The Generation Of Human Neural Crest Cells From Induced Pluripotent Stem Cells From Urine-Derived Stem Cells

Objective To investigate whether the urine cells which used to transform into induced pluripotent stem cells from Urine(Ui PSCs) are urine-derived stem cells(USCs), and whether induced pluripotent stem cells(i PSC)can be transformed from urine-derived stem cells by transfection with episomal plasmids under feeder-free, virus-free condition, and then differentiate into neural crest cells. Sequentially to establish a practical protocol for neural crest cells induction from i PSCs which reprogrammed from urine-derived stem cells.Methods According to the method used in the Ui PSCs, namely collection and expansion of urine cells by REGM? Renal Epithelial Cell Growth Medium. After amplification, identifying whether the urine cells possess the ability of pluripotency, containing evaluation the expression of the common markers of USCs, CD34, CD45, CD29, CD73 and CD146, by flow cytometry and differentiation into adipocyte, osteocyte and smooth muscle cells. To electroporate the urine cells with p CXLE-EGFP by Nucleofector R II, and observing the expression of fluorescence after 1 week to 4 week. By electroporated with p CXLE-h OCT3/4-shp53-F 、p CXLE-h SK 、 p CXLE–hΜL and p CXLE-EGFP under feeder-free, virus-free condition, the urine cells were reprogrammed into i PSCs. Then identified the pluripotency of i PSCs from the urine cells. At last, differentiated the i PSCs into neural crest cells, and identified the expression of mark of neural crest cells, AP2, p75, HNK1 and Fox D3.Results Flow cytometry results showed that the urine cells which collected and expanding by Clonetics? REGM? Renal Epithelial Cell Growth Medium expressed the positive markers of urine-derived stem cells, CD29, CD73 and CD146, and did not express the negative markers of urine-derived stem cells, CD34 and CD45. Among them, expression rate of CD29 and CD73 were more than 95%, CD34 and CD45 were less than 3%. After adipogenic differentiation, the cells contained a large number of lipid droplets which can be watched by oil red O staining. After osteogenic differentiation, under the ordinary optical microscope, we saw calcified nodules, and after alizarin red staining, the calcified nodules turned into red. Moreover, after differentiated into smooth muscle cells, we compare the expression of specific markers of smooth muscle cells, SM-22α, SMMHC, SM-α-actin and calopnin, between before and after induction by quantitative PCR. The results showed that the expression of SM-22α、SM-α-actin and calopnin were significantly higher after induction, and the difference had the statistically significance, P <0.05. After 24 hours of electroporated with p CXLE-EGFP, the cells expressed the fluorescence, and the expression of fluorescence decreased gradually as time goes by. Finally, without feeder, we got 37 clones from 4.8×105 urine-derived stem cells. The results of pluripotent identification were as follow: alkaline phosphatase staining, immunofluorescence(OCT4, SOX2, NANOG and SSEA4) and flow cytometry(SSEA4 and TRA-1-60) were identified as positive. After digestion, the i PSCs mass which cultured suspended became spherical by themselves. The sizes of the embryoid bodies became bigger, and the color deeper as time goes by. When the embryoid bodies attached to tissue culture and grew, different types of cells appeared. Compared to the undifferentiated human i PSCs, the expression of SOX17, endoderm markers, MSX1, mesoderm marker, and MAP2, ectoderm markers, have significantly increased(P <0.05) by quantitative PCR. The teratoma experiment confirmed that the cells had the ability to differentiate into all three germ layers of different cells in vivo, such as glands(endoderm), muscle(mesoderm), neural tube(ectoderm). After differentiated into neural crest cell, the cell morphology changed. The morphology were almost triangular or polygonal, and had many thin and long projections which interacted to form a mesh. Compared to the undifferentiated human i PSCs, the expression of specific markers of neural crest cells, AP2, p75,HNK1 and Fox D3, have significantly increased by quantitative PCR, and further increased as time goes by, and the difference had statistically significance, P <0.05.Conclusion The urine cells which collected and expanding by Clonetics? REGM? Renal Epithelial Cell Growth Medium, not only expressed the mark of USCs, but also possessed the differentiation potential in vitro which is similar to that USCs. Namely, the urine cells were USCs. Moreover, the i PSCs can be reprogrammed from the urine cells by electroporation with episomal plasmids under feeder-free, virus-free condition, and then differentiate into neural crest cells. Sequentially, we established a practical protocol for neural crest cells induction from induced pluripotent stem cells which reprogrammed from urine-derived stem cells, and provided a complete as well as feasible method for future research on the diseases about neural crest and the cells which differentiated from neural crest, such as peripheral nervous system diseases, carotid artery disease.