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Directed differentiation of functional astroglial subtypes from human pluripotent stem cells

Nature Protocols volume 6, pages 1710–1717 (2011)Cite this article

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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.

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

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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).

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Authors and Affiliations

  1. Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Robert Krencik & Su-Chun Zhang

  2. Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Robert Krencik & Su-Chun Zhang

  3. Department of Neuroscience, University of Wisconsin-Madison, Madison, Wisconsin, USA

    Su-Chun Zhang

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

    Su-Chun Zhang

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  1. Robert Krencik
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Contributions

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

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Correspondence to Su-Chun Zhang.

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The authors declare no competing financial interests.

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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

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