人類羊水間葉幹細胞（hAFSCs）為多能性胎兒細胞，具有能夠分化成特定細胞的能力，包括代表性的三個胚層。AFSCs可能成為幹細胞在再生醫學和組織工程更合適的來源。然而，由於幹細胞的特性，如適當的分化和多能性的維持，不只調節幹細胞本身，也可利用細胞的培養環境。此外，像是調整細胞培養基材的物理特性如軟硬度，可能會影響幹細胞分化的命運。在這個研究裡，調配在基材上不同的軟硬度，並固定有細胞外基質衍生的寡肽的細胞培養基材培養hAFSCs的成骨分化的效率。利用調配具有不同的軟硬度的polyvinylalcohol-co-itaconic acid（PVA-IA）在培養皿上，通過控制交聯劑的交聯時間製備。將PVA-IA培養皿通過N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (（EDC）和N-hydroxysuccinimide（NHS）去接枝上細胞外基質（ECM）衍生的寡肽。在這項研究中選擇了5種不同類型的寡肽，維持 AFSCs的多能性，藉由測定qRT-PCR測量，利用多能性基因的表現（Nanog的，Oct4和Sox2的）找出細胞培養基質的最佳軟硬度。為了分析hAFSCs的成骨分化特性，在兩週和四周利用誘導培養液誘導分化後進行alkaline phosphatase（ALP），alizarin Red S staining和von Kossa staining。利用物理特性如培養材料的軟硬度以及細胞外基質成分的生物特性可以誘導並決定hAFSCs分化成成骨細胞。;Human amniotic fluid-derived stem cells (hAFSCs) are pluripotent fetal cells capable of differentiation into multiple lineages, including representatives of the three embryonic germ layers. 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 regulated not only by the stem cells themselves but also by their microenvironment. Furthermore, physical characteristics of cell culture substrates such as substrate elasticity may influence the fate of stem cell differentiation. I investigated the efficiency of osteogenic differentiation of hAFSCs cultured on cell culture substrates which have different elasticities and are immobilized with extracellular matrix-derived oligopeptides. The dishes coated with polyvinylalcohol-co-itaconic acid (PVA-IA) films having different elasticities were prepared by controlling the crosslinking time in crosslinking solution that contains glutaraldehyde. The PVA-IA dishes were grafted with extracellular matrix (ECM)-derived oligopeptides through N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry in an aqueous solution. Five different types of oligopeptides were selected in this study. Pluripotent gene expressions (Nanog, Oct4 and Sox2) were evaluated by the qRT-PCR measurements. There was an optimal elasticity of cell culture matrix to keep pluripotency of AFSCs for their culture. To characterize osteogenic differentiation of hAFSCs, alkaline phosphatase (ALP) activity, alizarin Red S staining and von Kossa staining were evaluated after two weeks and four weeks of culture in induction media. 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 differentiation of hAFSCs into osteoblasts.
