| Background and Objective:Embryonic stem cell is a pluripotent cell derived from the inner cell mass of early mammalian embryo or primordial germ cells. It is capable of unlimited, undifferentiated and proliferative in vitro. It has potential applications in treating a wide array of diseases in the brain, kidney, bone and many other tissues. So the key point of embryonic stem cell in basic research and clinical application is how to induce it into a specific cell line. The orientation of embryonic stem cell is in addition to cellular differentiation of various definitive transcription factors, extracellular material inhibition mediated by endogenous factors is also essential. Now, inducing embryonic stem cell differentiation in vitro by co-culture hasbeen a very emerging and promising technology in tissue engineering. It has success reports in many medical fields, such as in the field of cardiovascular, respiratory and liver. In the field of urinary system, the recent researches showed that embry onic stem cell could differentiate into a renal lineage via embryoid bodies. But could embryonic stem cell differentiate into renal-like cell types by co-culturing with a particular type of cell had not reported yet.So we developed an efficient method by which embryonic stem cell could be induced to differentiate into a renal lineage especially the renal ancester cells by co-culturing with the rat meta-nephric mesenchymal cells in Transwell system, which will lay the foundation for the next step in the progression of future renal therapy by the induced renal-like cells.Methods:(1) The mouse embryonic fibroblast cells (MEFs) were isolated from the mouse embryos of 13.5 day gestation through primary tissue digestion. The morpholo-gy, growth curve and Mitotic Index of MEFs in vitro was investigated. The optional concentration of mitomycin C and the time treated with mitomycin C on MEFs were determined by MTT assay.(2) The blastocysts (4 dpc) from Kunming species mouse were collected, cultured on the third passages of embryonic fibroblast cell feeder layers treated with mitomycin C. The mESC cells which were pluripotential cells had been identified by morphology of cells, alkaline phosphatase staining, expression of OCT-4 and NANOG by RT-PCR, karyotype analysis, the formation of embryoid body and teratoma which are typical characteristics of ES cells.(3) The rat metanephric mesenchymal cells were isolated and cultured from the Sprague Dawley rats embryos of 13.5 day gestation. The cells had been identifi-ed by morphology of cells, immunocytology and electron-microscope.(4) ESCs were induced to form embryoid bodies by suspension culture. The cells from embryoid bodies were co-cultured with metanephric mesenchymal cells in Transwell system for differentiation into renal lineage. The treated ESCs were detected by expressing marker molecules characteristic for initiation of nephrogenesis (WT-1,Wnt-4,pax-2,c-Ret) and terminally differentiated renal cell types (podocalyxin, Nephrin).Result:(1)MEFs in vitro was a kind of adherent cell with good ability for proliferation,especially in passage 3, but from the 5th passage, MEFs was going to be senescence and distortion. The proliferation of MEFs could be efficiently repressed by mitomycin C.When mitomycin C concentrated on 10ug/ml and acted for 2.5-4 hours or on 20ug/ml and acted for 1-2.5hours, it worked best to inhibit the proliferation of MEFs.(2)Most of these cells had typical characteristics of mESC.These cells aggregated to form colonies with a clear borderline between colonies and feeder layer cells. The Colonies showed the high activity of AKP, expressed OCT-4 and NANOG by RT-PCR and had a normal karyotype. They also formed embryoid bodies by non-adherent culture in vitro and formed teratoma in severe combined immunodeficiency (SCID) mice in vivo.(3) The rat metanephric mesenchymal cells are cells with growing adherencely.They growed fast and showed spiral-like growth. The cells were small and they had little cytoplasm, big nucleus and prominent nucleoli.They were vimentin positive and keratin negative. In electron-mieroscope, the cells were irregular with big nuclei, cellular organelle inside and microvilli.(4) By RT-PCR analysis, after 7 days of co-culturing with metanephric mesenchymal cells, the embryoid body cells gave rise to the cell population expressing the above marker molecules characteristic for nephrogenesis. Conclusion:Embryonic stem cell could be induced into renal-like cells by co-culturing with metanephric mesenchymal cells. However, the induced cells were merely pre-progenitor cells, rather than specific renal cells. Therefore, how to get a specific kind of kidney cells and purify these cells in order to apply to in the treatment of renal repair in experimental models of acute and chronic renal failure is our next step.
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