Postconfluent day 4 B4G12 cells were serum starved for 24 hours, followed by pretreatment with various concentrations of LY294002 or Y27632 for an additional 2 hours and treatment with LPA (20 mol/l) for another 4 hours. HCECs for transplantation and cell therapy. Introduction Facing the aqueous humorCcontaining anterior chamber, the corneal endothelium regulates stromal hydration and subsequent corneal transparency through the expression of the tight junction component ZO-1, which forms barriers,1 and partly through the expression of Na/K-ATPases, which act as pumps.2 In contrast to the situations in other species, human corneal endothelial cells (HCECs) retain only a very limited proliferative potential both expansion of HCECs, growth factors such as bFGF can be used11; however, EnMT is often activated.10 On the other hand, downregulation of p120-catenin using siRNA in both contact-inhibited HCECs10 and retinal pigment epithelial cells12 uniquely promotes proliferation by activating trafficking of p120-catenin to the nucleus, thus relieving the repression of the cell cycle by nuclear Kaiso without inducing EnMT.10 This nuclear p120/Kaiso signaling is associated with activation of the RhoA/ROCK signaling and inhibition of the Hippo pathway, but without activation of Tacrine HCl the Wnt/-catenin signaling.10,13,14 To prevent potential biohazards related to off-target effects induced by RNA silencing, we aimed to develop an alternative strategy for expansion of HCECs for clinical applications. The Hippo pathway was identified through genetic screens of and is highly conserved in mammals. This pathway is usually involved in controlling organ size and regulating embryonic development15,16 and is also a regulator of contact inhibition, 17 which plays crucial roles in regulating cell proliferation and apoptosis.18,19 The transcriptional coactivator yes-associated protein (YAP) is an important mediator of the Hippo pathway. Upon formation of cellular contacts, culture for 7 days (Physique 1a). In the HCEC monolayers, close cellCcell contacts and a polygonal cell morphology were established and preserved, mimicking those observed = 3; **< 0.01). (d) The suspension culture of HCECs showed a fibroblast-like morphology and expression of SMA fiber, but weak expression of ATPase and ZO-1 in the margin of cells, demonstrating the specificity of antibodies and an EnMT phenotype. The cell nuclei were counterstained with Hoechst 33342 (blue). Exogenous expression of YAP promoted proliferation in contact-inhibited HCECs YAP has been reported to promote proliferation in miscellaneous types of cells.25C28 To understand the effect of YAP on inducing proliferation in HCECs, HCEC monolayers were transfected with the pCMV6-YAP vector (pCMV6-YAP) for 72 hours, and monolayers transfected with the pCMV6-AC-GFP vector (pCMV6-control) served as controls. Subsequently, immunofluorescence revealed expression of YAP and BrdU-labeling, showing colocalization in cells transfected with pCMV6-YAP, suggesting an induction of proliferation by YAP in contact-inhibited HCEC monolayers (Physique 2a). On the other hand, EnMT was not induced in the HCEC monolayers, as there was positive Tacrine HCl immunostaining for Na/K-ATPase and ZO-1, whereas SMA staining was unfavorable (Physique 2b). Tacrine HCl Open in a separate FAAP24 window Physique 2 Overexpression of YAP leads to proliferation in contact-inhibited human corneal endothelial cells (HCECs). (a) HCEC monolayers were transfected with either the pCMV6-YAP vector (pCMV6-YAP) or the pCMV6-AC-GFP vector (pCMV6-control), as a control. After transfection, the HCEC monolayers were further cultured in HCEC growth medium for 2 days. The cultures were first starved for 2 hours, then fixed and immunostained Tacrine HCl with YAP (green) and BrdU (red; smaller physique in the right panel). Immunofluorescence images of YAP, BrdU, and nuclei (Hoechst 33342) were merged, and the colocalization of YAP and BrdU appeared as white Tacrine HCl color. Expansion culture of HCEC aggregates exhibited confluent monolayer cells under DIC microscopy (lower panel). In the pCMV6-YAP group, BrdU labeling was significantly increased. Colocalization of YAP and BrdU indicated that proliferation in contact-inhibited HCECs was promoted by YAP (= 3; **< 0.01). (b) HCEC monolayers transfected with the.