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Matthew G. Trese, Murilo B. Abud, Sr., Caio V. Regatieri, Michael J. Young; Human Retinal Pigment Epithelium Cell Secretions Promote Human Retinal Progenitor Cell Differentiation into Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1124.
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© ARVO (1962-2015); The Authors (2016-present)
This in vitro study developed a co-culture system that mimicked outer retinal architecture and exposed human retinal progenitor cells (hRPCs) to the apical secretions of a mature retinal pigment epithelium (RPE). In doing so we were able to establish a relationship between RPE cell secretions and hRPC differentiation.
In 20% oxygen conditions, ARPE19 cells were differentiated on the underside of 12 well tissue culture inserts, until they attained a transepithelial resistance (TER) value that was consistent with functional maturity. Mature ARPE19 cell cultures were added to hRPC cultures for a period of seven days. At the end of the co-culture period, hRPCs and ARPE19 cell cultures were analyzed using immunocytochemistry (ICC) quantification and semi-quantitative reverse transcriptase polymerase chain reaction (SQ-PCR). This study was repeated in both 20% and 3% oxygen tensions because environmental oxygen has a profound effect on the proliferative capacity of RPCs.
This study showed that a functionally mature RPE monolayer can be derived in both 3% and 20% oxygen (TER values p< .05). Additionally, we demonstrated the viability of a complex co-culture system as evidenced by cellular survival and function throughout the co-culture period. Finally, this study showed that the apical secretions of ARPE19 cells altered the gene and protein expression of hRPCs resulting in the up regulation of the photoreceptor markers CRX, Recoverin, Rhodopsin and Opsin Blue. Conversely, the gene and protein expression of the immature markers PAX6, SOX2, KI-67, CMYC and nestin decreased between control and co-culture groups. As expected cellular proliferation markers increased in 3% oxygen when compared to 20% oxygen.
This study shows that mature RPE cell secretions promote the differentiation of human RPCs into photoreceptors. Additionally this study provides a spring board for future investigations into the specific trophic factors that most efficiently drive photoreceptor specific differentiation.
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