Pax 6 Controls Neural Crest Potential of Limbal Niche Cells to Support Self-Renewal of Limbal Epithelial Stem Cells

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.

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

“…CPVL immunopositive cells were found in the limbal stroma next to the limbal basal epithelial cells, with a small minority coexpressing TP63 ( Figure S6B-D). This cluster also showed high expression of PAX6 (Table S1), a transcription factor expressed in the limbal niche cells in the stroma (Chen et al, 2019), whose function is to maintain the phenotype of neural crest progenitors. In view of this, we speculated that cluster 10 may represent the limbal neural crest derived progenitors (LNCPs).…”

KIT ligand produced by limbal niche cells under control of SOX10 maintains limbal epithelial stem cell survival by activating the KIT/AKT signalling pathway

“…These results extend previous observations supporting a role for LNCs and KITL signalling in maintaining LESCs. 21,22 The above notion is based on multiple observations. First, both in That SOX10 is particularly important for LNC development and function is supported by its wider role in the physiology of neural crest cells.…”

“…LESCs are limbal niche cells (LNCs). [13][14][15][16][17][18][19][20][21] Previous results have shown that LNCs support self-renewal of LESCs, 21 and LNCs transplants prevent LSCD in an alkali burn rabbit model. 22 They likely do this by secreting paracrine factors including KITL, 22 but the detailed mechanisms of KITL regulation in LNCs and how KIT, KITL and its downstream signal pathways are involved are not clearly understood.…”

“…Interestingly, in vivo track systems based on SOX10-cre-driven YFP fluorescence have shown that SOX10-positive NCCs are the major contributors to the corneal stroma. 24 Given the above importance of SOX10 in NCCs and the fact that LNCs are essential for supporting self-renewal of LESCs, 21 we here focused on the role of SOX10 in LNCs for LESC maintenance both in vivo and in vitro. Using mice heterozygous for a Sox10 mutation and isolated LNCs as well as their conditioned mediums, we show that a gene dose reduction of Sox10 significantly impairs the ability of LNCs to support LESC maintenance and that SOX10 acts through KITL to activate the KIT-AKT signalling cascade in LESCs.…”

Process Development and Safety Evaluation of ABCB5+ Limbal Stem Cells as Advanced-therapy Medicinal Product to Treat Limbal Stem Cell Deficiency

“…Immuno uorescence characterization of unsegregated cultured limbal cells revealed a marker expression pro le that corresponded, for the very most part, to the pro le expected for LSCs. Speci cally, we detected heterogenous expression of the wellrecognized LSC marker p63 [42] and homogenous expression of PAX6, which has been shown to play a critical role in limbal stem cell fate determination [51][52][53]. The absence of the lament proteins CK3/12, which are, due to their speci c expression in corneal epithelial cells and limbal suprabasal but not basal cells, regarded as markers of corneal epithelial differentiation [17,54,55], indicates that the cultivated LSCs have maintained their undifferentiated nature throughout the expansion process.…”