Coculture Of Mesenchymal Stem Cells Induced Endothelial-like Cells And Cell Sheet

Objective To explore the coculture of BMSC sheet and BMSC induced endothelial-like cells,exploring the feasibility of them to promote blood vessel formation in vitro.Meanwhile,by implanting cocultured cell sheet in ischemic free flap model,we access blood vessel formation in vivo.Methods 1,Extraction,culturing and identification of bone marrow mesenchymal cells(BMSC): with the adoption SPF male Sprague Dawley rats of 4 weeks old,extraction marrow rinses after execution,the whole bone marrow adherent culture method,the difference between the in vitro culture of rat bone marrow mesenchymal stem cell(BMSC).The passage 4 BMSCs are digested and then identidied by FACS to detect the surface markers CD90,CD73,CD11 b,CD45,CD31.2,Ascorbic acid induced BMSCs forming cell sheet: The Passage 4 BMSCswere cultured in DMEM which containing Ascorbic acid for 10 days later.the BMSCs formed a sheet in culture dish taht can be scrape off,respectively,the BMSCs sheet were induced to differentiate into osteoblasts and adipocytes.the proliferation of BMSCs were detected by CCk-8 in two groups contains ascorbic acid or not.as well as histological structure of cell that observed by HE and Masson staining.3,Induced BMSCs to the endothelial like cells and its indentification:BMSCs were induced to differentiate into endothelial like cell by cultured in complete medium contains vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(b FGF),then we has determinated cell marker CD31,Flt-1 by immunofluorescence.The expression of surface markers was identified by flow cytometry,and morphological changes after endothelial like cells wre planted in Matrigel.4,Fluorescence labeling of BMSC cells and endothelial cells and coculture in vitro and in vivo: we used red fluorescent protein(RFP)marked BMSCs and induced then to endothelial like cells,which coculture with green fluorescent protein(GFP)marked BMSCs sheet.Tracing the morphological changes and migration of marked cells by fluorescence microscope after coculture.cell morphological changes and migration.Moreover,coculture cells were planted under the skin of SD rat for 2weeks,after postoperative death,tissue specimens were collected,histological observation and immunofluorescence were observed for the distribution of CD31 positive cells.Results The original generation BMSC are long spindle and grow as a colony,by the flow cytometry shows that CD73 CD90 marker is positive,CD11 b CD45 CD31 is negative.By adding ascorbic acid in medium,BMSCs shows incresing of proliferation,as well as expression of type I collagen than the control group,After ten days of culturing BMSCs forme a integrated sheet,after HE and Masoon histologic sections show ascorbic acid group has more collagen deposition than the control group between cells.After induction,oil red O and alizarin red staining are positive for BMSCs sheet.After endothelial induction,we observed that cells gradually become short spindle,as cell density increses,they formed pavement structure,2 weeks after induced,the flow cytometry identification showed CD31 positive rate is 91%.When the endothelial-like cells grown in Matrigel,they extend pseudopod after 4 hours and formed grid structure,gradually gathered to form multiple colony after 8h.RFP markers of endothelial-like cells with GFP marker of BMSC after mixed culture with fluorescence microscope,visible pseudopod extend endothelial cells after 4h,a long spindle,and mutual connection,the growth in good condition.After inplanted in vivo for two weeks,we observed microtubule formation.Conclusion : 1.Ascorbic acid induced BMSCs cell sheet can provide supportive structure to endothelial-like cells that differentied from BMSCs for developing and forming blood vesssels;2.BMSCs cell sheet cocultured with endothelial-like cells has the potential role of rescuing skin flap and tissue ischemia and remodeling microvessels.