Reprogramming of mouse renal tubular epithelial cells to induced pluripotent stem cells.

Kidney disease has reached epidemic proportions and is associated with high mortality and morbidity rates. Stem cell-based therapy may effectively treat kidney damage by cell transplantation. The breakthrough discovery using a combination of four transcription factors to reprogram genetically somatic cells into induced pluripotent stem (iPS) cells was a milestone in stem cell therapy. The lentivirus was packaged containing OCT4, SOX2, c-MYC and KLF4 transcription factors and then transfected mouse renal tubular epithelial cells (RTECs). The colonies were picked up at 21 days and were tested by cytochemistry, immunofluorescence assay and quantitative real-time polymerase chain reaction. Viral transgene expression levels were also assessed by quantitative analysis. Additionally, embryoid bodies from iPS cells were formed, and immunofluorescence and teratoma assays were performed. Karyotype analysis of mouse RTEC-derived iPS cells was also performed. The iPS cells were indistinguishable from mouse embryonic stem cells with respect to colony morphology, the expression of pluripotency-associated transcription factors and surface markers, embryoid body-mediated differentiation potential and teratoma assays. Quantitative polymerase chain reaction demonstrated that the lentiviral transgenes were largely silenced. The mouse RTEC-derived iPS cells exhibited a normal karyotype of 40,XY. iPS cells can be produced using mouse RTECs, which would be helpful in investigations to ameliorate the symptoms of kidney disease and to slow the progression of kidney disease by in vitro and in vivo animal studies.

[Effects of erythropoietin on mesenchymal stem cells' function of differentiation and secretion cultured under acute kidney injury microenvironment].

OBJECTIVE: To explore the effects of erythropoietin (EPO) on the differentiation and secretion of cultured bone marrow-derived mesenchymal stem cells (BM-MSC) in the microenvironment of acute kidney injury (AKI). METHODS: C57BL/6 murine BM-MSC (mBM-MSC) were successfully isolated by the methods of Percoll density gradient centrifugation and adherence cultivation. The AKI murine model was induced by ischemia/reperfusion (I/R). The homogenate supernatants were prepared for normal and I/R murine kidney. P3-mBM-MSC were treated differently: Group A: low glucose DMEM medium with 10% fetal bovine serum, Group B: normal murine kidney homogenate supernatant intervention, Group C: I/R kidney homogenate supernatant intervention, Group D: I/R kidney homogenate supernatant plus different concentrations of EPO (1, 5, 10, 50 U/ml). Each group was incubated for 1, 3, 5 and 7 days. Inverted microscope was used to observe the morphological changes of these cells and their ultrastructural changes were observed by transmission electron microscope. Cytokeratin-18 was detected by flow cytometry. The levels of bone morphogenetic protein-7 (BMP-7), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were detected by ELISA in culture medium. RESULTS: The cells yielded a high expression of CD29 and CD44 and a low expression of CD34 and CD45. Compared with Groups A and B, the cells of Group C presented oval and short fusiform shapes. After the intervention of EPO, Group D showed a cobble appearance. More organelles were also found. A trace expression of CK18 was found in Groups A and B. A positive expression of CK18 was significantly higher in Groups C and D than Groups A and B (P < 0.01). The expression of EPO 50 U/ml at Day 5 and 7 was higher than Group C of the same time (5 d: 35.22 ± 4.04 vs 8.72 ± 0.38, 7 d: 42.00 ± 5.39 vs 13.20 ± 1.14, both P < 0.01). The results of ELISA showed that the levels of BMP-7, HGF and VEGF in Group C decreased significantly (P < 0.01 or P < 0.05). CONCLUSION: The intervention of EPO may boost the differentiation of mBM-MSC but reverse its low secretion.