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Ruby Shalom-Feuerstein, Stephanie De La Forest Divone, Isabelle Petit, Laura Serror, Avraham Solomon, Daniel Aberdam; Pluripotent Stem Cells As A Model For Corneal Development, Physiopathology And Therapy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1219.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a robust in vitro model that recapitulates corneal-epithelial development using human pluripotent (embryonic (ES) cells and induced pluripotent (iPS)) stem cells and in addition, to evaluate the potential of this system for studying corneal development, physiopathology and as an alternative source for corneal therapy.
Pluripotent stem cells were produced from hair follicle-derived keratinocytes by lentiviral infection of a polycistronic cassette of pluripotent genes (oct4, sox2, klf4 and c-myc). hES cells or iPS cells were cultivated on collagen IV-coated dishes in epithelial media that was conditioned by corneal fibroblasts for two weeks. Committed cells collected at different time points of culture were subjected to FACS analysis, immunofluorescent staining, microRNA profiling and air-liquid interface culture. The functional role of two specific microRNA molecules was further investigated in ES-derived corneal-epithelial commitment, in clonogenicity assay of adult primary corneal epithelial cells and in the cornea ex vivo.
Pluripotent stem cells efficiently differentiated into ectodermal progenitors expressing K18 and pax6 that appeared in early culture days. These cells further differentiated into corneal epithelial-like mature cells expressing various corneal putative markers (K3/K12, pax6, and Cnx43). Transplantable corneal tissues were successfully reconstituted by organotypic assay. Of interest, 85% of previously listed corneal miRs were significantly enhanced during differentiation, suggesting a high degree of similarity between hES/iPS-derived corneal-epithelial cells and adult corneal cells in vivo. We identified one specific microRNA molecule that controls both corneal-epithelial lineage commitment and stem/progenitor phenotype, and a second microRNA molecule that specifically targets pax6, a master gene of eye development. Preliminary data showed that these miRs were deregulated in some corneal diseases.
This model that recapitulates the major steps of corneal-epithelial embryogenesis, will serve as a valuable tool for the in vitro study of corneal development and corneal diseases. Indeed, by using this system, we have identified two microRNA molecules that play an important role in corneal embryogenesis and homeostasis. The therapeutic potential of pluripotent stem cell-derived tissues needs to be determined in animal studies.
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