Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells

Nature Protocols volume 8, pages 1670–1679 (2013)Cite this article

Subjects

Abstract

Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Timeline of forebrain GABA interneuron generation.
Figure 2: Step-by-step illustration of neuroepithelial differentiation.
Figure 3: Characteristics of MGE progenitors and forebrain GABA interneurons.
Figure 4: Human PSC-derived forebrain GABAergic interneurons are functional in vitro.
Figure 5: Maturation of GABA interneuron subtypes.
Figure 6: Pasteur pipette technique for triturating MGE-like neurospheres.

Similar content being viewed by others

References

  1. Le Magueresse, C. & Monyer, H. GABAergic interneurons shape the functional maturation of the cortex. Neuron 77, 388–405 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Goulburn, A.L., Stanley, E.G., Elefanty, A.G. & Anderson, S.A. Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells. Stem Cell Res. 8, 416–426 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Nobrega-Pereira, S. et al. Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors. Neuron 59, 733–745 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Fishell, G. & Rudy, B. Mechanisms of inhibition within the telencephalon: “where the wild things are”. Annu. Rev. Neurosci. 34, 535–567 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kriegstein, A.R. & Noctor, S.C. Patterns of neuronal migration in the embryonic cortex. Trends Neurosci. 27, 392–399 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Marin, O. & Rubenstein, J.L. Cell migration in the forebrain. Annu. Rev. Neurosci. 26, 441–483 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Letinic, K., Zoncu, R. & Rakic, P. Origin of GABAergic neurons in the human neocortex. Nature 417, 645–649 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Jakovcevski, I., Mayer, N. & Zecevic, N. Multiple origins of human neocortical interneurons are supported by distinct expression of transcription factors. Cereb. Cortex 21, 1771–1782 (2011).

    Article  PubMed  Google Scholar 

  9. Sussel, L., Marin, O., Kimura, S. & Rubenstein, J.L. Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126, 3359–3370 (1999).

    CAS  PubMed  Google Scholar 

  10. Danjo, T. et al. Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals. J. Neurosci. 31, 1919–1933 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Campbell, K. Dorsal-ventral patterning in the mammalian telencephalon. Curr. Opin. Neurobiol. 13, 50–56 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Olsson, M., Campbell, K., Wictorin, K. & Bjorklund, A. Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence. Neuroscience 69, 1169–1182 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Ma, L. et al. Human embryonic stem cell–derived GABA neurons correct locomotion deficits in quinolinic acid–lesioned mice. Cell Stem Cell 10, 455–464 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhao, Y. et al. The LIM-homeobox gene Lhx8 is required for the development of many cholinergic neurons in the mouse forebrain. Proc. Natl. Acad. Sci. USA 100, 9005–9010 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Flandin, P. et al. Lhx6 and Lhx8 coordinately induce neuronal expression of Shh that controls the generation of interneuron progenitors. Neuron 70, 939–950 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Manabe, T. et al. L3/Lhx8 is involved in the determination of cholinergic or GABAergic cell fate. J. Neurochem. 94, 723–730 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Maroof, A.M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nicholas, C.R. et al. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 12, 573–586 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu, Y. et al. Medial ganglionic eminence-like cells derived from human embryonic stem cells correct learning and memory deficits. Nat. Biotechnol. 31, 440–447 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Li, X.J. et al. Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells 26, 886–893 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hu, B.Y. & Zhang, S.C. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat. Protoc. 4, 1295–1304 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li, Y. et al. Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons. J. Neurosci. 15, 2888–2905 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pankratz, M.T. et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells 25, 1511–1520 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cooper, O. et al. Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid. Mol. Cell Neurosci. 45, 258–266 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. LaVaute, T.M. et al. Regulation of neural specification from human embryonic stem cells by BMP and FGF. Stem Cells 27, 1741–1749 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Reilly, J.O., Karavanova, I.D., Williams, K.P., Mahanthappa, N.K. & Allendoerfer, K.L. Cooperative effects of sonic hedgehog and NGF on basal forebrain cholinergic neurons. Mol. Cell Neurosci. 19, 88–96 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Krencik, R. & Zhang, S.C. Directed differentiation of functional astroglial subtypes from human pluripotent stem cells. Nat. Protoc. 6, 1710–1717 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ogura, A., Morizane, A., Nakajima, Y., Miyamoto, S. & Takahashi, J. Gamma-secretase inhibitors prevent overgrowth of transplanted neural progenitors derived from human-induced pluripotent stem cells. Stem Cells Dev. 22, 374–382 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported in part by the US National Institutes of Health (NS045926, MH099587, NS076352), the Busta Family Foundation, the Bleser Family Foundation and the US National Institute of Child Health and Human Development (P30 HD03352).

Author information

Authors and Affiliations

  1. Waisman Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA

    Yan Liu, Huisheng Liu, Conall Sauvey, Lin Yao & Su-Chun Zhang

  2. Department of Anesthesiology, University of Wisconsin, Madison, Wisconsin, USA

    Ewa D Zarnowska

  3. Neuroscience Training Program, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA

    Su-Chun Zhang

  4. Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA

    Su-Chun Zhang

  5. Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA

    Su-Chun Zhang

Authors
  1. Yan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huisheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Conall Sauvey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lin Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ewa D Zarnowska
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Su-Chun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the design of the experiments, analysis of data and writing of this paper.

Corresponding author

Correspondence to Su-Chun Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Y., Liu, H., Sauvey, C. et al. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells. Nat Protoc 8, 1670–1679 (2013). https://doi.org/10.1038/nprot.2013.106

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.106

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing