Abstract
Background: Phase I/II clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE as potential sources of materials for cell-based therapy to treat degenerative retinal diseases have been carried out during the past decade. Challenges for successful translational cell-based therapy include cell manufacture, cell quality, cell storage, and cell behavior in vivo. In this study, we investigated the culture-induced changes in passaged fetal RPE, hESC-RPE and hiPSC-RPE cells in vitro and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on those RPE cells. Methods: A total of 9 fetal RPE cell lines, hESC-RPE and hiPSC-RPE cell lines were set up using previously described methods. The culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method and repeated passaging were studied by microscope, real-time quantitative PCR, western blot and immunofluorescent assays. Gene and protein expression and functional characteristics of fRPE, hESC-RPE and hiPSC-RPE incubated with ATRA at different concentration were also evaluated.Results: Compared with fRPE, hESC-RPE and hiPSC-RPE showed decreased gene and protein expression of RPE markers. Passage 3 RPE of all three types seeded at a density of 6×105 and 9x105 cells/mL in basal medium maintained pigmented polygonal, cobblestone-like morphology. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. fRPE, hESC-RPE and iPSC-RPE treated by ATRA at different concentrations showed increased expression of RPE markers such as RPE65, bestrophin (BEST) and CRALBP, and increased expression of negative complement regulatory proteins (CRP) including complement factor H (CFH), CD46, CD55 and CD59, and increased transepithelial resistance (TER) as well.Conclusion: Although hESC and hiPSC-derived RPE are morphologically similar to fRPE, and also have the tendency to undergo epithelial-to-mesenchymal transition (EMT) changes during the culturing and passaging process in vitro, differences in protein and gene expression among three RPE types exist. Moreover, ATRA can increase RPE markers expression, as well as to increase the expression levels of CRPs gene and protein in fRPE and stem cell-derived RPE.
Arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RAR -positive leukemic stem cells
