Abstract
An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.
Highlights
The corneal epithelium forms the outermost layer of the cornea and acts as a barrier against infection and injury
Miyashita et al developed a novel culture system supplemented with keratinocyte growth factor (KGF) and Y-27632, and demonstrated by immunohistochemical staining that limbal epithelial cells (LECs) cultured in this system could maintain high protein expression of PAX6 and several corneal epithelial cells (CECs)-specific markers such as keratin 3 (KRT3) and KRT1225
To investigate how gene expression of LECs varies across different culture systems, we followed this system and performed a comprehensive promoter analysis of LECs cultured in two distinct conditions: in the presence of Epidermal growth factor (EGF), and in the presence of KGF and Y-27632
Summary
The corneal epithelium forms the outermost layer of the cornea and acts as a barrier against infection and injury. Its availability is limited by a global donation shortage[4], and it sometimes fails as a result of immunorejection[5] To overcome these problems, alternative CECs are differentiated from different sources such as ex vivo-expanded limbal epithelial cells (LECs)[6, 7], oral mucosal epithelial cells[8, 9], and induced pluripotent stem cell (iPSC)-derived CECs10–12. The molecular mechanism underlying the effect of different culture media on LECs remains poorly understood To elucidate this mechanism, here we performed a comprehensive gene expression analysis of LECs cultured in these two distinct conditions, by cap analysis of gene expression (CAGE)[26, 27]. Our results provide new insights in the regulation of the characteristic gene expression of CECs and support the development of alternative CECs with better conditions for the treatment of corneal disorders
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