Differentiation of human pluripotent stem cells into corneal epithelial like cells

Abstract

Cornea is the clear outermost protective layer of the eye which enables transmission of light onto the retina. The corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in limbal stem cell deficiency (LSCD). Transplantations of ex vivo expanded autologous LSCs from patient’s healthy eye onto the affected eye have provided a successful treatment for unilateral LSCD. This however is not applicable to patient with total bilateral LSCD, whose both eyes are affected. This thesis investigated the potential of human induced-pluripotent stem cell (hiPSCs) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD, and tested the engraftment of the differentiated cells in LSCD mouse model. Combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid (RA) and epidermal growth factor (EGF) for the first nine days of differentiation followed by cell-replating on collagen-IV coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESCs) to corneal epithelial progenitors and mature corneal epithelial-like cells. Differences in the ability of hiPSCs lines to undergo differentiation to corneal epithelial-like cells were observed. These were dependent on the level of endogenous BMP signalling and could be restored via activation of this signalling pathway by a specific TGFβ inhibitor (SB431542). The hESC and hiPSCs-derived corneal epithelial cells were transplanted into a LSCD mouse model where they survived up to 14 days, but failed to provide long term engraftment and corneal surface regeneration. The findings showed a differential ability of hESCs and hiPSCs lines to generate corneal epithelial cells which is underlined by the endogenous BMP signalling pathway activity. However, the engraftment and functionality of the differentiated cells in the LSCD animal model has yet to be improved.