| Background:The chronic kidney disease (CKD) has been up to 10% among the adult nowadays. It's characterized by the excessive deposition and fibrosis of extracellular matrix (ECM), which deteriorates real function and leads to the end stage renal disease (ESRD). At present, only peritoneal dialysis, hemodialysis and kidney transplantation are available for patients. However, the dialysis can only partly substitute the function of the kidney. And the kidney transplantation also exists lots of problem such as side effects of anti-rejection drug, chronic rejection and the shortage of kidney. It's now very argent to find a better way to treat CKD.Recent researches have suggest the plasticity and ability of bone marrow mesenchymal stem cells (BMSCs) which could differentiate into several functional cell types. The embryo could induct BMSCs to be differentiated into renal stem cells or resident cells in vivo, and xenobiotic growing embryo also has the similar potential. However, inducing the BMSCs to be differentiated into renal stem cells in embryo is still impossible for clinical application, because the purification of the renal stem cells from the embryo is not so easy. If the BMSCs could be induced to differentiate into renal stem cells in vitro, the induced cells could be easily isolated and purified.Previous studies have proved the differentiation of stem cells would depend on the microenvironment. The metanephric mesenchyme stem cells (MMSCs) are distinguished by their self-renewal ability and multipotency of differentiation. In the development of metanephron, the MMSCs would be differentiated into nephron and matrix cells, except the renal collecting tubule which oriented from ureteric bud (UB), and the embryonic renal stem cells could develop into resident cells even in vitro, which suggest the embryonic kidney tissue perform the feature of stem cell niches. Based on these researches mentioned above, our study intends to induce the BMSCs to be differentiated into renal stem cells with the help of soluble factors released by growing embryonic kidney rudiments containing MMSCs and UB by Transwell system. And we also assessed the function of differentiation of BMSCs in a functional co-culture assay with ureteric bud. The purpose of the study is to provide non-immunogenicity renal seeding cells for the renal tissue engineering and differentiation of BMSCs as the renal stem cell substitutes which could form new nephron in chronic kidney disease.Methods:1. The rat BMSCs were isolated and cultured with density gradient centrifugation combined with attachment culture method. The cell morphology was observed by phase contrast microscope, cell proliferative potential by methylthio tetrazole(MTT), cell inter structure by transmission electron microscopy, the cell cycle and the CD44, CD45, CD90 expressed on cell surface by flow cytometry, the expression of Vimentin, Fibronectin, Keratin, CD34 and E-cadherins by immunochemistry. We also make the cultured cells to be differentiated into adipocytes and osteoblasts. After the cell identification, the cells were suspended in the extracellular matrix gel and cultured at he interface of air and medium. The cell morphology change in ECM gel was observed by phase contrast microscope and laser scanning confocal microscope.2. The rat embryo was isolated from the pregnant Sprague–Dawley rats at gestation day 13.5(The plug day was designated as day 0.5). The rat embryonic metanephroi free from ureteric bud was primary cultivated with tissue culture methods. The morphological change was observed by phase contrast microscope. Proliferation and renewal of cells was measured by means of MTT. Cell inter and outer structure was observed by transmission electron microscopy and scanning electron microscope. The cell cycle and phenotypic protein (CD44, CD45, CD90) was examined via flow cytometry. We also assay the phenotypic protein expression of Vimentin, Fibronectin, Keratin ,CD34 and E-cadherins by immunochemistry staining. Differentiation potential was examined by culturing these cells under favorable conditions for adipogenic and osteogenic differentiation. After the cell identification, the cells were suspended in the extracellular matrix gel and cultured at he interface of air and medium. The cell morphology change in ECM gel was observed by phase contrast microscope and laser scanning confocal microscope. 3. Kidney rudiments were dissected from timed pregnant Sprague–Dawley rats at gestation day 13.5 (The plug day was designated as day 0.5). The UB and the MMSCs were co-cultivate by the transwell chamber system. The ECM gel was applied on top of the Transwell insert. Isolated UB was suspended in the ECM gel and cultured at the interface of air and medium. The morphology of UB was observed by phase contrast microscope and laser scanning confocal microscope.4. Establishment of the co-culture Model of BMSCs differentiation into renal stem cells in vitro. There are five groups: Group A: BMSCs and MMSCs. Group B: BMSCs and UB. Group C: BMSCs, MMSCs and UB. Group D: BMSCs. Group E: control. The morphology change of the induced BMSCs was observed by phase contrast microscope, the cell cycle and the CD44, CD45, CD90 expressed on cell surface by flow cytometry, the expression of Vimentin, Fibronectin, Keratin and E-cadherins by immunochemistry. We observed the function of differentiation of BMSCs co-cultured with UB by laser scanning confocal microscope.Results:1. Primary BMSCs demonstrated fibroblast colony-forming units by phase contrast microscopy. After the passage culture, Cells exhibit a spindle-shaped morphology and cells have strong proliferative and self-renewal potential. Transmission electron microscopy results showed that the nucleus is irregular-shaped with 1-2 nucleolus. There were ribosome and mitochondrion in cytoplasm, while other organelle seldom existed. The cell cycle analysis showed that more than 90% of BMSCs was at G0/G1 phase. The results of flow-cytometry showed that BMSCs strongly expressed CD44 and CD90, while CD45 was negative.The result of immunohistochemistry indicates that BMSCs strongly expressed Vimentin and Fibronectin, while Keratin , E-cadherin and CD34 were negative in contrast. BMSCs could differentiate into adipocytes and osteoblasts under certain condition. In three-dimensional culture system, rat BMSCs grew well.2. The cultured cells showed shuttle shape with single nuclear in each one. Transmission electron microscopy results showed that cells are irregular-shaped or spindle-shaped with lots of microvillus on the surface. There were rich smooth endoplasmic reticulum with secretory granule in cytoplasm, while other organelle seldom existed. The scanning electron microscope results revealed that cells were spindle-shaped or polygon with an obvious nucleolus and lots of microvillus on the surface. The cell cycle shows that more than 90% cells stay in the G0/G1 phase. Results of flow cytometry indicates that almost all cells strongly express CD44 and CD90, and none of they express CD45. Results of immwtcrowpy showed rat MMSCs strongly expressd vimentin, Fibronectin, while keratin, E-cadherin and CD34 are not expressed. Cultured cells could be induced to differentiate to adipocytes and ostepcytes in special induction condition.The result of observing the three-dimensional cultivated rat MMSCc by inverted microscope shows that cells on different layers are spindle-shaped. After cultivated for 2-3 days, the proliferation is enhanced. The result of the three-dimensional cultivated rat MMSCs by laser scanning confocal microscope showed that vimentin and fibronectin are expressed, while keratin, E-cadherin and CD34 aren't expressed. In three-dimensional culture system, rat MMSCs grew well.3. At gestational day 13.5, rat UB is a''T''-shaped epithelial tubule which undergoes repeated dichotomous branching suspended in ECM gel. Staining with lectins indicated that the tubular structures remained UB-derived and epithelial in character. Both cell proliferation and branching morphogenesis appeared to occur simultaneously.In ECM gel, UB epithelial tissues started to increase in size and developed small protrusions from the T-shaped ureteric bud. After 3-4 days of culture, those protrusions started to elongate, and the tips of the elongated structure started to branch dichotomously. As the time prolonged, this single-branched epithelial tubule underwent repeated dichotomous branching and forms the''tree''-shaped collecting system4. The inducedof rat BMSCs showed shuttle shape and the cells close tightly without gap under phase contrast microscopy. The results of cell cycle by flow cytometry showed that more than 90% cells stay in the G0/G1 phase. The result of flow cytometry detecting the cell surface marker revealed that most of the cells express CD44 and CD90; nearly none of them express CD45. The result of immunofluorescence discovered that rat BMSCs express Vimentin and Fibronectin strongly, while the Keratin and E-cadherin were negative. The induction by induced BMSCs to rat UB was observed by laser scanning confocal microscope, which displayed that the UB of group A, B, D don't change much, the induced UB of group C undergo morphogenesis. Conclusion:1. We successfully isolated the rat BMSCs and MMSCs which have typical phenotype of mesenchymal cells and stem cells, and they could be induced to differentiate into adipocytes and osteoblasts.2. We isolated, cultivated, identified rat UB successfully.3. We established the model of rat BMSCs differentiated into renal stem cell in vitro. And the induced rat BMSCs in vitro could initiate the rat UB dichotomous branching.
|
PDF Full Text Request