Rat Limbal Niche Cells Prevent Limbal Epithelial Stem/progenitor Cells From Differentiation And Proliferation Via Inhibiting Notch Signaling Pathway In Vitro

Purpose:Limbal niche cells(LNCs)were reported to play a pivotal role in regulating limbal epithelial stem/progenitor cells(LESCs).This study was conducted to investigate whether Notch signaling is involved in LESCs regulation by LNCs.Methods:Rat limbus was digested by dispase Ⅱ and collagenase A respectively.Limbal niche cells(LNCs)were isolated by serial passage of collagenase-digested cells on coated Matrigel.Dispase-isolated cells,with or without LNCs,were seeded on 3D Matrigel in modified embryonic stem cell medium(MESCM).The effects of Notch inhibition(by DAPT or Notch 1-siRNA)and activation(by Jagged 1)on LESCs were analyzed by quantitative RT-PCR,immunostaining,and Western blot.Results:Dispase-isolated limbal sheets consisted of PCK+ limbal epithelial cells(LECs).Collagenase-isolated limbal clusters consisted of LECs and Vim+ LNCs.LNCs were purified and expanded with expression of Oct4,Rexl,Sox2,Nanog,SSEA4,N-cadherin and CD34 on coated Matrigel.On 3D Matrigel,rat LECs alone(LECs group)failed to form clusters for 7 days,and rat LNCs(P3)exhibited an asterism-shaped growth.When mixed rat LECs with LNCs at a ratio of 4:1(LECs+LNCs group),LECs reunited with LNCs homogeneously expressed p63a.LNCs reunited with p63a+ LESCs to form clusters and prevented their differentiation on 3D Matrigel.Notch receptor(Notchl)and ligands(Deltal and Jaggedl)were unactivated in rat corneal and limbal in vivo,but activated in cultured LECs in vitro.LNCs significantly inhibited the Notch signaling of LECs in culture.The Notch inhibition and activation experiments were conducted on LECs,both DAPT and Notch1-siRNA significantly decreased the expression of Notch signaling in LECs,and Jagged1 significantly increased the expression of Notch signaling in LECs.Notch inhibition(by DAPT or Notch1-siRNA)increased p63α expression and decreased CK12 expression in LECs.Notch activation(by Jaggedl)decreased p63α expression and increased CK12 expression in LECs.The expression levels of p63α and CK12 in DAPT-treated LECs were similar to that in LECs+LNCs.Notch inhibition by DAPT decreased Ki67 expression in LECs,Notch activation by Jagged1 increased Ki67 expression in LECs,and the expression level of Ki67 in DAPT-treated LECs was similar to that in LECs+LNCs.Conclusions:Rat LNCs prevent LESCs from differentiation and proliferation mainly via inhibiting the Notch signaling in vitro.Manipulating the Notch signaling may help to preserveLESCs for corneal epithelial tissue engineering.