| 摘要: | 人類多能幹細胞，包括人類胚胎幹細胞和人類誘導多能幹細胞，在再生組織工程領域裡是相當有前景的來源。起初，人類多能幹細胞需要培養在動物飼養層細胞或動物源蛋白塗佈的培養皿上進行培養，例如小鼠胚胎成纖維細胞（MEF）或Matrigel，這些異種源細胞所分泌細胞外間質（ECM）有利於幹細胞進行附著進而增長，但因為這些異種源細胞與蛋白質含有未知的化學組成，對於外來的臨床試驗增添許多不確定性。因此，可靠的、無異源的生物材料──合成胜肽，不僅提供確定的化學成分，還提供了可重複培養的條件。
在我們之前的研究中已成功的將人類多能幹細胞培養在寡玻連蛋白（KGGPQVTRGDVFTMP）嫁接的聚乙烯醇-衣康酸水膠上。但是，一旦我們將誘導人類多能幹細胞進一步分化，與培養在其他細胞外間質相比，人類多能幹細胞較容易從寡玻連蛋白胜肽嫁接的聚乙烯醇-衣康酸水膠上脫附。因此本研究之目的，希望能設計出新胜肽不僅提供早期的細胞增殖，還可以協助後期的細胞分化。所以本實驗在層粘連蛋白（LN）發現了可能的新胜肽，並將新衍生的胜肽嫁接在聚乙烯醇-衣康酸水膠上來研究其中關鍵的細胞與基材結合機制，此次新設計的層粘連蛋白衍生之胜肽（PASYRGDSC和PMQKMRGDVFSP），其中序列裡的RGD基序提供細胞貼附的主要識別系統且有望用於進一步的支持細胞分化。一方面，本研究同時製造幾種不同的設計的層粘連蛋白衍生胜肽，設計不同長度的節鏈結來仿生原來的細胞外間質，評估鏈接的長度和結構效應。另一方面，在胜肽的末端使用或不使用酰胺修飾（-CONH2），用以比較細胞附著的效率。此外，在多能幹細胞培養於合成胜肽嫁接表面上後，評估其四種多能基因（Oct4，Sox2，SSEA-4和Nanog）的表現量。最後檢驗人類胚胎幹細在不同設計的胜肽上對分化能力的影響，從幹細胞分化成間業幹細胞（MSC）和心肌細胞（CM）。
我們的結果將有助於理解現階段培養人類多能幹細胞中細胞與基材結合機制，進而針對不同的使用條件客製專屬的、有利的增長環境。
;Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are attractive source for regenerative tissue engineering. Typically, hPSCs are used to culture on animal feeder cells or secreted protein-coated dishes, such as mouse embryonic fibroblast (MEF) or Matrigel, respectively which secrete extracellular matrices (ECMs) and growth factors for cell attachment but provide uncertain chemical compositions for cell growth. On the other hand, reliable biomaterials such as xeno-free synthetic peptides-immobilized surface, offer not only a chemically defined composition but also reproducible conditions for hPSC cultivation.
In previous studies in our laboratory, hPSCs were cultured on polyvinyl alcohol-co-itaconic acid (PVA-IA) hydrogels, which were conjugated with oligo-vitronectin (KGGPQVTRGDVFTMP) stably. However, once I induced the hPSCs for further differentiation, hPSCs detached much easier comparing to hPSCs cultured on other ECM-coated surface. In this study, I designed new peptides from Laminin (LN), which was a remarkable ECM for cell differentiation in previous studies where the key cell binding mechanism was investigated by using PVA-IA hydrogels. Several new designs of laminin-derived peptide (PASYRGDSC and PMQKMRGDVFSP) were grafted on PVA-IA hydrogels, which provided RGD binding motifs and constituted the major recognition system for hPSC adhesion. These synthetic peptides were investigated by comparing the ability to support long term hPSCs cultivation and differentiation into mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). The different lengths and sequences of joint segment, mimicking the original ECM, were evaluated to investigate the length effect and the effect of structures on hPSC culture and differentiation. Moreover, the C-terminus of peptides were treated with and without amide modification (-CONH2) to compare the efficiency of hPSC attachment. Furthermore, after hPSC cultivation on those synthetic peptides-grafted surface, hPSCs were evaluated for the expression level of four pluripotent protein (Oct4, Sox2, SSEA-4 and Nanog) expression. These results will help understanding of the cell-substrate binding mechanisms in sustaining hPSC culture and differentiation . |
