Ihmisalkion kantasoluista erilaistettujen verkkokalvon pigmenttisolujen kiinnittyminen ja erilaistuminen erilaisilla pinnoitemateriaaleilla

Age-related macular degeneration (AMD) and some other retinal diseases are associated with degeneration of retinal pigment epithelium (RPE) cells. A potential cure could involve cell transplantation therapy using RPE cells differentiated from pluripotent human embryonic stem cells (hESCs). A big challenge is to produce a homogeneous population of hESC-derived RPE (hESC-RPE) cells, and coating materials are one potential alternative to promote the differentiation. The aim of this study was to evaluate the cell attachment and differentiation of hESC-RPE cells on different coating materials and culture conditions. hESCs were derived and maintained without serum and animal feeder cells. This study consisted of three parts. In the first part, hESCs were cultured on different xeno-free coating materials and in a culture medium that stimulated the differentiation towards RPE cells. CELLstart™, collagen IV, and VitroCol™ were the best coating materials to support the cell attachment. The differentiation of the hESCs towards RPE cells was analyzed by visual pigment observation, immunocytochemistry, and quantitative real time PCR. There were no significant differences in the differentiation of the cells cultured on CELLstart™, collagen IV, or VitroCol™. In the second part, confocal microscopy wells were tested. hESCs differentiating to RPE cells and enzymatically degraded hESC-RPE cells attached and grew best in collagen IV coated Ibidi wells. In the third part, hESC-RPE cells were cultured in BD BioCoat™ collagen IV cell culture inserts in culture mediums containing basic fibroblast growth factor (bFGF) or not. RPE cells attached and grew well in the inserts with or without bFGF. The good attachment of hESCs differentiating towards RPE cells to CELLstart™, collagen IV, and VitroCol™ coatings was a remarkable result, since to my knowledge no publication of hESC differentiation towards RPE cells with a xeno-free coating material and a serum-free culture medium in adherent cell culture exists. Collagen IV can be recommended as a coating material for hESCs differentiating towards RPE cells, since it is cheaper than CELLstart™ and VitroCol™ and easy to use. In the future, the effects of growth factors and other supplements on RPE differentiation could be studied. Collagen IV can also be recommended for Ibidi confocal microscopy wells for hESC-RPE cells, at least for short-term experiments. Longer-term effects should be further studied. BD BioCoat™ collagen IV cell culture inserts can be utilized in research purposes, but they are not optimal since they contain xeno-products. In the future, the ability of bFGF to promote RPE cell cluster distribution and to transdifferentiate RPE cells could be further studied. /Kir1