Differentiation Of Human Adipose - Derived Mesenchymal Stem Cells Into Renal Cell

Objective:The chronic kidney disease (CDK) has been increased nowadays. It deteriorates real function and leads to the end stage renal disease (ESRD), which can not be repaired by kidney itself. At present, hemodialysis, peritoneal dialysis and kidney transplantation are available for patients. However, the dialysis can only partly substitute the function of the kidney. Kidney transplantation also exists lots of problem such as immunological rejection, the shortage of kidney and the side effects of anti-rejection drug. It is very important to find a better way to treat the disease.Adipose-derived mesenchymal stem cells (AD-MSCs), known as multipotent and low-immunogenic cells, appear to be an ideal type of cells for tissue engineering and cell therapy. Recent researches suggest that the embryo can induce stem cells to be differentiated into renal lineage cells both in vitro and vivo. Whereas inducing AD-MSCs to differentiate into renal lineage cells in vivo is still impossible for clinical application, because the purification of induced cells is difficult. If AD-MSCs can be induced to differentiate into renal lineage cells in vitro, it can not only solve the important scientific problem, but also provide new treatments for repair of renal damage.At first, we study the biological characteristics and differentiation potential of human Adipose-derived mesenchymal stem cells (hAD-MSCs). The hAD-MSCs in our study can be induced to differentiate into adipocytes and osteoblasts, appear to be an ideal type cells for tissue engineering. Second, we imitate the embryonic kidney microenvironment by coculture and conditioned medium to investigate its induction effects on the renal differentiation of hAD-MSCs. Third, we imitate the embryonic kidney development by adding factors in sequence to investigate its induction effects on the renal differentiation of hAD-MSCs, in order to use it for clinical application.Methods:1. The hAD-MSCs were isolated from human adipose tissue. The cell morphology was observed by microscope. The cell phenotypic protein was examined by flow cytometry. And we induced the cultured cells to differentiate into adipocytes and osteoblasts, in order to examine the differentiation potential of the cultured cells.2. The mouse metanephric mesenchymal (MM) and ureteric bud (UB) were isolated and cultured from the CD-I mouse embryos of 13.5 day gestation. The hAD-MSCs were cocultured with MM and UB in Transwell systerm or with the conditioned medium of MM and UB for differentiating into renal lineage. The expression of renal cell markers was evaluated by RT-PCR and immunofluorescence.3. We imitate the embryonic kidney development by adding Activin A, retinoic acid (RA), bone morphogenetic protein (BMP7) and Wnt-4 in sequence to investigate its induction effects on the renal differentiation of hAD-MSCs. The expression of renal cell markers was evaluated by RT-PCR and immunofluorescence.Results:1. Primary hAD-MSCs demonstrated fibroblast colony-forming units. After the passage culture, cells exhibit a spindle-shaped morphology and have strong proliferative and self-renewal potential. The results of flow cytometry showed that hAD-MSCs strongly expressed CD29, CD44, CD105 and Flk-1, while CD34, CD106, CD31 and HLA-DR were negative. The hAD-MSCs could differentiate into adipocytes and osteoblasts under certain condition.2. After coculturing and culturing with the conditioned medium, the results of RT-PCR and immunofluorescence showed that hAD-MSCs expressed the marker characteristic for nephrogenesis.3. After inducing with special factors, the results of RT-PCR and immunofluorescence showed that hAD-MSCs expressed the marker characteristic for nephrogenesis.Conclusions:1. We successfully isolate the hAD-MSCs which have typical phenotype of mesenchymal stem cells, they are multipotent.2. The metanephric cells microenvironment could induce hAD-MSCs to differentiate into renal cells, and regulate the expression of its development related genes.3. hAD-MSCs can be induced into renal cells by adding inducing factors in vitro.