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Samuel McLenachan, Dana Zhang, Fred Chen; Retinal Pigment Epithelial Cell Differentiation in Primary Human Limbal Neurosphere Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1008.
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© ARVO (1962-2015); The Authors (2016-present)
The human corneoscleral limbus contains multipotent stem cells that can be isolated and cultured for clinical applications, such as the treatment of limbal stem cell deficiency. In culture, limbal stem cells can be induced into the neural linage to produce cells displaying the characteristics of photoreceptors. Here, we have examined the hypothesis that retinal pigment epithelial (RPE) cells can be produced from primary human limbal cultures.
Human corneoscleral rims were incubated with collagenase to facilitate the removal of the limbal epithelium (LE). LE was dissociated non-enzymatically and cultured in the presence of EGF, FGF2 and Noggin to produce floating neurospheres (LiNS). LiNS were plated on a geltrex matrix to form adherent colonies. In parallel, primary limbal cells were cultured in keratinocyte media to produce adherent LE cell monolayers. Primary LE and LiNS cultures were examined by microscopy and immunocytochemical analysis.
Two different populations of neurospheres were evident in primary human LiNS cultures; pigmented and non-pigmented. Comparison of primary keratinocyte and neurosphere cultures revealed the predominance of non-pigmented LiNS in cultures with stromal keratocyte contamination. Pigmented LiNS expressed the ocular transcription factors PAX6, OTX2 and MITF, and contained cells expressing the RPE specific markers RPE65 and ZO1.
Culture of human limbal epithelial stem cells in the presence of EGF, FGF2 and Noggin leads to the induction of LiNS containing pigmented cells expressing the RPE cell markers MITF, RPE65 and ZOI.
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