Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells

Abstract

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.

Figure 1. Optimization of a method to generate non-integrated iPS cells from UCs.

A. UCs from healthy (UC-012) and diseased donors (listed in Table 1 and Table 2). B. Left: EdU imaging of representative UC. Right: EdU positive percentages of 5 UCs. Error bars are standard deviation of the mean, n = 3. C. Phase contrast and fluorescent photographs of UC-012 and UC-015 electroporation with episomal plasmid pCEP4-EGFP and cultured for 24 h in UC medium. D. Growth curves of UC-012 and UC-015 in UC medium, defined medium E8 and mTeSR1, respectively. *** indicates P<0.001. E. Schematic representation of iPS cell generation. The defined medium can be either mTeSR1 or E8. F. Different reprogramming factor combinations for iPS cell generation of UCs and skin fibroblasts from the same donor. −: failure, +: success. G. Representative phase contrast photographs of a successful iPS cell generation process: (1) Emerging iPS cell colony generating by our optimized method in E8 from UC-012 at different time points. (2)A picked colony. (3) The established iPS cell line from this colony. P8: passage 8. H. Top: AP staining of iPS cells generated from UC-012, UC-015 and UC-041 in 6 well plates. I. Bottom: reprogramming efficiencies of UC-012, UC-015 and UC-041. n = 3.

Figure 2. Characterization of a typical non-intergrated iPS cell line generated from UC-012.

A. Non-integrating analysis of eipsomal DNA in the iPS cells. −: negative control. +: positive control, UCs transfected with indicated episomal vectors. UC-012: urine-derived cells 12. UiPSC-012 C2P19 and UiPSC-012 C6P22: iPS cell lines, C: colony number, P: passage. B. G-band analysis of the iPS cells derived from UC-012 shows normal karyotype. C. qPCR for endogenous human ES cell specific transcription factors of UC-012 and two derivative iPS cell lines. UC-015 and UC-041 and derivative iPS cell lines were also shown. Values were referred to 10⁶ copies of ACTIN. Human ES cell line H1 was used as control. n = 3. P value is referred to UCs. ** indicates P<0.01. D. Expression of human ES markers OCT4 and SSEA4 of UiPSC-012 C2P19 by flow cytometry. E. Immunofluorescence for human ES markers of UiPSC-012 C2P18. F. Methylation status of OCT4 and NANOG promoters in UC-012 and UiPSC-012 C2P19. G. Top: EB formed from UiPSC-012 C2P18. Bottom: qPCR analysis of human ES cell markers and markers for the three germ layers relative to iPSCs. n = 3. * indicates P<0.05. H. HE-staining of the teratomas from UiPSC-012 C2P17.