Abstract
Purpose :
In our previous studies, we have found that corneal epithelial cells could be dedifferentiated and retained the undifferentiated phenotype in ES microenvironment. Furthermore, this culture system could maintain the stemness of human limbal stem cells (LSCs) via telomerase/p21/mitochondria axis and the activation of FAK/Wnt signaling pathways.Studies have shown that microRNAs could regulate the functional properties of adult stem cells. Based on our previous work, we propose to explore the molecular mechanism of ES micro-environment regulates the self-renewal and differentiation in LSCs.
Methods :
The LSCs were cultured in different media, either CnT-20 medium or CnT-20 +20% ES culture supernatant (ESC-CM). Colony formation assay was used to analyse cell proliferation. miR-31 mimics and Antagomir-31 were transfected into the cells via the Ribo FECT TM transfection system. Then the cells were harvested to investigate the changes of the expressions of the mRNA and proteins.
Results :
We observed that LSCs LSC cultured in ESC-CM had an increased proliferative capacity, greater serial passage capacity, higher colony-forming efficiency (CFE) and higher levels of stem cell associated marker than those cultured in CnT-20. Compared with CnT-20, the level of miR-31 in ESC-CM was relative lower. We investigated the role that miR-31 and FIH-1 play in regulating the functional properties in LSCs. We used antagomirs (antago) to reduce the level of miR-31 in LSCs, and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced p21 expressional level in LSCs compared to irrelevant-antago treatment. The down regulation of miR-31 in LSCs promotes the maintenance of stemness.
Conclusions :
Our results demonstrate that ESC-CM regulates the fate of LSCs partially via Notch signaling, controlled through a miR-31/FIH-1/p21 axis. This study may have high impact and clinic implication on the expansion of LSC in regenerative medicine, especially for ocular surface reconstruction.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.