人類多能性幹細胞分化心肌細胞培養於熱敏性高分子塗佈細胞外間質;Differentiation of Human Pluripotent Stem Cells into Cardiomyocytes Cultured on Thermo-Responsive Polymer Coated with Extracellular Matrix

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题名:	人類多能性幹細胞分化心肌細胞培養於熱敏性高分子塗佈細胞外間質;Differentiation of Human Pluripotent Stem Cells into Cardiomyocytes Cultured on Thermo-Responsive Polymer Coated with Extracellular Matrix
作者:	蘇煥喬;Su, Huan-Chiao
贡献者:	化學工程與材料工程學系
关键词:	熱敏性聚合物;人類多能性幹細胞衍生心肌細胞;細胞外基質;embryonic stem cell;induced pluripotent stem cell;differentiation;thermo-responsive polymer;cardiomyocyte;extracellular matrix
日期:	2019-08-19
上传时间:	2019-09-03 12:33:36 (UTC+8)
出版者:	國立中央大學
摘要:	人類多能性幹細胞對於組織再生醫學是具有吸引力的來源。人體多能性幹細胞包括人體胚胎幹細胞與人體誘導多能性幹細胞已被使用在心血管研究包括 (a) 人類心臟發育 (b) 藥物發現 (c) 心臟毒性篩查和 (d) 疾病模型。此外，人體胚胎幹細胞分化的心肌前驅細胞已被用於臨床實驗中，用於治療心肌梗塞的患者。近年來，一直迫切需要在無異種條件下使用化學成分確定的培養基，發展再現性高且高效率的人體多能性幹細胞分化成心肌細胞的方法。特別是，一直以來都難以從培養皿上的未分化細胞中分離已分化或前驅的細胞。在這項研究中，熱敏性聚合物，如聚N-異丙基丙烯酰胺（polyNIPAAm），聚（N-異丙基丙烯酰胺-丙烯酸丁酯）（polyNIPAAm-co-BA）其在20 ℃ 時具有低臨界溶液溫度(LCST)，作為與幾種細胞外間質（ECM）綴合的細胞培養皿上的表面塗層聚合物。使用這個【智能生物材料】，開發出具有細胞分選功能的培養皿，可以分離或純化人體多能性幹細胞分化的心肌細胞在這個具有熱敏性的表面上。比起用其他細胞外間質(LN-511（層粘連蛋白-511）、纖連蛋白、第一型膠原蛋白或重組玻連蛋白)塗層於聚N-異丙基丙烯酰胺表面上，在塗有LN-521（層粘連蛋白-521）的聚N-異丙基丙烯酰胺表面上發現人體多能性幹細胞分化的心肌細胞具有較強的貼附能力。在低臨界溶液溫度(LCST)下，從熱敏感性表面收集的分離細胞其表面標誌物cTnT（心肌肌鈣蛋白T，心肌細胞標記物）表達高於未分離細胞。此外，發現人體多能性幹細胞分化的心肌細胞再次利用熱敏性表面分選後其cTnT的表現量可以高於90%。此外，人體多能性幹細胞分化的心肌細胞在塗覆有細胞外間質的熱敏性聚合物皿顯示出比直接塗覆有細胞外間質的細胞培養皿有著更好的細胞附著力。還證明了人體多能性幹細胞分化成心肌細胞的過程中，在用重組玻連蛋白（rVN）塗覆的聚N-異丙基丙烯酰胺共聚物表面上有更高的存活率比起直接塗覆重組玻連蛋白（rVN）的表面。可以得出結論，塗有LN-521的熱敏性聚合物表面可以用作細胞分選培養皿，並且可以在無異種細胞培養條件下分離具有高純度的人體多能性幹細胞分化的心肌細胞。;The human pluripotent stem cells (hPSCs) are attractive source for tissue regeneration medicine. hPSCs including hiPSCs (human induced pluripotent stem cells) and hESCs (human embryonic stem cells) have been used in the cardiovascular investigation of (a) human cardiac development, (b) drug discoveries, (c) cardiotoxicity screening, and (d) disease modeling. Furthermore, hESC-derived cardiac progenitors have started to be used in clinical trials for the treatment of myocardiac infarction patients. The development of reproducible and highly efficient protocols for hPSC differentiation into cardiomyocytes under xeno-free conditions using chemically defined medium has been in great demand in recent years. Especially, it is difficult to isolate differentiated/progenitor cells from undifferentiated cells on culture dishes. In this study, thermo-responsive polymers such as poly-N-Isopropylacrylamide (polyNIPAAm) and Poly(N-isopropylacrylamide-co-butylacrylate) (polyNIPAAm-co-BA), which have lower critical solution temperature (LCST) around 20 degrees, are selected as surface coating polymers on the cell culture dishes coated with several extracellular matrices (ECMs). Using these smart biomaterials, the cell sorting dishes were developed to isolate/purify hPSCs derived cardiomyocytes (hPSCs-derived-CMs) on the thermoresponsive surface in this study. It was found that the polyNIPPAm surface coated with LN-521 (laminin-521) had stronger affinity to hPSCs-derived-CMs than the surface coated with other ECMs (LN-511 (laminin-511), fibronectin, collagen type I, or recombinant vitronectin (rVN)). The cTnT (cardiac Troponin T, cardiomyocyte marker) was expressed higher on the detached cells from the thermoresponsive surface than non-detached cells, which were collected below LCST. Moreover, cTnT expression of hPSCs-derived-CMs were found to be higher than 90% after resorting on the thermoresponsive surface. The thermoresponsive polymeric dishes coated with ECMs showed better cell attachment of hPSCs-derived-CMs than tissue culture polystyrene (TCPS) dishes coated with ECMs directly. It was also demonstrated that hPSC-derived-CMs had higher survival rate on polyNIPAAm copolymer surface coated with rVN compared to dishes directly coated rVN during differentiation stage into CMs. It is concluded that the thermoresponsive polymer surface coated with LN-521 can serve as the cell sorting dishes and can isolate hPSCs-derived-CMs with high purity in xeno-free cell culture conditions.
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