Skip to main content

Advertisement

SpringerLink
Log in

Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAMpos and Tra-1-60neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Schwartz, P. H., Brick, D. J., Stover, A. E., Loring, J. F., & Muller, F. J. (2008). Differentiation of neural lineage cells from human pluripotent stem cells. Methods, 45(2), 142–158.

    Article  PubMed  CAS  Google Scholar 

  2. Falk, A., Koch, P., Kesavan, J., et al. (2012). Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PloS One, 7(1), e29597.

    Article  PubMed  CAS  Google Scholar 

  3. Nemati, S., Hatami, M., Kiani, S., et al. (2011). Long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors. Stem Cells and Development, 20(3), 503–514.

    Article  PubMed  CAS  Google Scholar 

  4. Nistor, G., Siegenthaler, M. M., Poirier, S. N., et al. (2011). Derivation of high purity neuronal progenitors from human embryonic stem cells. PloS One, 6(6), e20692.

    Article  PubMed  CAS  Google Scholar 

  5. Cohen, M. A., Itsykson, P., & Reubinoff, B. E. (2007). Neural differentiation of human ES cells. Curr Protoc Cell Biol. Chapter 23, Unit 23 27.

  6. Iacovitti, L., Donaldson, A. E., Marshall, C. E., Suon, S., & Yang, M. (2007). A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo. Brain Research, 1127(1), 19–25.

    Article  PubMed  CAS  Google Scholar 

  7. Koch, P., Opitz, T., Steinbeck, J. A., Ladewig, J., & Brustle, O. (2009). A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proceedings of the National Academy of Sciences, USA, 106(9), 3225–3230.

    Article  CAS  Google Scholar 

  8. Kumar, M., Bagchi, B., Gupta, S. K., Meena, A. S., Gressens, P., & Mani, S. (2007). Neurospheres derived from human embryoid bodies treated with retinoic Acid show an increase in nestin and ngn2 expression that correlates with the proportion of tyrosine hydroxylase-positive cells. Stem Cells and Development, 16(4), 667–681.

    Article  PubMed  CAS  Google Scholar 

  9. Lim, U. M., Sidhu, K. S., & Tuch, B. E. (2006). Derivation of motor neurons from three clonal human embryonic stem cell lines. Current Neurovascular Research, 3(4), 281–288.

    Article  PubMed  Google Scholar 

  10. Ma, W., Tavakoli, T., Derby, E., Serebryakova, Y., Rao, M. S., & Mattson, M. P. (2008). Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Developmental Biology, 8, 90.

    Article  PubMed  Google Scholar 

  11. Schuldiner, M., Eiges, R., Eden, A., et al. (2001). Induced neuronal differentiation of human embryonic stem cells. Brain Research, 913(2), 201–205.

    Article  PubMed  CAS  Google Scholar 

  12. Schulz, T. C., Palmarini, G. M., Noggle, S. A., Weiler, D. A., Mitalipova, M. M., & Condie, B. G. (2003). Directed neuronal differentiation of human embryonic stem cells. BMC Neuroscience, 4, 27.

    Article  PubMed  Google Scholar 

  13. Swistowski, A., Peng, J., Liu, Q., et al. (2010). Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells, 28(10), 1893–1904.

    Article  PubMed  CAS  Google Scholar 

  14. Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., & Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotechnology, 19(12), 1129–1133.

    Article  PubMed  CAS  Google Scholar 

  15. Bauwens, C. L., Peerani, R., Niebruegge, S., et al. (2008). Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells, 26(9), 2300–2310.

    Article  PubMed  Google Scholar 

  16. Sachlos, E., & Auguste, D. T. (2008). Embryoid body morphology influences diffusive transport of inductive biochemicals: a strategy for stem cell differentiation. Biomaterials, 29(34), 4471–4480.

    Article  PubMed  CAS  Google Scholar 

  17. Axell, M. Z., Zlateva, S., & Curtis, M. (2009). A method for rapid derivation and propagation of neural progenitors from human embryonic stem cells. Journal of Neuroscience Methods, 184(2), 275–284.

    Article  PubMed  Google Scholar 

  18. Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27(3), 275–280.

    Article  PubMed  CAS  Google Scholar 

  19. Erceg, S., Lainez, S., Ronaghi, M., et al. (2008). Differentiation of human embryonic stem cells to regional specific neural precursors in chemically defined medium conditions. PloS One, 3(5), e2122.

    Article  PubMed  Google Scholar 

  20. Gerrard, L., Rodgers, L., & Cui, W. (2005). Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells, 23(9), 1234–1241.

    Article  PubMed  CAS  Google Scholar 

  21. Joannides, A. J., Fiore-Heriche, C., Battersby, A. A., et al. (2007). A scaleable and defined system for generating neural stem cells from human embryonic stem cells. Stem Cells, 25(3), 731–737.

    Article  PubMed  CAS  Google Scholar 

  22. Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., et al. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell, 3(5), 519–532.

    Article  PubMed  CAS  Google Scholar 

  23. Lee, S. H., Lumelsky, N., Studer, L., Auerbach, J. M., & McKay, R. D. (2000). Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nature Biotechnology, 18(6), 675–679.

    Article  PubMed  CAS  Google Scholar 

  24. Watanabe, K., Ueno, M., Kamiya, D., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25(6), 681–686.

    Article  PubMed  CAS  Google Scholar 

  25. Okabe, S., Forsberg-Nilsson, K., Spiro, A. C., Segal, M., & McKay, R. D. (1996). Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mechanisms of Development, 59(1), 89–102.

    Article  PubMed  CAS  Google Scholar 

  26. Itsykson, P., Ilouz, N., Turetsky, T., et al. (2005). Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Molecular and Cellular Neuroscience, 30(1), 24–36.

    Article  PubMed  CAS  Google Scholar 

  27. Bianco, P., Kuznetsov, S. A., Riminucci, M., & Gehron Robey, P. (2006). Postnatal skeletal stem cells. Methods in Enzymology, 419, 117–148.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  29. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–676.

    Article  PubMed  CAS  Google Scholar 

  30. He, J. Q., Ma, Y., Lee, Y., Thomson, J. A., & Kamp, T. J. (2003). Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circulation Research, 93(1), 32–39.

    Article  PubMed  CAS  Google Scholar 

  31. Si-Tayeb, K., Noto, F. K., Nagaoka, M., et al. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology, 51(1), 297–305.

    Article  PubMed  CAS  Google Scholar 

  32. Mallon, B. S., Park, K. Y., Chen, K. G., Hamilton, R. S., & McKay, R. D. (2006). Toward xeno-free culture of human embryonic stem cells. The International Journal of Biochemistry & Cell Biology, 38(7), 1063–1075.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Dr. Sergei Kuznetsov and Dr. Pamela Robey of the National Institute for Dental and Craniofacial Research for providing the bone marrow stromal cells from which the iPSC line was derived. We would also like to thank Dr. Ron McKay and Dr. Josh Chenoweth of the Lieber Institute for Brain Development for helpful discussions. This research was supported by the Intramural Research Program of the NIH, NINDS.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Biology, Division of Intramural Research, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA

    O. A. Kozhich

  2. NIH Stem Cell Unit, Division of Intramural Research, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA

    R. S. Hamilton & B. S. Mallon

Authors
  1. O. A. Kozhich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. S. Hamilton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. S. Mallon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. S. Mallon.

Electronic Supplementary Materials

Below is the link to the electronic supplementary material.

Supplementary Figure 1

Thawed neural precursor cells. A&B) H1-derived NPCs p10, 0.5 well frozen and thawed in the presence of 10 μM Y27632 - A) 4 h post thaw; B) 24 h post thaw; C&D) SCU-i10-derived NPCs p5, 1 well frozen and thawed in the presence of 10 μM Y27632 - C) 4 h post thaw; D) 24 h post thaw. Scale bars = 100 μm. (PPT 3105 kb)

Supplementary Figure 2

Immunostaining of SCU-i10 NPCs differentiated for 11 days with antibodies to MAP2 (top row; red) and TuJ1 (second row; green). Merged images of both immunostains with Hoechst nuclear stain (third row; blue) are shown in the bottom row. Scale bars = 100 μm. Three differentiation media were compared - standard Neurobasal medium containing BDNF and GDNF (left column), mTeSR1 (center column) and NDM (right column). (PPT 3859 kb)

Supplementary Figure 3

Characterization of the SCU-i10 human iPSC line. A) FACS analysis shows SCU-i10s are positive for SSEA-4, Tra-1-60 and Tra-1-81 and negative for SSEA-1; B) Karyotype is normal at p40 (performed by Cell Line Genetics, Madison, WI); C) Immunostaining of cells differentiated to endodermal lineage with antibodies to hepatocyte markers HNF4A (red) and albumin (green) with Hoechst nuclear stain (blue). Scale bar = 100 μm; D) Image captured from Supplementary Movie 1 which shows spontaneously beating area of differentiated cells. (PPT 1022 kb)

(MOV 15341 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kozhich, O.A., Hamilton, R.S. & Mallon, B.S. Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells. Stem Cell Rev and Rep 9, 531–536 (2013). https://doi.org/10.1007/s12015-012-9357-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-012-9357-8

Keywords

Search

Navigation