title: Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin β1-FAK-PI3K/Akt pathway.
abstract: Adult stem cells are important cell sources in regenerative medicine, but isolating them is technically challenging. This study employed a novel strategy to generate stem-like corneal epithelial cells and promote the functional properties of these cells by coculture with embryonic stem cells. The primary corneal epithelial cells were labelled with GFP and cocultured with embryonic stem cells in a transwell or by direct cell-cell contact. The embryonic stem cells were pre-transfected with HSV-tk-puro plasmids and became sensitive to ganciclovir. After 10 days of coculture, the corneal epithelial cells were isolated by treating the cultures with ganciclovir to kill the embryonic stem cells. The expression of stem cell-associated markers (ABCG2, p63) increased whereas the differentiation mark (Keratin 3) decreased in corneal epithelial cells isolated from the cocultures as evaluated by RT-PCR and flow cytometry. Their functional properties of corneal epithelial cells, including cell adhesion, migration and proliferation, were also enhanced. These cells could regenerate a functional stratified corneal epithelial equivalent but did not form tumors. Integrin β1, phosphorylated focal adhesion kinase and Akt were significantly upregulated in corneal epithelial cells. FAK Inhibitor 14 that suppressed the expression of phosphorylated focal adhesion kinase and Akt inhibited cell adhesion, migration and proliferation. LY294002 that suppressed phosphorylated Akt but not phosphorylated focal adhesion kinase inhibited cell proliferation but had no effect on cell adhesion or migration. These findings demonstrated that the functional properties of stem-like corneal epithelial cells were enhanced by cocultured embryonic stem cells via activation of the integrin β1-FAK-PI3K/Akt signalling pathway.
authors.affiliation: State Key Laboratory of Ophthalmology, Sun yet-sen University, Guangzhou, Guangdong, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhou
authors.forename: Fen
authors.initials: F
authors.lastname: Chen
authors.forename: Jianhui
authors.initials: J
authors.lastname: Xiao
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Ding
authors.forename: Pengxia
authors.initials: P
authors.lastname: Wan
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Wang
authors.forename: Junqi
authors.initials: J
authors.lastname: Huang
authors.forename: Zhichong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000328:Adult; D000818:Animals; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism*; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction; D014162:Transfection
chemical_list: D019012:Integrin beta1; D019869:Phosphatidylinositol 3-Kinases; D051418:Focal Adhesion Kinase 2; D051057:Proto-Oncogene Proteins c-akt