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Ruby Shalom-Feuerstein, Laura Serror, Daria Putin, Eshkar Nir, Edith Aberdam, Daniel Aberdam; MicroRNA-184 and microRNA-450b are essential regulators of corneal epithelial fate and ocular development. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6039.
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To identify new microRNAs involved in corneal epithelial lineage commitment and pathophysiology.
Induced pluripotent stem cells (iPSCs) were differentiated into corneal epithelial cells on collagen IV-coated dishes in the presence of epithelial medium. Following microRNA profiling two selected microRNAs was examined in human and mouse tissues by in situ hybridization and real time PCR analyses. Knock-down and over expression of two specific microRNAs was further investigated in iPSCs, human limbal cultures and mouse embryos. Target genes were identified by in silico analysis and confirmed by molecular approaches.
The efficient differentiation of iPSCs recapitulated major steps of corneal embryogenesis in the dish. We identified miR-450b as a new repressor of Pax6, a major regulator of eye development. MiR-450b and Pax6 were reciprocally expressed during corneal differentiation of iPSCs and during ocular and epidermal lineage specification of the surface ectoderm. Interestingly, miR-450b inhibited Pax6 expression and corneal epithelial fate in vitro and in vivo, altogether suggesting that by repressing Pax6, miR-450b triggers epidermal specification of the ectoderm while its absence allows corneal epithelial fate. Likewise, miR-184 was elevated during early iPSC differentiation and was highly expressed by the developing lens vesicle and presumptive cornea of mouse embryos. In the adult cornea, miR-184 was preferentially expressed by corneal progenitors and not by limbal cells. The knockdown or over expression of miR-184 resulted in defects in limbal epithelial cell differentiation and stratification. Interestingly, we found out that miR-184 directly repressed the stem cell marker cytokeratin 15 and induces differentiation.
We identified two microRNAs that play a significant role in eye development and homeostasis. Our findings suggest that miR-450b modulates the dosage of Pax6 expression while miR-184 induces an escape from stemness of limbal epithelial cells. This data is interesting in light of the fact that point mutations in miR-184 were linked with familial keratoconus with cataract. Finally, these data indicate that iPSCs are valuable for modeling corneal development, and may pave the way for future cell-based therapy.
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