Metanephric Cells Microenvironment Induces Differentiation Of Embryonic Stem Cells Toward Renal Cells

Objective:Embryonic stem cells (ESCs) are pluripotent and possess the remarkable ability of self-renew, which makes ESCs become a new resource of seed cells in regenerative medicine, and has drawn wide attention of the scientific community. The research concerning about renal differentiation of ESCs starts relatively late, relevant reports only showed up in the recent years. It was proved in vitro that some growth factors could induce ESCs to differentiate toward some definite types of renal cells, and researches in vivo indicated that metanephric cells microenvironment plays an important role in the differentiation of ESCs into renal cells. Here we imitated the metanephric cells microenvironment by coculture to investigate its induction effects on the renal differentiation of ESCs, which could provide a new experiment method for the research in this field, and provide experimental data for the exploration of key factors that determine the renal differentiation of ESCs in the metanephric cells microenvironment.Methods:1. Mouse embryonic stem cell line D3 was cultured on the primary mouse embryonic fibroblast cell feeder layers. The pluripotent markers, such as Oct4, Nanog and Sox2 in ESCs were detected by reverse transcription polymerase chain reaction (RT-PCR).2. The embryoil bodies (EBs) formation was induced using hanging-drop method, and then the EBs were divided into coculture group and natural differentiation group. The EBs in the coculture group were non-contact cocultured with the metanephric cells derived from embryonic day 12.5 fetal mouse in the Transwells system. The EBs in the natural differentiation group were cultured in the tradition way to let them natural differentiate.3. The effects of metanephric cell microenvironment on the expression of renal tubular cell marker Pax2 and renal podocyte marker WT1 were evaluated by immunofluorescence.4. The effects of metanephric cell microenvironment on the expressions of kidney develoment related genes (Pax2, WT1, GDNF, Emx2, BMP-7, Nephrin and KSP) and kidney progenitor cell marker CD24 in the EBs were detected by RT-PCR.Results:1. ESCs presented a clone-like growth on the primary mouse embryonic fibroblast cell feeder layers. RT-PCR result showed that ESCs had a strong expression of pluripotent marker Oct4, Nanog and Sox2, which indicated that the ESCs were in an undifferentiated state.2. Immunofluorescence analysis showed that in the coculture group, the positive cells of Pax2 in the EBs emerged in the 3rd day, with the prolongation of coculture time, the positive cells number increased. And in the EB outgrowths of the coculture group there are some positive cells assemble to distinct ring-like structure. In the natural differentiation group, the positive cells of Pax2 appeared in the 7th day. According to the statistical analysis, there were more positive cells of Pax2 in the coculture group than in the natural differentiation group,and the difference was significant (p<0.05). The positive cells of WT1 also emerged in the 3rd day of coculture, and with the prolongation of the coculture time, the positive cells increased gradually, but in the natural differentiation group there were no positive cells of WT1 detected. These results indicated that the metanephric cells microenvironment imitated by coculture promoted the differentiation of ESCs toward renal tubular cells and podocytes.3. The results of RT-PCR showed that in the early phase of coculture, the metanephron development early stage related genes Pax2, WT1, the middle stage related genes Emx2, GDNF, BMP7 and the Kidney progenitor cell marker CD24 mRNA levels in the EBs were promoted in the metanephric cells microenvironment, with the prolong of the coculture time, the latter stage related genes Nephrin and KSP expressions enhanced, though the early stage and middle stage related gene expressions were reduced. The results suggested that the metanephric cells microenvironment imitated by coculture could regulate the expression of metanephric development related genes in the induced ESCs.Conclusion:The metanephric cells microenvironment imitated by non-contact coculture could induce ESCs to differentiate toward renal cells, and regulate the expression of its metanephric development related genes.