Effects Of TGF-β Receptor Inhibitor Compound C On Directed Differentiation Of Human Embryonic Stem Cells Into Retinal Pigment Epithelial Cells

ObjectiveTransplantation of human embryonic stem cells (hESC) derived retinal pigment epithelium (RPE) cells to replace damaged or lost cells is a promising approach for the patients suffered from retinal degeneration disease. However, a long-term differentiation period with low efficiency and high cost are still the bottleneck in the present study. RPE derives from neuroectoderm. Promoting hESC differentiation into neuroectoderm can enhance the differentiation efficiency of ESC into RPE. It has been reported that TGF-β receptor inhibitor Compound C suppresses mesoderm, endoderm, and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in hESC. In this research, we observe the effect of Compound C on the directed differentiation of hESC into RPE cells and to characterize the hESC derived RPE(hESC-RPE) cells.MethodsH1hESC were divided into control group and Compound C group. When the hESC reached overconfluence, the medium was changed to KOSR medium without bFGF to induce RPE differentiation. The Compound C group was supplemented with1μM TGF-β receptor inhibitor Compound C at the first six days of induction. Realtime-PCR was carried out to examine the expression of PAX6, MITF, CRALBP, RPE65in both groups at the w1,3,5of the induction process. HESC-RPE cells were isolated mechanically and purified. Realtime-PCR,Western blot, Immunofluorescence and phagocytosis assay were used to characterize the purified hESC-RPE cells.Results1、Pigmented colonies were observed in Compound C group at the w4of the induction process, while no pigmented colony could be detected in the control group. Mutiple pigmented colonies were observed in Compound C group at the w5of the induction process,while pigmented colony could just be detected in the control group.2、Compared with the control group, Compound C group showed higher PAX6expression at the w1,3of the induction process (t=28.498,3.141, P<0.05).MITF, CRALBP and RPE65, as the marker genes of RPE cells, were detected in both groups at the w3,5of the induction process. Compound C group showed significantly higher expression of these markers than the control group (MITF:t=8.866,10.111, P<0.05; CRALBP:t=5.293,5.394, P<0.05; RPE65:t=9.263,9.504, P<0.05)3、All the purified pigmented cells from Compound C group showed polygonous morphology full with pigment granules.4、Compared with the hESC and ARPE19cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(PAX6:1=9.154,7.605, P<0.05; MITF:t=17.284,16.770, P<0.05; CRALBP:t=18.204,18.190, P<0.05; RPE65:t=44.194, t=44.190, P<0.05)5、High expression level of PAX6, MITF, CRALBP and RPE65proteins were observed in hESC-RPE cells.6、Immunofluorescence verified the expression of PAX6, MITF, CRALBP, RPE65and ZO-1in hESC-RPE cells.7、Phagocytosis assay showed that hESC-RPE cells can phagocytose polystyrene microspheres and have characteristics of phagocytosis ability.ConclusionsTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE. HESC-RPE cells possess the typical polygon and pigment appearance of RPE cells, which can highly expressed the RPE marker genes and proteins and have characteristics of phagocytosis ability. RPE marker genes and proteins are highly expressed in hESC-RPE cells than in ARPE19cell line. HESC-RPE cells are more suitable for cell transplantation in the treatment of retinal degeneration diseases and can provide a better cell platform for the research of these disease.