Small molecule induction promotes corneal epithelial cell differentiation from human pluripotent stem cells

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Abstract

Purpose

Corneal epithelium is maintained by limbal stem cells, and their transplantation has been used to treat limbal stem cell deficiency (LSCD). However, this is only possible if enough healthy limbal tissue is available. Thus, novel cell sources for treating LSCD are needed. Human induced pluripotent stem cells (hiPSCs) provide unique opportunities for differentiation of limbal and corneal epithelial cells for cell transplantations.

Methods

In order to improve the efficiency and reproducibility of hiPSCs differentiation towards corneal epithelial progenitor cells, signaling cues active during ocular surface ectoderm development was replicated with two small-molecule inhibitors in combination with growth factors. The extent of differentiation was evaluated by following the expression of key markers using immunocytochemistry and qPCR at several time-points.

Results

Small-molecule induction down-regulated the expression of pluripotency marker OCT4 while up-regulating the eye-field transcription factor PAX6. Protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63-positive after five weeks of differentiation. Finally, after a total of six weeks in differentiation culture, the two markers specific to terminally differentiated corneal epithelium, cytokeratins 3 and 12, were expressed in an average of 35% and 71% of cells, respectively.

Conclusion

In contrast to all earlier studies, corneal epithelial cells were differentiated in serum-free culture conditions without the use of amniotic membrane or other undefined culture substrates. This highly efficient differentiation method could potentially be used for treating LSCD in the future.

Conclusion

Commercial interest

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