Abstract
It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.
Highlights
The cornea is a convex and highly transparent tissue providing approximately 75% of the ocular refractive power needed for normal visual acuity (DelMonte and Kim, 2011)
It is well known that the mouse corneal epithelial cells (mCEC) were poorly adherent when cultured in vitro (Kawakita et al, 2004), but by the third day, most of the cells in the 6C treatment group adhered and the proliferation rate far exceeded that of the other four treatment groups (Figure 1A)
Cell growth curves were generated to determine if culturing mCEC exposed to the 6C cocktail promoted their adherence and increased cell proliferation (Figure 1B)
Summary
The cornea is a convex and highly transparent tissue providing approximately 75% of the ocular refractive power needed for normal visual acuity (DelMonte and Kim, 2011). Corneal Epithelial Cells Culture Paradigm corneal transparency They provide a physical barrier against pathogenic infiltration and injury to layers beneath the tissue’s outer surface (Ouyang et al, 2014). Preservation of these tight junctional and barrier functions is dependent on the ability of the epithelial cells to undergo continuous renewal to replace terminally differentiated cells in the uppermost layers. The epithelial cells of the full thickness epithelium originate from a small stem cell population located in microenvironments at the junction between the corneal periphery and the adjoining conjunctiva This unique domain is requisite for perpetuating these slow cycling cells which give rise to a continuous supply of proliferating progenitor cells. Studies focused on clarifying the underlying mechanisms controlling this chain of events are relevant for delineating the factors optimizing this renewal process
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