Study Of Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Tubular Epithelial-like Cells In Vitro

AIM: To explore the possibility of mesenchymal stem cells(MSCs) differentiating into tubular epithelial-like cells in vitro, and to identified the contribution of MSCs to the renal repair of ischemia-reperfusion in vivo,and to investigate differentiation of rat bone marrow mesenchymal stem cells into tubular epithelial-like cells under different conditions.METHODS: MSCs were obtained from rats. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified, The surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were respectively cultured in DMEM medium with fetal bovine serum (blank group), DMEM medium with fetal bovine serum and all-trans retinoic acid and ischemic reperfusion injured kidney tissue homogenate (ATRA control group), DMEM medium with fetal bovine serum and epidermal growth factor and ischemic reperfusion injured kidney tissue homogenate (EGF induction group), DMEM medium with fetal bovine serum and hepatocyte growth factor and ischemic reperfusion injured kidney tissue homogenate (HGF induction group), DMEM medium with fetal bovine serum and bone morphogenetic protein-7 and ischemic reperfusion injured kidney tissue homogenate (BMP-7 induction group), DMEM medium with fetal bovine serum and erythropoietin and ischemic reperfusion injured kidney tissue homogenate (EPO induction group),DMEM medium with fetal bovine serum and ischemic reperfusion injured kidney tissue homogenate and four factors above-mentioned (combined induction group). After 7 days, the cells were collected for alkaline phosphataestaining,cytokeratin-18 immunocytochemical analysis, E-cadherin immunofluorescence analysis, and the mRNA of E-cadherin was detected by RT-PCR.RESULTS: (1)Cultured rat MSC at the passage 3 were positive for CD44,CD90 and CD29 (98.7%,98.1%,96.1%,respectively), but negative for CD11b/c and CD34 (14.7% and 1.98%, respectively). (2)Compared with blank group, the cells of ATRA control group and single factor induction group changed from spindle alike cells to round-like cells and elliptic cell. The cells of combined induction group became cobblestone-like cells after 7 days. (3)During differentiation of the tubular epithelial-like cell from the MSC, the counts of positive cells determined by AKP staining was increased. (4)Compared with blank group, the ratio of cytokeratin-18 positive cell in ATRA control group,EGF induction group,HGF induction group,BMP-7 induction group,EPO induction group and combined induction group were 27.40%±2.70%;29.60±4.51%;26.20±3.70%;26.80±5.00%;27.80±3.03%;44.00%±3.16%(P<0.01 vs blank group), the combined induction group increased obviously.(5)The mean of E-cadherin positive cell in ATRA control group,EGF induction group,HGF induction group,BMP-7 induction group,EPO induction group and combined induction group were 16.40±2.69 ; 18.25±3.50 ; 19.24±3.41 ;16.06±2.00;19.33±3.57;30.26±5.16 (P<0.01 vs blank group), the combined induction group increased obviously. (6)the mRNA expression of E-cadherin was increased obviously in combined induction group detected by RT-PCR.CONCLUSION: MSC may differentiate into tubular epithelial-like cells under the induction condition of ischemic reperfusion injured kidney tissue homogenate and growth factors in vitro.