Toward the defined and xeno-free differentiation of functional human pluripotent stem cell–derived retinal pigment epithelial cells

摘要

Purpose: The production of functionalretinal pigment epithelium (RPE) cells from human embryonic (hESCs) andhuman induced pluripotent stem cells (hiPSCs) in defined and xeno-freeconditions is highly desirable, especially for their use in celltherapy for retinal diseases. In addition, differentiated RPE cellsprovide an individualized disease model and drug discovery tool. Inthis study, we report the differentiation of functional RPE-like cellsfrom several hESC lines and one hiPSC line in culture conditions,enabling easy translation to clinical quality cell production underGood Manufacturing Practice regulations. Methods: Pluripotent stem cells werecultured on human fibroblast feeder cells in serum-free medium. Thedifferentiation toward RPE was induced by removing basic fibroblastgrowth factor and feeder cells from the serum-free conditions. RPEdifferentiation was also achieved using xeno-free and defined cultureconditions. The RPE cell morphology and pigmentation of the cells wereanalyzed and the expression of genes and proteins characteristic forRPE cells was evaluated. In vitro functionality of the cells wasanalyzed using ELISA measurements for pigment epithelium derived factor(PEDF) secretion and phagocytosis of photoreceptor outer segments(POS). The integrity of the generated RPE layers was analyzed usingtransepithelial electric resistance measurements. Results: We generated putative RPE cellswith typical pigmented cobblestone-like morphology. The expression ofRPE-specific markers was confirmed at the gene and protein level. Thedifferentiated cells were able to phagocytose POS and secrete PEDFcharacteristic of native RPE cells. In addition, cultured cells formeda polarized epithelium with high integrity and exhibited excellenttransepithelial electric resistance values, indicating wellestablished, tight junctions. Moreover, we introduced an improvedmethod to generate functional putative RPE cells withoutxeno-components under defined conditions. Conclusions: We have developed aprogressive differentiation protocol for the production of functionalRPE-like cells from hESCs and hiPSCs. Our results demonstrate thatputative hESC-RPE and hiPSC-RPE express genes and proteinscharacteristic for RPE cells, as well as being able to phagocytose POSand secrete PEDF. Furthermore, our results show that RPE-like cells canbe differentiated in xeno-free and defined culture conditions, which ismandatory for Good Manufacturing Practice-production of these cells forclinical use.