Abstract
Purpose:
Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells, which has the ability to function like embryonic stem (ES) cells. Unlike ES cells, iPS cells do not carry ethical issues and are easy to be applicable for ther¬apy and research. Limbal stem cell deficiency is a painful blinding disease characterized by epithelial defects, corneal opacity, and corneal vasculization. The use of iPS cells can be able to generate limbal progenitor cells that could provide a suitable cell source for corneal epithelial cells. Herein we demonstrate that iPS from human dermal fibroblast can be induced to differentiate towards limbal progenitor cells.
Methods:
Human dermal fibroblast cells were reprogrammed using a doxycycline inducible lentiviral vector containing Klf4, Sox2, Oct4, and c-myc in culture using an radiation-treated mouse embryonic fibroblast (MEF) feeder layer in ES culture medium. Limbal progenitor cells from these iPS cells by co-culture with PA6 feeder cells on limbal specific media the BMP4 and WNT treatment. Differentiated cells were analyzed by PCR and immunocytochemistry and cultured in the transwell with corneal epithelial medium.
Results:
Human dermal fibroblast-derived iPS cells are able to give rise to the limbal progenitor cells in vitro. Moreover, we obtained multilayered corneal epithelial cells from these limbal progenitor cells by air-lift culture assay.
Conclusions:
The present study demonstrate the successful reprogramming strategy for limbal progenitor cells differentiation from human dermal fibroblast derived iPSC using a mix of 4 factors and further suggests the therapeutic application of limbal progenitor cells from iPS cells for the limbal stem cell deficiency in the future.
Keywords: 482 cornea: epithelium •
721 stem cells