Corneal Stromal Stem Cells Cultured In 3-Dimensional Gelatin Micro-scaffolds And The Effect On Limbal Injury In Mice Cornea

ObjectiveTo gain cell tools for tracing corneal matrix microenvironment in vivo,corneal stromal stem cells(CSSCs)were extracted from stroma of m Tm G mice,using fluorescence-activated cell sorting(FACS)-side populations(SP)technique.The cells labeled with red fluorescence protein(RFP).RFP-CSSCs were co-cultured with Gelatin Micro-scaffolds(GMs)to construct corneal stromal microtissue in vitro.Furthermore,microtissue was used to treat the corneal limbal epithelial and stromal injury induced by alkali burn in mice.The therapeutic effect and the healing mechanism after limbal alkali burn by corneal stromal microtissue with exogenous CSSCs were investigated.We hope that the corneal stromal microtissue technology would be applied to treat limbal stem cells deficiency,and to improve the biological healing of ketatoprosthesis and tissue-engineered cornea in the future.Methods1.The locations of CSSCs were observed using immunofluorescence confocal microscope based on the expression of the adult stem cell marker ABCG2 in mice.The ABCG2 positive cells(stem cells)were isolated based on FACS-SP technique.It is remarkably useful in the identification and isolation of stem cells due to its ability to efflux Hoechst 33342,a fluorescent dye that binds DNA.The stem cell markers of cultured CSSCs were detected via flow cytometry,which combined positive markers CD44,CD90,CD29 and negative CD31,CD117 of mouse MSC.Then,CSSCs were identified by markers above mentioned with flow cytometry and corneal stem cell marker PAX6 with immunofluorescence confocal microscope.2.The RFP-CSSCs were cultured in GMs to construct corneal stromal microtissues.The activity,phenotype and functional characteristics of CSSCs were evaluated.3.Corneal stromal microtissue was used to treat the epithelial and stromal damage by corneal alkali burn in mice.The therapeutic effects and possible mechanism were evaluated.Results1.CSSCs with stable stem cell phenotype and red fluorescence protein(RFP)with labeled endogenous fluorescent maker were obtained successfully.2.The ABCG2-positive stromal cells in mice were located in the whole corneal stroma,and were mainly distributed near the endothelial cells.The ABCG2-positive stromal cells extended backward through the endothelial cell gap,and contact directly with the endothelial cells and the anterior chamber.3.RFP-CSSCs were cultured in 3D microgel carrier GMs.It was found that this culture method not only promoted the expansion of CSSCs,but also maintained their stem cell activity.CSSC cells were more likely to release vesicles in the 3D state,suggesting that this culture method was more benefical to the maintenance and function of stem cells.4.The constructed corneal stromal microtissue was used to treat corneal limbal alkali burn in mice.Compared with the fresh amniotic membrane control group and blank control group,the microtissue group showed more inhibition on corneal opaqueness and neovascularization(NV)score of P14(P<0.05),and the effects were confirmed histologically.5.Vesicles released from CSSCs in the stromal microtissues covering the lesion can appeared under two-photon microscopy.ConclusionsThis study demonstrated endogenous fluorescent labeled RFP-CSSCs with stable stem cell phenotype were obtained based on FACS-SP technique,which provides a cell tool for the study of corneal matrix microenvironment.RFP-CSSCs were co-cultured with GMs in vitro and successfully construct corneal stromal microtissues.Those microtissues have certain effect on treating epithelial and stromal damage of corneal limbal alkali burn in mice.The healing mechanism of corneal stromal microtissues with exogenous CSSCs after limbal alkali burn may be related to paracrine.