Abstract
Purpose: :
To investigate the potential of human parthenogenetic embryonic stem cells (hPESCs) to differentiate into retinal pigment epithelium (RPE) cells, and identify development-regulating microRNAs (miRNAs).
Methods: :
RPE cells were drived from hPESCs. The expression of markers, miRNA expression profiles and positional information of RPE ‘Signature’ miRNAs during differentiation were studied by using real-time RT-PCR, western blot and miRNA expression array at three time points. Human fetal RPE (hfRPE) cells were also analyzed. Then target genes of candidate miRNAs were validated.
Results: :
RPE cells can be derived from hPESCs with an efficiency similar to human embryonic stem cells (hESCs). hPESCs-derived RPE cells exhibited similar morphology and pigmentation to hfRPE cells. However, the expression of markers (Oct4, Pax6, ZO-1, RPE65, MERTK) during retinal differentiation indicated that hPESCs-derived RPE cells were in an immature state. Most specific miRNAs played a role at some point in differentiation and maturation of RPE from hPESCs, with the exception of only two miRNAs (miR-204 and the miR-302s family). miR-204 showed an up-regulation, and miR-302 showed a down-regulation throughout the process. CTNNBIP1 and TGFBR2 were confirmed to be the target genes of miR-204 and miR-302 respectively, involved in mediating the activation of the Meis2/Pax6, Wnt/beta-catenin and TGFβ/Nodal pathways.
Conclusions: :
hPESCs can develop into RPE-like cells, which provides an additional promising source for RPE cells in cell therapy. miR-204, 302s and their targets are involved in regulating directed differentiation during the full course, which contribute to the search for a new method of improving the differentiation efficiency using miRNAs.
Keywords: retinal pigment epithelium • differentiation • gene/expression