Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells Into Corneal Epithelial-like Cells In Vitro

ObjectiveThe corneal epithelium is a rapidly regenerating stratified epithelium,which plays a critical role in maintaining corneal transparency and integrity of the ocular surface.The maintenance of the corneal epithelial cell mass is achieved by limbal stem cells(LSCs) located in the basal epithelium of the limbus.The partial or total deficiency of the LSCs population may greatly affect the ocular surface integrity and stability.After almost 20 years of studies,there are still no reliable therapies for severe forms of LSCs dysfunction,especially when LSCs deficiencies are bilateral.It is important to test the suitability of other stem cell lines for the reconstruction of a stem cell-deficient ocular surface.Due to their great expansion and differentiation potential,particular attention has been focused on mesenchymal stem cells(MSCs).MSCs are self-renewing, multipotent stem cells that mainly present in bone marrow(BM).Numerous reports have indicated that MSCs can give rise to a broad spectrum of tissues,including bone, cartilage,adipose tissue,and muscular tissue both in vivo and ex vivo.This type of cross-lineage differentiation is known as transdifferentiation.It has been indicated that MSCs can generate epithelial cell types in skin,lung,and other tissues in vivo.A Recent report suggested that transplantation of human mesenchymal stem cells(hMSCs) could reconstruct the damaged cornea.The therapeutic effect of the transplantation may be associated with the inhibition of inflammation and angiogenesis.These evidences suggest that MSCs may have the ability to transdifferentiate into corneal epithelial cells. To exam whether MSCs could differentiate into corneal epithelial cells,we reconstructed the damaged rabbit cornea with grafted rabbit mesenchymal stem cells (Rb-MSCs) in fibrin gel.We found that transplanted Rb-MSCs reached to the basal layer of corneal epithelium and expressed corneal epithelial cell molecular marker: cytokeratin 3(publication in progress).Data are rare,however,about the feasibility that MSCs could differentiate into corneal epithelial ceils,and there is still great controversy on MSCs plasticity because of the phenomenon that MSCs could fuse with the target tissue cells.Therefore,further studies are essential to examine whether MSCs can differentiate into corneal epithelial cells in vivo and ex vivo with or without cell fusion.In this research,examining whether MSCs could differentiate into corneal epithelial cells and avoid cell fusion,we cultured Rb-MSCs in four different independent culture systems in vitro.We found that the Rb-MSCs rapidly differentiated into cells with both morphological and molecular phenotype of corneal epithelial cells in all culture systems.Furthermore,we found epidermal growth factor(EGF) may be one of the important chemical factors in Rb-MSCs differentiation.Our study provides the first line of evidence that MSCs could differentiate into corneal epithelial phenotypic cells in vitro and presents a new promising source of cells for cell-replacement therapy treatment of corneal disorders.Methods1.Rabbit MSCs(Rb-LSCs) were obtained from the femurs of adult New Zealand white rabbits as previously described.In brief,10ml bone marrow was diluted 1:2 with PBS and loaded over 5ml Percoll(1.073g).Cells were harvested from the interface after centrifugation and resuspended in DMEM containing 5%fetal bovine serum. Cells were plated at 10~4cells/cm~2 in cell culture dishes and incubated at 37℃.To obtain MSCs expressing BrdU for later identification,10μmol/l BrdU containing media were added to 60%～70%confluent cultures for 24 hours.2.Rabbit limbal stem cells(Rb-LSCs) used for cell-suspension culture were isolated from New Zealand white rabbit limbal tissues by a previously described method.In brief,rabbit limbal tissues were washed in PBS containing 100U/ml penicillin,50μg/ml gentamicin.After careful removal of corneal endothelium,iris, excessive sclera,conjunctiva,the limbal rings were exposed to DispaseⅡ(1.2IU/ml) at 4℃for 18 hour.The loosened epithelial sheets were removed with a cell scraper and separated into single cells by 0.25%trypsin + 0.02%EDTA for 10 minutes at 37℃. Cells were pelleted at 1000 rpm for five minutes and resuspended in SHEM medium. Cells were plated at 10~4cells/cm~2 in cell culture dishes and incubated at 37℃.3.Expression of CD29,CD34 were detected with flow cytometry analysis to examine the phenotype of Rb-MSCs.4.Expression of CK3,Integrinβ1 and p63 were detected with immunofluorescent to examine the phenotype of Rb-LSCs.5.To induce differentiation in vitro,Brdu labeled Rb-MSCs were divided into four groups and incubated at 37℃for 3 day.(MSCs cultured in normal medium served as negative control)Group A:Rb-MSCs were established using 0.45um MILLICELL-HA membrane Transwell culture system.A suspension of Rb-LSCs was added to the upper membrane surface.Then inserts were positioned in the culture wells which had been incubated with Rb-MSCs.Group B:Rb-MSCs were cultured with conditioned medium.Conditioned medium were the supernatants medium which had been used to culture Rb-LSCs and filtered with 0.45um filter.Group C:Rb-MSCs were cultured directly with fresh SHEM medium.Group D:Rb-MSCs were cultured with normal DMEM medium containing 10ng/ml EGF.6.Expression of cytokeratin 3(CK3) in differentiated Rb-MSCs of different groups was examined by immunofluorescence,flow cytometric analysis.7.Brdu labeled Rb-MSCs were incubated in four different independent culture systems for 3 days which culture mediums supplemented with Tyrosine kinase inhibitors(AG1478).MSCs cultured in normal medium served as negative control. Immunofluorescence and flow cytometric analysis were used to examine the expression of CK3 in differentiated Rb-MSCs of the different groups.8.Results are expressed as the mean±SD%.Data were analyzed by analysis of variance(ANOVA) where appropriate.(P values of less than 0.05 were considered).Results1.Morphology of Rb-MSCs and Rb-LSCsRb-MSCs were successfully established from bone marrow collected from 10 anesthetized gilts(n=10).Most of the non-adherent cells were removed during the first media change at 24 hours.Colonies of fibroblast-like cells attached to the plastic were evident at day four-five after initial seeding.Most cell lines were composed of cells with a characteristic spindle shape.The number and size of the colonies reached 80% confluens by day 12～15.Within 24 hours after seeding,most Rb-LSCs attached to plastic and started spreading.Small colonies including approximately 8～10 cells were formed day 3～4 after inoculation.The cell morphology appeared to be compact,uniform,and small polygon in shape.By day 10～14,colonies began to fuse and formed a monolayer on confluence.2.Molecular phenotype of Rb-MSCs and Rb-LSCs(1)Molecular phenotype of Rb-MSCs:Flow cytometry analyses confirmed that 97.05%of the cells expressed CD29 antigen;5.15%of the cells expressed CD34 antigen.(2)Cell-surface markers of Rb-LSCs:Immunofluorescent revealed that Rb-LSCs were positive for the cell surface marker p63,integrinβ1,and negative for CK3.3.Differentiated analysis of Rb-MSCs in vitro(1)Morphological and molecular phenotype of differentiated Rb-MSCs:We found a subset of the adherent Rb-MSCs in all groups rapidly lose their characteristic fibroblast morphology and became broad and flattened with an epithelial shape.The expression of CK3 was observed in differentiated Rb-MSCs by immunofluorescence. Rb-MSCs of CK3~+ were first observed in two hours and there was little difference between different groups.The morphological and phenotypic differentiated Rb-MSCs remained unchanged in all groups within three days.(2)Differentiated ratio analysis of different groups:FCAS showed that only 3.46±1.9%of cells expressed CK3 in Group A at day one,and increased to 7.24±3.8% at day two,and slightly decreased(5.5±3.33%) at day three.The proportion of CK3 in Group B was 4.09±1.84%at day one,and rose to 9.31±5.92%after 24 hours,but fell significantly(4.37±2.61%) at day three.Changes in Group C and Group D showed a similar pattern:increased in day two(Group C from 3.48±1.39%to 5.61±3.82%; Group D from 1.84±0.47%to 4.73±3.94%),but decreased in day three(Group C 2.54±2.01%;Group D 1.68±0.88%).The mean difference is significant between each group and negative control(p＜0.05),but is not significant between groups(p＞0.05).4.Differentiated analysis of Rb-MSCs in vitro after Tyrosine kinase inhibitors treatment.(1)Morphological and molecular phenotype of differentiated Rb-MSCs:We found a subset of the adherent Rb-MSCs in Groups A and Group B became broad and flattened with an epithelial shape.The expression of CK3 was observed in differentiated Rb-MSCs by immunofluorescence in two hours and remained unchanged within three days.The expression of CK3 was not observed in Groups C and Group D.(2)Differentiated ratio analysis of different groups:FCAS showed 1.44±0.18%of cells expressed CK3 in Group A at day 1,and was increased(1.88±0.69%) at day 2,but was slightly decreased(1.76±0.15%) at day 3.The proportion of CK3 in Group B was 1.3±0.28%at day 1,and rise to 2.73±0.45%at day 2,but fell slightly(2.3±1.11%) after 24 hours.The proportion of CK3 was steady in Group C and Group D(Group C: 0.78±0.16%at day 1,0.68±0.14%at day2,0.73±0.16%at day 3;Group D: 0.62±0.11%at day 1,0.7±0.12%at day 2,0.56±0.1%at day 3).The mean difference is significant between Group A(or B) and negative control(p＜0.05),but not significant between Group A and Group B(p＞0.05).It is not significant between Group C and Group D(p＞0.05),and the same results were observed between Group C(or D) and negative control(p＞0.05).However,the means of CK3 in Group C and Group D significantly decreased compared with Group A and Group B(p＜0.05).ConclusionsMSCs could differentiate into corneal epithelial-like cells in vitro.EGF was one of the important factors in this process.