L-Phenylalanine high efficiently induces differentiation of human embryonic stem cells into retinal pigment epithelial cell

Abstract

Objective To investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. Methods H1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. Results On the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P<0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P<0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P<0.01) at the stimulus intensity of 3.0 cd·s/m2. Conclusions L-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models. Key words: Phenylalanine/drug effects; Embryonic stem cells; Cell differentiation; Retinal pigment epithelium