Preliminary Study On The Dedifferentiation Of Mature Corneal Epithelial Cells To Stem Cells

Purpose: To investigate the feasibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells ex vivo.Methods: Donor human corneas were obtained from the Tongji Hospital Eye Bank and managed in accordance with the Declaration of Helsinki and approved by Tongji Ethics Committee.Intact central or limbal epithelial sheets were isolated by digestion of central corneas or limbal segments with Dispase II in MESCM to generate MCECs sheets or limbal epithelial sheets(LEPCs).Limbal segments digested with collagenase A in MESCM generate clusters.The epithelial sheets were fixed with paraformaldehyde and embedded into paraffin for sections.MCECs,limbal LEPCs or limbal clusters were further digested with 0.25% trypsin/1 m M EDTA(T/E)to yield single cells.Limbal niche cells with collagenase A digestion methods were expanded on 5% Matrigel coated plastic.Single MCECs obtained from dispase-isolated epithelial sheets were seeded on 50% Matrigel with or without LNC.Expression of CK12,p63α,PCK,Vim was analyzed by immunofluorescence staining.Result: All of the MCECs(100%)express CK12,with 22.76±2.66% expressing p63α,which were mainly localized in the basal layer.In contrast,the positive rate of CK12 in LEPC is 38.73±23.28% and the positive rate of p63α is 84.42±2.33%.Single cells from MCEC sheets tended to aggregate on 3D Matrigel(3D-MCEC group)within 3 days after seeded.Only a few cells aggregated into spheres,which are irregular in shape.The diameter was between 52.01μm~180.32μm and the averaged 109.39±34.85μm.After 10 days,the expression of CK12(24.06±8.85%)reduced(P=0.005)but the expression of p63α also reduced to zero than those MCECs.MCECs mixed with LNC at 4:1.MCECs and LNC reunited as well generating round spheres.The diameter was between 129.06μm~306.20μm and the averaged 212.24±57.91μm,which are bigger in size than 3D-MCEC group(P=0.000).Besides,more cells expressing p63α(35.58±8.11%)than that in 3D-MCEC group(P=0.043)and less cells expressing CK12(52.95±20.99%)(P=0.000).Nevertheless,there was no significant difference between 3D-MCEC group and 3D-MCEC+LNC group for the expression of CK12(P=0.051).Spheres in 3D-MCEC+LNC group were consisted of both PCK+/Vim-epithelia cells and PCK-/Vim+ LNCs.Conclusion: Human MCECs tends to be induced to dedifferentiate into corneal epithelial stem cells in 3D Matrigel with the help of LNC.