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Dhruv Sareen, Karoline Rostamiani, Mehrnoosh Saghizadeh, Alexander V. Ljubimov, Yaron S. Rabinowitz, Clive N. Svendsen; Generation Of Induced Pluripotent Stem Cells From Human Limbal Epithelial Cells And Re-differentiation Into Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1712.
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© ARVO (1962-2015); The Authors (2016-present)
Cultured limbal epithelial stem cell (LESC) transplantation is a promising treatment for LESC injury or deficiency. However, these cultures have variable production time and feeder quality, present viral transmission risks from feeders, and in total LESC deficiency cannot be established from affected patient tissues. The purpose of this study was to obtain consistent and quality controlled corneal epithelial cells by generating human induced pluripotent stem cells (iPSCs) from donors and then differentiating them back into corneal epithelial cells, which could then be used for transplantation. Recent evidence implies that human iPSCs retain residual epigenetic memory of their tissue of origin, which may facilitate iPSC differentiation toward parental cell lineages.
Explant cultures were generated from donor limbal rims. LESC-enriched cultures were expanded feeder-free on fibronectin-collagen-laminin substrata and were positive for putative LESC markers, such as ΔNp63, and keratins 15, 17, and 19. Cultures were reprogrammed to iPSC (CNL-iPSCs) using a non-integrating technique to generate episomal-iPS cell lines with pluripotency reprogramming factors, OCT3/4, SOX2, KLF4, L-MYC, and LIN28, and shRNA to p53. Colonies with the best rounded morphology were picked and transferred to BD Matrigel™ Matrix for feeder-free growth in mTeSR®1 medium.
CNL-iPSCs were successfully generated. They acquired markers of pluripotency and were able to differentiate into the three embryonic germ layers using in vitro embryoid body formation and in vivo teratoma formation assays. Quantitative RT-PCR analysis confirmed the absence of residual transgene expression. Cultures of CNL-iPSCs on basement membrane proteins showed the emergence of epithelial-like differentiated cells with significantly elevated gene expression of several corneal epithelial markers by quantitative RT-PCR, especially, keratin 3, suggesting differentiation into corneal epithelial cells.
iPSCs from human limbal cultured cells were generated and differentiated into cells with elevated expression of corneal epithelial markers. Such cells could be potentially useful in future as a source of limbal progenitor cells for clinical applications. Culture conditions including special substrata and media supplementation for corneal differentiation are currently being optimized.
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