Abstract
Purpose :
Limbal epithelial stem cells (LESCs) play key roles in corneal epithelial homeostasis. When LESCs are compromised, delayed wound healing and persistent corneal epithelial defects often occur. Transplantation of ex vivo limbal epithelial cultures has been used to treat LESC deficiency and the success rate is significantly associated with the numbers of LESCs in the ex vivo cultures. Therefore, it is clinically significant to understand the signaling pathways that are central for maintenance of stem cells and which could be pharmacologically targeted to enhance ex vivo LESC viability.
Methods :
To elucidate novel signaling pathways involved in LESC homeostasis, single cell RNA (scRNA) seq was used to transcriptionally analyze the rare population of limbal stem/early TA epithelial cells. Gene expression was validated using immunoblotting, qPCR and immunostaining. For functional studies, gene expression was silenced via siRNA knockdown and knockout mouse models. Diet-induced obesity (DIO) mice and Akita mice were used as diabetic mouse models.
Results :
ScRNA seq and immunostaining revealed that ID3, a key regulator for maintaining stemness in several non-ocular tissues, was preferentially expressed in the stem/early TA population of limbal epithelium. Loss of ID3 in human limbal epithelial cells markedly reduced the expression of putative LESC markers (e.g., N-cad, ABCB5). In the limbal epithelium of diabetic patients, as well as the DIO and Akita mice, we observed a reduction of ID3 expression, which was accompanied by a depletion in LESCs. This suggests that loss of ID3 may contribute to the LESC defect reported in diabetic corneas. RNA seq, immunostaining and biochemistry revealed that leucine rich repeat kinase 1 (LRRK1), which is preferentially expressed in limbal epithelium, positively regulates ID3 expression in vitro and in vivo. Similar to ID3, loss of LRRK1 diminished the expression of putative LESC markers and altered the ability of LESCs to respond to corneal epithelial wounding in mice.
Conclusions :
Our findings indicate that ID3/LRRK1 forms a novel signaling axis, which plays a critical role in maintenance of LESCs. Delivery of ID3 mRNA via nanotechnology has therapeutic potential to preserve stem cells in ex vivo limbal epithelial cultures.
This is a 2020 ARVO Annual Meeting abstract.