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H. Vaajasaari, T. Ilmarinen, H. Hongisto, R. Suuronen, H. Uusitalo, H. Skottman; Adherent Differentiation of Human Embryonic Stem Cells Promotes Generation of Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2658. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells can provide a cell model for various retinal diseases or drug discovery and essentially, a regenerative cure for vision-threatening diseases. The utilization of hESC derived RPE cells for these purposes requires an efficient differentiation method. In this study, the efficiency of the adherent differentiation was compared to suspension (EB) differentiation in the generation of the RPE cells from hESCs.
Human ESCs (Regea 08/023) were derived and maintained without serum and animal feeder cells. The differentiation was performed by plating the hESCs on protein coating or by culturing them as cell aggregates in suspension using a serum-free medium without any growth factors. The differentiation efficiency of the methods was compared by using qRT-PCR. The maturation of the cells was confirmed by RT-PCR and immunocytochemistry.
First pigmented cells were observed by day 14 with both differentiation methods. qRT-PCR data demonstrated that the adherent differentiation of the hESCs enhanced the expression of the essential genes for RPE development (PAX6, RAX, SIX3, MITF) compared to EB differentiation. Furthermore, the expression of neural retinal marker, CHX10, was down-regulated in adherent differentiation compared to EB differentiation. The RPE cell maturation was confirmed at gene level (MITF, RPE65, BEST, PEDF, PMEL, tyrosinase) and at protein level (MITF, CRALB, Bestrophin, ZO1).
The adherent differentiation method was more efficient for the generation of RPE cells than the EB method. The constituents responsible for the differences between the two methods are currently under investigation.
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