Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts.
Keywords: BMP, bone morphogenetic protein; BrdU, bromodeoxyuridine; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitors; DMEM, Dulbecco's modified Eagle's medium; E2F2; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EMT, endothelial mesenchymal transition; ESC, embryonic stem cell; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; HBSS, Hanks’ balanced salt solution; HCEC, human corneal endothelial cell; ID, inhibitor of differentiation; ITS, insulin-transferrin-sodium selenite; JAK, Janus kinase; JAK1; LEF1, lymphoid enhancer-binding factor 1; LIF; LIF, leukemia inhibitory factor; MESCM, modified embryonic stem cell medium; NC, neural crest; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBS, phosphate-buffered saline; RPE, retinal pigment epithelial cells; Rb, retinoblastoma tumor suppressor; SHEM, supplemental hormonal epithelial medium; STAT3; STAT3, signal transducer and activator of transcription 3; ZO-1, Zona occludens protein 1; bFGF, basic fibroblast growth factor; contact inhibition; corneal endothelium; iPSCs, induced pluripotent stem cells; p120, p120 catenin; p16INK4a; proliferation; scRNA, scramble RNA; siRNA, small interfering ribonucleic acid.