寡肽嫁接具有硬度/彈性表面的材料,其分化能力及多能性之研究;Proliferation and Differentiation of Amniotic Fluid-Derived Stem Cells on Oligopeptide-Grafted Surface Having Different Elasticity

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Title:	羊水間葉幹細胞培養於細胞外間質寡肽嫁接具有硬度/彈性表面的材料,其分化能力及多能性之研究;Proliferation and Differentiation of Amniotic Fluid-Derived Stem Cells on Oligopeptide-Grafted Surface Having Different Elasticity
Authors:	王品毓;Wang,Pin-Yu
Contributors:	化學工程與材料工程學系
Keywords:	多能性幹細胞;羊水幹細胞;pluripotent stem cell;Amniotic Fluid-Derived Stem Cells
Date:	2014-06-27
Issue Date:	2014-08-11 18:14:17 (UTC+8)
Publisher:	國立中央大學
Abstract:	從人類羊水來源的幹細胞是一種多能性幹細胞，因為其具備分化多種譜系的能力，包括代表性的三個胚層。因此，羊水來源幹細胞可能成為一個更適合的幹細胞在再生醫學和組織工程。此外，細胞培養基材的物理特性會影響幹細胞分化的命運。然而，在過去研究報告上沒有探討出最佳的組合以結合物理線索中的彈性及生物線索中的細胞培養基質，以保持幹細胞的多能性和長時間細胞的培養。因此，在我的研究中，為了探討維持羊水幹細胞的多能性以及如何使羊水幹細胞有效地分化。因為我將羊水來源幹細胞培養於具有不同軟硬度的水凝膠且固定細胞外基質衍生的寡肽以提升羊水幹細胞的多能性基因且有效調節細胞的分化能力。一開始，準備polyvinylalcohol-co-itaconic acid (PVA-IA)塗層於細胞培養層上，藉由不同交聯時間已達成控制PVA-IA具有不同的軟硬度，經過活化的程序，便能將細胞外基質衍生的寡肽固定在PVA-IA塗層上，最後將羊水幹細胞培養於這些盤子上，以便後續的分析及探索。經過細胞經過數天的培養後，將種於不同條件的羊水幹細胞取其檢測多能性基因：Oct4, Nanog, 和Sox2，和分化基因:Nestin, Sox17, 和Runx2。在最後的檢測結果指出vitronectin來源寡肽固定於低彈性的PVA-IA塗層具有較大的潛能以提升羊水幹細胞的多能性；而且在不同軟硬度的PVA-IA塗層能使羊水分化成相似於基材軟硬度的細胞類型，例如：當羊水幹細胞培養於較低彈性的PVA-IA嫁接塗層，會使的羊水幹細胞偏好分化成神經類型細胞；然而，當細胞培養於較高彈性的PVA-IA嫁接途層，將使的細胞分化成成骨幹細胞型態，這項研究結果報導出與先前研究一樣的結果。因為這項研究中指出，結合培養材料的鋼度以及細胞外基質成份的生物線索可以引導和決定幹細胞多能細和分化的譜系。另一方面，我們進一步發現，不同軟印度和細胞外基質寡肽之間的合作將有效地調節羊水幹細胞分化成不同譜系的承諾。 ;Stem cells derived from human amniotic fluid (AFSCs) are pluripotent fetal cells capable of differentiating into multiple lineages, including representatives of the three embryonic germ layers. Therefore, AFSCs may become a more suitable source of stem cells in regenerative medicine and tissue engineering. However, stem cell characteristics, such as proper differentiation and maintenance of pluripotency, are notregulated only by the stem cells themselves but also by their microenvironment. Furthermore, physical characteristics of cell culture substrate influence the fate of stem cell differentiation. However, there have been no reports from our database study that investigates the optimal elasticity to keep pluripotency of stem cells for a long time and studies the optimal combination of physical cue and biological cue on cell culture substrates. Here I report pluripotent maintenance and differentiation efficiency of AFSCs cultured on cell culture substrates immobilized extracellular matrix-derived oligopeptides, which have different elasticity. We prepared dishes coated with polyvinylalcohol-co-itaconic acid (PVA-IA) films having different elasticity by controlling the crosslinking time in crosslinking solution containing glutaraldehyde, and grafted with several ECM-derived cell-adhesion peptides though N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDC) and N-hydroxysuccinimide (NHS) chemistry in an aqueous solution. qRT-PCR measurements suggest that pluripotent genes, Nanog, Oct4 and Sox2, were kept on PVA-IA hydrogels grafted with oligopeptide derived from vitronectin (oligoVN) and fibronectin (oligoFN) having moderate elasticity around 12-25kPacompared toAFSCs on conventional tissue culture dishes. I found that there is an optimal elasticity of cell culture matrix to promote pluripotency of AFSCs for their culture. Furthermore, early differentiation marker of osteoblasts (Runx2) were found on AFSCs cultured on stiffer PVA-IA hydrogels grafted with oligopeptides derived from Vitronectin (oligoVN) in expansion medium without any induction (differentiation) components, suggesting stiffer culture substrates grafted with oligoVNguide AFSCs into osteoblast lineage, whereas early neural differentiation marker of nestin were more expressed on softer PVA-IA hydrogels grafted with oligoFN in the expansion medium, suggesting softer culture substrates grafted with oligoFN and oligoVN promote differentiation into neural cell lineages. It is suggested that physical cues such as stiffness of culture materials as well as biological cues of extracellular matrix components can guide and decide pluripotency and differentiation lineages of stem cells. On the other hands, I further discoveredthatAFSCs cultured on thecooperation between ECM-ligands and stiffness matrices have been engaged into different lineages commitments.
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