LIF-JAK1-STAT3Signaling Delays Contact Inhibition Of Human Corneal Endothelial Cells

Purpose:To determine the mechanism involved in sustained expansion of human corneal endothelial cells (HCECs) in modified embryonic stem cell medium (MESCM).Methods:BrdU labeling of HCEC monolayers cultured in supplemented hormonal epithelial medium (SHEM), ESCM, ESCM+LIF, ESCM+bFGF and MESCM were compared at day7,21,35and42. Their mRNA expression of embryonic stem cell (ESC) markers, neural crest (NC) markers, bone morphogenetic protein (BMP) ligands and receptors and cell cycle related genes were assessed by qRT-PCR. The cytolocalization of p120, pNFkB, pSmad1/5/8, F-actin, β-catenin, LEF1, Na/K-ATPase, N-cadherin, ZO-1, S100A4, p-Rb and pTyr705-STAT3were determined by confocal imaging.Results:The BrdU labeling was negative in the endothelial cells cultured in SHEM at day21, but still positive in the cells cultured in ESCM+LIF and MESCM at day35. In addition, supplement of LIF in ESCM not only retained upregulation of all the seven ESC and NC markers in the cells cultured in MESCM, but also further upregulated expression of their ESC markers, Oct4, Rex1and SSEA4. Addition of LIF or bFGF or both in ESCM was not sufficient to activate canonical and non-canonical pNF-κB BMP signaling as evidenced by negative nuclear translocation of pSmad1/5/8and pNF-κB. Further analysis indicated that nuclear translocation of pSTAT3was apparent only in the cells cultured in ESCM+LIF and MESCM but not in ESCM+bFGF or ESCM alone, suggesting that LIF activates LIF-JAK1-STAT3signaling pathway in HCECs. Finally, BrdU labeling and upregulation of positive G1/S phase transition genes and nuclear translocation of p-Rb in HCECs cultured in LIF-containing media at day35were nullified by Janus kinase (JAK1) or STAT3siRNA knockdown, indicating that the delay of contact inhibition is controlled by LIF-JAK1-STAT3signaling at this stage.Conclusions:Compared with SHEM, MESCM may further expand the size of HCEC monolayers by delaying re-establishment of contact inhibition in HCEC monolayers. LIF, but not bFGF in MESCM plays a pivotal role in delaying contact inhibition of HCEC monolayers through upregulating expression of positive G1/S phase transition genes in the late phase (D35) of culture by activating LIF-JAK1-STAT3signaling pathway.