Regulation Of TGF-? Signaling Pathway On The Proliferation And Differentiation Functions Of Corneal Epithelial Stem/progenitor Cells

Transforming growth factor ?(TGF-?)signaling is one of the most important signaling pathways regulating cell behavior in ocular tissues.Its functions are mainly linked to tissue fibrosis and inflammatory responses in ophthalmology.In epithelial cells,however,the growth inhibitory activity of TGF-? was reported in both nonocular and ocular tissues.Since TGF-? is a bifunctional regulator that either inhibits or stimulates cell proliferation according to the specific context,we examined the effect of inhibition of TGF-? receptor(T?R)Imediated signaling on primary corneal epithelial cells(CECs)in serum-and feeder-free conditions.The mouse CECs were isolated from the eyeballs of 6–8 weeks old female C57BL/6 mice using dispase and trypsin separately,cultivated in defined Keratinocyte serum-free medium(KSFM)with supplements(the complete medium)without feeder layer.Cells were divided into three groups,those cultured in complete medium additionally supplemented with 10 ?M SB-431542,a specific inhibitor of T?R-I,were SBCECs;those cultured in complete medium additionally supplemented with 10 ng/ml SRI-011381,a TGF-beta signaling agonist,were SRI-CECs;those cultured in complete medium without SB-431542 or SRI-011381 were control CECs.The growth rate and morphology were analyzed by light microscopy.The identity and stemness of cells was investigated through marker staining of p63,inhibitor of differentiation 1(ID1),cytokeratin 12(K12),cytokeratin 14(K14),PAX6,p Smad3,alpha smooth muscle Actin(?SMA)and E-cadherin(Ecad);Real time quantitative PCR(RT-PCR)analysis of p63;Western blot analysis of ID1;as well as colony forming assay,sphere forming assay,healing wound in vitro assay and air-lifting interface assay.The results showed SBCECs subcultured steadily,achieved sustained expansion,and expanded almost thrice faster than control CECs.Expanded SB-CECs exhibited smaller and more compact morphology,up-regulated p63 and ID1,as well as better performed colony-forming capacity,sphereforming capacity,in vitro wound healing capacity,and the capacity to stratify and differentiate on air-lifting interface.Preliminary tests on human limbal epithelial cells(HLECs)showed the same results as mouse CECs.Interestingly,the ID1 expression pattern was almost identical to p63,the typical marker for corneal epithelial stem/progenitor cell(CESC/CEPC),in cultured CECs and normal corneal sections.Since ID1 has been proven to be regulated negatively by TGF-? signaling in epithelial cells and plays a role in blocking cell differentiation,its derepression by T?R-I inhibitor could be,at least in part,the underlying cause of CESC/CEPC expansion and the synchronously up-regulated expression of p63 in SB-CECs.In conclusion,inhibition of T?R-I-mediated signaling,CESCs/CEPCs achieved efficient long-term expansion in a feeder-and serum-free condition in vitro.And derepression of ID1 could be the underlying cause.Meanwhile,ID1 could serve as a marker for CESC/CEPC.These results may advance the basic and clinical CESC/CEPC research.