Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo

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.

Commentary: Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity In Vitro and In Vivo

“…Moreover, the 6C culture system conduces to construct tissue-engineered corneal epithelium and promotes healing of corneal epithelial wounds in mice. These smallmolecule combinations regulate mCEC proliferation involving in cAMP, TGF-β, BMP, Notch, Wnt/β-catenin, and Rho/ROCK signaling (An et al, 2021). Remarkably, 6C seem to maintain the limbal proliferating stem and progenitor cell phenotype in vivo, which demonstrates that these signaling modulators might regulate LSC functions.…”

“…Interestingly, each compound of 5C has been proved to improve corneal epithelium homeostasis. This study by An et al (2021) compared the effects of 5C and 6C (combined Y-27632 and 5C) on the mouse CECs (mCECs) and found that 6C could increase mCEC proliferation, sustain the expression levels of the progenitor cell function gene, as well as suppress epithelial-mesenchymal transition. The 6C culture method may be applied for improving the availability of CECs to treat LSCD in clinical practice.…”