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Generation of integration-free neural progenitor cells from cells in human urine

Nature Methods volume 10, pages 84–89 (2013)Cite this article

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Abstract

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.

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Figure 1: Generation and expansion of integration-free NPCs from human urine cells.
Figure 2: Global gene expression analysis of hUiNPCs.
Figure 3: Differentiation of hUiNPCs in vitro.
Figure 4: hUiNPC-derived neurons are functional in vitro.
Figure 5: In vivo transplant of hUiNPCs.

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References

  1. Aboody, K., Capela, A., Niazi, N., Stern, J.H. & Temple, S. Translating stem cell studies to the clinic for CNS repair: current state of the art and the need for a Rosetta stone. Neuron 70, 597–613 (2011).

    CAS  PubMed  Google Scholar 

  2. Breunig, J.J., Haydar, T.F. & Rakic, P. Neural stem cells: historical perspective and future prospects. Neuron 70, 614–625 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    CAS  Google Scholar 

  4. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  PubMed  Google Scholar 

  5. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  PubMed  Google Scholar 

  6. Hu, B.Y. et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl. Acad. Sci. USA 107, 4335–4340 (2010).

    CAS  PubMed  Google Scholar 

  7. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Israel, M.A. et al. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature 482, 216–220 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Yoo, A.S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Caiazzo, M. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227 (2011).

    CAS  PubMed  Google Scholar 

  13. Ambasudhan, R. et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9, 113–118 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Son, E.Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Qiang, L. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell 146, 359–371 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang, P. et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475, 386–389 (2011).

    CAS  PubMed  Google Scholar 

  17. Sekiya, S. & Suzuki, A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 475, 390–393 (2011).

    CAS  PubMed  Google Scholar 

  18. Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D.A. In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 455, 627–632 (2008).

    CAS  PubMed  Google Scholar 

  19. Zhou, T. et al. Generation of induced pluripotent stem cells from urine. J. Am. Soc. Nephrol. 22, 1221–1228 (2011).

    PubMed  PubMed Central  Google Scholar 

  20. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Liao, B. et al. MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J. Biol. Chem. 286, 17359–17364 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).

    CAS  PubMed  Google Scholar 

  23. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Shi, Y. et al. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2, 525–528 (2008).

    CAS  PubMed  Google Scholar 

  25. Lluis, F. et al. Periodic activation of Wnt/β-catenin signaling enhances somatic cell reprogramming mediated by cell fusion. Cell Stem Cell 3, 493–507 (2008).

    CAS  PubMed  Google Scholar 

  26. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    CAS  PubMed  Google Scholar 

  27. Burdon, T., Stracey, C., Chambers, I., Nichols, J. & Smith, A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev. Biol. 210, 30–43 (1999).

    CAS  PubMed  Google Scholar 

  28. Yu, J., Chau, K.F., Vodyanik, M.A., Jiang, J. & Jiang, Y. Efficient feeder-free episomal reprogramming with small molecules. PLoS ONE 6, e17557 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hao, J. et al. In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors. ACS Chem. Biol. 5, 245–253 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl. Acad. Sci. USA 108, 7838–7843 (2011).

    CAS  PubMed  Google Scholar 

  31. Reynolds, B.A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).

    CAS  PubMed  Google Scholar 

  32. Zhang, X. et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90–100 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu, H. & Zhang, S.C. Specification of neuronal and glial subtypes from human pluripotent stem cells. Cell. Mol. Life Sci. 68, 3995–4008 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hu, B.Y., Du, Z.W., Li, X.J., Ayala, M. & Zhang, S.C. Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Thier, M. et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479 (2012).

    CAS  PubMed  Google Scholar 

  36. Ring, K.L. et al. Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor. Cell Stem Cell 11, 100–109 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Han, D.W. et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10, 465–472 (2012).

    CAS  PubMed  Google Scholar 

  38. Sheng, C. et al. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors. Cell Res. 22, 208–218 (2012).

    CAS  PubMed  Google Scholar 

  39. Buganim, Y. et al. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150, 1209–1222 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Pan, G. & Pei, D. Order from chaos: single cell reprogramming in two phases. Cell Stem Cell 11, 445–447 (2012).

    CAS  PubMed  Google Scholar 

  41. Esteban, M.A. et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6, 71–79 (2010).

    CAS  PubMed  Google Scholar 

  42. Esteban, M.A. et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J. Biol. Chem. 284, 17634–17640 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Esterban for helpful suggestions, Z. Li for providing support in the initial phase of this work and members of our labs for their kind help. This work is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDA01020202 and XDA01020401); National Basic Research Program of China, 973 Program of China (2012CB966503 and 2012CB966802); National S&T Major Special Project on Major New Drug Innovation (2011ZX09102-010); and National Natural Science Foundation of China (31200970 and 91213304). D.P. and G.P. are supported by the 100 Talents Project of Chinese Academy of Sciences, China.

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  1. Huanxing Su

    Present address: Present address: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China.,

Authors and Affiliations

  1. Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Lihui Wang, Linli Wang, Wenhao Huang, Huanxing Su, Yanting Xue, Zhenghui Su, Baojian Liao, Haitao Wang, Xichen Bao, Dajiang Qin, Guangjin Pan & Duanqing Pei

  2. Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Lihui Wang, Linli Wang, Wenhao Huang, Huanxing Su, Yanting Xue, Zhenghui Su, Baojian Liao, Haitao Wang, Xichen Bao, Dajiang Qin, Guangjin Pan & Duanqing Pei

  3. Department of Pathology, Dalian Medical University, Dalian, China

    Lihui Wang

  4. School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.,

    Yanting Xue & Zhenghui Su

  5. Department of Rehabilitation Science, Hong Kong Polytechnic University, Hong Kong, China

    Jufang He

  6. Department of Anatomy, Hong Kong University, Hong Kong,, China

    Wutian Wu & Kwok Fai So

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  1. Lihui Wang
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Contributions

G.P., Lihui, W. and D.P. conceived hypotheses and designed the experiments. Lihui, W. and W.H. performed the experiments and generated data in all figures. In addition, Linli, W., D.Q., Y.X. and Z.S. performed experiments for Figure 1 and Supplementary Figure 2; H.S., W.W. and K.F.S. participated in experiments and analysis for Figure 5; X.B. provided reagents and experimental assistance for miR302–367; B.L. performed the experiments for Figure 1; and H.W. and J.H. performed the experiments for Figure 4. G.P. and D.P. wrote the paper.

Corresponding authors

Correspondence to Guangjin Pan or Duanqing Pei.

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Supplementary Figures 1–3 and Supplementary Tables 1–4 (PDF 8827 kb)

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Wang, L., Wang, L., Huang, W. et al. Generation of integration-free neural progenitor cells from cells in human urine. Nat Methods 10, 84–89 (2013). https://doi.org/10.1038/nmeth.2283

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