Abstract
On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.
Highlights
Www.nature.com/scientificreports lack of eyes and nose and dies soon at birth[18,19]
To determine whether there was any difference between limbal niche cells (LNC) and CSC in the expression of Pax[6] immediately after isolation, we isolated them from epithelium-containing limbal stroma and epithelially denuded corneal stroma from the same donor using collagenase digestion as reported[2]
LNC and CSC were expanded on coated Matrigel in a modified serum-free ESC medium (MESCM)[26] and compared to CSC expanded on plastic in Dulbecco’s Modified Eagle’s Medium (DMEM)/10%FBS27 or in neural stem cell expansion medium (NSCM)[12,28]
Summary
Www.nature.com/scientificreports lack of eyes and nose and dies soon at birth[18,19]. Expression of Pax[6] is dosage dependent as mutation or missing allele leads to aniridia in humans (Review in16) and the small eye (sey, Pax6+/−) in mouse animal model[20]. We found the expression and nuclear localization of Pax[6] differentiates LNC from CSC and causally correlates with the neural crest progenitor status regarding marker expression, neurosphere formation, and neuroglial differentiation. Such a phenotype is crucial to endow LNC with the capability of supporting self-renewal of limbal epithelial SCs by suppressing corneal epithelial differentiation and maintaining holoclone formation
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