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Heidi Hongisto, Tanja Ilmarinen, Alexandra Mikhailova, Heli Skottman; Xeno- and feeder-free differentiation of human pluripotent stem cells to ocular epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1143.
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© ARVO (1962-2015); The Authors (2016-present)
As human pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy applications, more standardized and xenogeneic-free culture and differentiation protocols for hPSCs are desired. We aimed to develop robust, xeno- and feeder cell-free culture system for undifferentiated hPSCs and efficient differentiation methods thereafter to produce ocular epithelial cells suitable for clinical transplantation.
Several human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) lines were transferred to the hPSC culture system based on the Essential 8TM (E8, Thermo Fisher Scientific) culture medium and human recombinant laminin matrix. Differentiation to retinal pigment epithelial cells (RPE) was performed with two xeno-free media: X-VIVOTM 10 (Lonza) and xeno-free differentiation medium (XF-RPEbasic) using both embryoid body and adherent differentiation methods on a combination of extracellular matrix (ECM) proteins. Cells were analyzed for differentiation efficiency, RPE gene (RT-PCR) and protein (immunofluorescence staining and confocal microscopy) expression, barrier properties (TER) and functional maturity (phagocytosis assay and ELISA).
Human PSCs were successfully maintained in the defined, xeno- and feeder-free culture system using single cell passaging. The hPSCs demonstrated correct undifferentiated morphology, hPSC marker expression, full pluripotency, and normal karyotype. Prolonged culture led to recurrent karyotype changes conveying culture and differentiation advantage. Both RPE differentiation media enabled efficient differentiation to RPE that was enhanced by small molecule induction in a media and cell line specific manner. A combination of ECM proteins mimicking the natural RPE basement membrane allowed for proliferation and maturation of tight epithelial monolayers with excellent TER values, correct expression and localization of RPE proteins and RPE functionality. Xeno-free cryopreservation protocols for both pluripotent hPSCs and hPSC-RPE were optimized for cell banking.
A robust and reproducible, xeno- and feeder-free culture method for hPSCs as well as RPE differentiation and cryobanking was established. The method allows for easy transition to Good Manufacturing Practice-quality for generation of clinical grade cells, and can also be used to generate corneal epithelial cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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