| 摘要: | 目前仍缺乏穩定可供應患者所需之器官或組織。人類多功能幹細胞(hPSCs)包含人類胚胎幹細胞(hESCs)及人類誘導多功能幹細胞(hiPSCs)，可分化成三胚層，並應用於再生醫學供作器官與組織來源。幹細胞臨床試驗僅應用下列疾病：(1)黃斑部退化；(2)急性心肌梗塞；(3)糖尿病；(4)脊髓損傷。目前的研究中缺乏將人類多功能幹細胞成功並具高再現率分化成心肌細胞之法。因此，本研究擬開發最佳細胞培養智能生醫材料，用於無異種環境將人類多功能幹細胞分化及純化心肌前驅細胞及心肌細胞。本研究擬針對（i）何種智能生物材料係為人類多功能幹細胞分化及純化心肌前驅細胞和心肌細胞所需及（ii）何種奈米片段或細胞外基質（ECMs）被固定於塗佈有熱敏性高分子聚合物（聚（N-異丙基丙烯酰胺），PNIPAAM共聚物）的生醫材料上，最適合用於培養和分化人類多功能幹細胞，以及在智能生醫材料上純化心肌前驅細胞和心肌細胞。本研究將開發以熱敏性高分子聚合物塗佈的智能生醫材料，並固定細胞外基質或細胞外基質衍生的寡肽，篩選出最適於幹細胞培養，並分化成心肌前驅細胞和心肌細胞之材料。並比較目前所發表與本研究室所開發之分化步驟之心肌細胞分化效率和純度，同時篩選出最佳的生醫智能材料用於分化和純化心肌前驅細胞和心肌細胞。 ;There is a lack of organs and tissues for patients in need. Human pluripotent stem cells (hPSCs), which encompass human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have been considered to be promising sources for regenerating damaged tissues and organs because of their ability to differentiate into cells from three embryonic germ layers. Currently, only for four main diseases are targeted for the clinical trials for stem cell therapy using hPSCs (mainly hESCs): (1) macular degeneration [age-related macular degeneration (AMD) and Stargardt’s macular dystrophy], (2) acute myocardial infarction (MI), (3) diabetes, and (4) spinal cord injury. The efficient and reproducible differentiation method of hPSCs into cardiomyocytes has not yet investigated from our database research. Especially, it is unclear what kinds of molecular, physical, and biological design of cell culture biomaterials are preferable for differentiation and purification (sorting) of hPSCs into cardiac progenitors and cardiomyocytes. Therefore, the development of sophisticated smart biomaterials, which are optimized for the differentiation and sorting of hPSCs into cardiomyocytes and cardiac progenitors are important for future stem cell therapies. In this study, we are going to develop optimal cell culture smart biomaterials for differentiation and sorting of hPSCs into cardiac progenitors and cardiomyocytes in xeno-free conditions. We will investigate (i) what type of smart biomaterials are desirable for differentiation of hPSCs into cardiomyocyte progenitors and cardiomyocytes and for sorting of hPSC-derived cardiomyocyte progenitors and caediomyocytes, and (ii) what kind of nanosegments or extracellular matrices (ECMs) should be immobilized on cell culture biomaterials coated with thermoresponsive polymer (poly(N-isopropylacrylamide), PNIPAAM copolymer) for culture and differentiation of hPSCs as well as sorting of hPSC-derived cardiac progenitors and cardiomyocytes on smart biomaterials. Our originality of the smart biomaterial development for stem cell culture, differentiation, and sorting is the combination of physical cues (thermoresponsive polymer-coated dishes to detach specific cells by reducing temperature of the culture medium) and biological cues (selection of specific extracellular matrix (ECM) or ECM-derived oligopeptide for recognition of cardiac progenitors and cardiomyocytes). We are going to develop cell culture smart biomaterials coated with thermoresponsive polymer (PNIPAAM copolymer), which are further immobilized with ECM or ECM-derived oligopeptides, which are suitable for stem cell culture, differentiation, and sorting into cardiac progenitors and cardiomyocytes in clinical usage in future. PNIPAAM copolymer is selected as the thermoresponsive polymer for the coating on the cell culture dishes, which has low critical solution temperature (LCST) around 20 degree. Several ECM proteins and oligopeptides are further immobilized on the PNIPAAM copolymer. hESCs and hiPSCs are cultured on the thermoresponsive dishes grafted with ECM and oligopeptides and are differentiated into cardiac progenitors and cardiomyocytes using several differentiation protocols from articles and from our development in this study. We evaluate the efficiency and purity of cardiomyocyte differentiation from the data and discuss the optimal cell culture smart biomaterials for specific differentiation and sorting of hPSCs into cardiac progenitors and cardiomyocytes. |
