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，而人類多能幹細胞可成為用於特定疾病的潛在療法。來自世界衛生組織的資料顯示老年性黃斑部病變是視力損害的第三大原因，且預計未來會變得越來越嚴重。80-90% 的老年性黃斑部病變主要由視網膜色素上皮的功能損壞而引起。幸運的是，移植人類多能幹細胞分化成的視網膜色素上皮細胞可以作為治癒老年性黃斑部病變疾病的方法。然而，來自人類多能幹細胞的視網膜色素上皮通常存在著純度不足和培養時間長的問題。因此，在本篇研究中有比較不同的培養製程和嘗試各種細胞外基質來評估哪些條件最適合人類多能幹細胞分化為視網膜色素上皮細胞。我測試了人類多能幹細胞分化為視網膜色素上皮細胞三種類型的培養製程（N2、NIC84 和Activin A 培養製程）。每個培養製程都經過進一步修改來提高人類多能幹細胞分化為成熟視網膜色素上皮的效率。在本研究中，我以原來的NIC84培養製成進行以下調整，(1) 降低 CTM 濃度；(2) 改變細胞繼代方式；(3) 減少製成前28天的培養液使用量。經過這些調整，可以用此改良後的培養製成高效地獲得從人類誘導多能幹細胞分化成的成熟視網膜色素上皮細胞。在此研究中，也有對這些細胞進行相關的驗證程序及檢測。視網膜色素上皮細胞因為有色素沉澱之功能，呈色為棕色，而我從人類誘導多能幹細胞誘導成的視網膜色素上皮細胞也呈棕色。加上這些視網膜色素上皮細胞有表達ZO-1和 RPE65 的這兩個代表成熟視網膜色素上皮細胞的標記。透過細胞顏色的轉變及特定抗體的標記，可證明改良後的NIC84培養製程可使人類多能幹細胞分化成視網膜色素上皮細胞。接著在不同的細胞外基質上，透過特定抗體的標記比例及細胞生長速率我發現擁有Matrigel 塗層和 LN-521 塗層之培養皿較擁有rVN 塗層、LN-511 塗層和 Synthemax II 塗層之培養皿更能支持人類多能幹細胞分化成視網膜色素上皮細胞。;Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), where hPSCs can be used for the potential cell therapy toward specific disease. Age-related macular degeneration (AMD), which ranks the third leading cause of vision impairment according to world health organization (WHO), is expected to become more and more serious in the future. 80-90% of AMD disease mainly results from the dysfunction of the retinal pigmented epithelium (RPE). Fortunately, transplantation of hPSCs-derived RPE can serve as a regenerative approach to cure AMD disease. However, RPE derived from hPSCs usually suffers from insufficient purity and long culture period. Therefore, different protocols and cell culture extracellular matrices (ECMs) were investigated to evaluate which conditions would be the most suitable for hPSCs to differentiate into RPE in this study. Three types of protocols (N2, NIC84, and Activin A protocols) for hiPSC differentiation into RPE were evaluated. Each protocol was further modified to improve the efficiency of differentiation into mature RPE. Mature RPE differentiated from hiPSCs can be obtained with high efficiency using the modified protocol of NIC84 with reducing concentration of CTM and modified medium in day 28-30 of differentiation, which was developed in this study. hiPSC-derived RPE showed brown color indicating pigmented cells (mature RPE) and expressed mature RPE marker of ZO-1 and RPE65. Matrigel-coated and LN-521-coated dishes supported high differentiation of hiPSCs into RPE compared to rVN-coated, LN-511-coated, and Synthemax II-coated dishes.
