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.

Directed differentiation of functional astroglial subtypes from human pluripotent stem cells

Abstract

Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are useful tools for understanding brain function, modulating disease processes and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated into neuroepithelial cells with or without exogenous patterning molecules (days 0–21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over a long-term expansion (days 21–90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated into functional astrocytes on removal of mitogens and the addition of ciliary neurotrophic factor (days >90). This method generates robust populations of functionally diversified astrocytes with high efficiency.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Timeline of astrocyte generation.
Figure 2: Stages of astroglial differentiation.
Figure 3: Confirmation of astroglial differentiation with astrocyte markers.

References

  1. Matyash, V. & Kettenmann, H. Heterogeneity in astrocyte morphology and physiology. Brain Res. Rev. 63, 2–10 (2010).

    Article  CAS  Google Scholar 

  2. Zhang, Y. & Barres, B.A. Astrocyte heterogeneity: an underappreciated topic in neurobiology. Curr. Opin. Neurobiol. 20, 588–594 (2010).

    Article  CAS  Google Scholar 

  3. Zhang, S.C. Defining glial cells during CNS development. Nat. Rev. Neurosci. 2, 840–843 (2001).

    Article  CAS  Google Scholar 

  4. Rao, M.S. & Mayer-Proschel, M. Glial-restricted precursors are derived from multipotent neuroepithelial stem cells. Dev. Biol. 188, 48–63 (1997).

    Article  CAS  Google Scholar 

  5. Carpenter, M.K. et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol. 172, 383–397 (2001).

    Article  CAS  Google Scholar 

  6. Liu, Y. et al. CD44 expression identifies astrocyte-restricted precursor cells. Dev. Biol. 276, 31–46 (2004).

    Article  CAS  Google Scholar 

  7. Morrison, R.S. & de Vellis, J. Growth of purified astrocytes in a chemically defined medium. Proc. Natl. Acad. Sci. USA 78, 7205–7209 (1981).

    Article  CAS  Google Scholar 

  8. Mi, H. & Barres, B.A. Purification and characterization of astrocyte precursor cells in the developing rat optic nerve. J. Neurosci. 19, 1049–1061 (1999).

    Article  CAS  Google Scholar 

  9. Kim, H.J., McMillan, E., Han, F. & Svendsen, C.N. Regionally specified human neural progenitor cells derived from the mesencephalon and forebrain undergo increased neurogenesis following overexpression of ASCL1. Stem Cells 27, 390–398 (2009).

    Article  CAS  Google Scholar 

  10. 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  Google Scholar 

  11. Reubinoff, B.E. et al. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19, 1134–1140 (2001).

    Article  CAS  Google Scholar 

  12. Itsykson, P. et al. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol. Cell Neurosci. 30, 24–36 (2005).

    Article  CAS  Google Scholar 

  13. Tabar, V. et al. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat. Biotechnol. 23, 601–606 (2005).

    Article  CAS  Google Scholar 

  14. Johnson, M.A., Weick, J.P., Pearce, R.A. & Zhang, S.C. Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J. Neurosci. 27, 3069–3077 (2007).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. Ruiz, S. et al. High-efficient generation of induced pluripotent stem cells from human astrocytes. PLoS One 5, e15526 (2010).

    Article  Google Scholar 

  17. Krencik, R., Weick, J.P., Liu, Y., Zhang, Z.J. & Zhang, S.C. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat. Biotechnol. 29, 528–534 (2011).

    Article  CAS  Google Scholar 

  18. Yuan, S.H. et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6, e17540 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Hu, B.Y., Du, Z.W. & Zhang, S.C. Differentiation of human oligodendrocytes from pluripotent stem cells. Nat. Protoc. 4, 1614–1622 (2009).

    Article  CAS  Google Scholar 

  21. Xia, X. & Zhang, S.C. Differentiation of neuroepithelia from human embryonic stem cells. Methods Mol. Biol. 549, 51–58 (2009).

    Article  CAS  Google Scholar 

  22. Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).

    Article  CAS  Google Scholar 

  23. Chojnacki, A. & Weiss, S. Production of neurons, astrocytes and oligodendrocytes from mammalian CNS stem cells. Nat. Protoc. 3, 935–940 (2008).

    Article  CAS  Google Scholar 

  24. Caldwell, M.A. et al. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat. Biotechnol. 19, 475–479 (2001).

    Article  CAS  Google Scholar 

  25. Li, X.J. et al. Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells. Development 136, 4055–4063 (2009).

    Article  CAS  Google Scholar 

  26. Rajan, P. & McKay, R.D. Multiple routes to astrocytic differentiation in the CNS. J. Neurosci. 18, 3620–3629 (1998).

    Article  CAS  Google Scholar 

  27. Davies, J.E. et al. Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury. J. Biol. 7, 24 (2008).

    Article  Google Scholar 

  28. Wilkinson, M., Hume, R., Strange, R. & Bell, J.E. Glial and neuronal differentiation in the human fetal brain 9–23 weeks of gestation. Neuropathol. Appl. Neurobiol. 16, 193–204 (1990).

    Article  CAS  Google Scholar 

  29. Pal, U., Chaudhury, S. & Sarkar, P.K. Tubulin and glial fibrillary acidic protein gene expression in developing fetal human brain at midgestation. Neurochem. Res. 24, 637–641 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the ALS Association, the National Institute of Neurological Disorders and Stroke (NS045926, NS057778, NS064578), the National Multiple Sclerosis Society (NMSS TR-3761), NYSTEM (C024406), the Bleser Family Foundation, the Busta Family Foundation, the Neuroscience Training Program (T32 GM007507) and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352).

Author information

Authors and Affiliations

Authors

Contributions

R.K. and S.-C.Z. designed the protocol and wrote the manuscript. R.K. collected the data for figures.

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

Krencik, R., Zhang, SC. Directed differentiation of functional astroglial subtypes from human pluripotent stem cells. Nat Protoc 6, 1710–1717 (2011). https://doi.org/10.1038/nprot.2011.405

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.405

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

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