TGF-β receptor inhibitor Compound C promotes the directed differentiation of human embryonic stem cells into retinal pigment epithelial cells

Abstract

Objective To observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells.Methods H1 hESC were divided into control group and experimental group.When the hESC reached over confluence,the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation.The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction.Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6),microphthalmia-associated transcription factor (MITF),cellular retinaldehyde blinding protein (CRALBP),and RPE65 in both groups at the 1,3,5 weeks of the induction process.hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified.Real-time PCR,Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells.Results Pigmented colonies were observed in experimental group at the 4 weeks of the induction process,while no pigmented colony could be detected in the control group.All the purified pigmented cells from experimental group showed polygons morphology.Experimental group showed significantly higher expression of RPE marker genes PAX6,MITF,CRALBP and RPE65 than the control group(P＜0.05).Compared with the hESC and ARPE-19 cells line,purified hESC-RPE cells showed much higher expression of PAX6,MITF,CRALBP and RPE65(P＜0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells.Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells.Conclusion TGF β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE. Key words: TGF-beta superfamily proteins/antagonists & inhibitors; Embryonic stem cells/transplantation; Retinal pigment epithelium/cytology