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.

Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells

Nature Protocols volume 10, pages 1143–1154 (2015)Cite this article

Subjects

Abstract

In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor–rich medium. Within 8 d, PSCs differentiate into paired box 6–positive (PAX6+) neural stem cells, which give rise to OLIG2+ progenitors by day 12. Oligodendrocyte lineage transcription factor 2–positive (OLIG2+) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4+) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4+ OPCs can be isolated by cell sorting for myelination studies, or they can be terminally differentiated to myelin basic protein–positive (MBP+) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4+ cells after only 55 d of culture.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Timeline of oligodendrocyte differentiation.
Figure 2: Fundamental steps of hPSC differentiation to oligodendrocytes.
Figure 3: Live imaging and quantification of O4+ OPCs.

References

  1. Kassmann, C.M. et al. Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes. Nat. Genet. 39, 969–976 (2007).

    Article  CAS  Google Scholar 

  2. Bartzokis, G. Brain myelination in prevalent neuropsychiatric developmental disorders: primary and comorbid addiction. Adolesc. Psychiatry 29, 55–96 (2005).

    PubMed  PubMed Central  Google Scholar 

  3. Irion, S., Nostro, M.C., Kattman, S.J. & Keller, G.M. Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb. Symp. Quant. Biol. 73, 101–110 (2008).

    Article  CAS  Google Scholar 

  4. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).

    Article  CAS  Google Scholar 

  5. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

    Article  CAS  Google Scholar 

  6. Nistor, G.I., Totoiu, M.O., Haque, N., Carpenter, M.K. & Keirstead, H.S. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49, 385–396 (2005).

    Article  Google Scholar 

  7. Izrael, M. et al. Human oligodendrocytes derived from embryonic stem cells: effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol. Cell. Neurosci. 34, 310–323 (2007).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. 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 

  10. Numasawa-Kuroiwa, Y. et al. Involvement of ER stress in dysmyelination of Pelizaeus-Merzbacher disease with PLP1 missense mutations shown by iPSC-derived oligodendrocytes. Stem Cell Rep. 2, 648–661 (2014).

    Article  CAS  Google Scholar 

  11. Stacpoole, S.R. et al. High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology. Stem Cell Rep. 1, 437–450 (2013).

    Article  CAS  Google Scholar 

  12. Wang, S. et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252–264 (2013).

    Article  CAS  Google Scholar 

  13. Liu, Y., Jiang, P. & Deng, W. OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation. Nat. Protoc. 6, 640–655 (2011).

    Article  CAS  Google Scholar 

  14. Sim, F.J. et al. CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells. Nat. Biotechnol. 29, 934–941 (2011).

    Article  CAS  Google Scholar 

  15. Boulting, G.L. et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011).

    Article  CAS  Google Scholar 

  16. Douvaras, P. et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Rep. 3, 250–259 (2014).

    Article  CAS  Google Scholar 

  17. Kang, J. et al. Granulocyte colony-stimulating factor minimizes negative remodeling of decellularized small diameter vascular graft conduits but not medial degeneration. Ann. Vasc. Surg. 27, 487–496 (2013).

    Article  Google Scholar 

  18. Fennema-Notestine, C. et al. In vivo evidence of cerebellar atrophy and cerebral white matter loss in Huntington disease. Neurology 63, 989–995 (2004).

    Article  CAS  Google Scholar 

  19. Behrendt, G. et al. Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men. Glia 61, 273–286 (2013).

    Article  Google Scholar 

  20. Bernstein, H.G., Steiner, J., Guest, P.C., Dobrowolny, H. & Bogerts, B. Glial cells as key players in schizophrenia pathology: recent insights and concepts of therapy. Schizophr. Res. 161, 4–18 (2015).

    Article  Google Scholar 

  21. 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  Google Scholar 

  22. Patani, R. et al. Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state. Nat. Commun. 2, 214 (2011).

    Article  CAS  Google Scholar 

  23. Miller, R.H. et al. Patterning of spinal cord oligodendrocyte development by dorsally derived BMP4. J. Neurosci. Res. 76, 9–19 (2004).

    Article  CAS  Google Scholar 

  24. Tomassy, G.S. & Fossati, V. How big is the myelinating orchestra? Cellular diversity within the oligodendrocyte lineage: facts and hypotheses. Front. Cell. Neurosci. 8, 201 (2014).

    Article  Google Scholar 

Download references

Acknowledgements

We thank M. Zimmer for excellent assistance with cell sorting and J. Goldman (Columbia University) for providing the O4 antibody. We are thankful to D. Paul and B. Corneo for their insightful comments. This work was supported by a New York Stem Cell Foundation (NYSCF)-Helmsley Early Career Investigator Award, The NYCSF, and The Leona M. and Harry B. Helmsley Charitable Trust. P.D. is a NYSCF-Druckenmiller postdoctoral fellow.

Author information

Authors and Affiliations

  1. The New York Stem Cell Foundation Research Institute, New York, New York, USA.,

    Panagiotis Douvaras & Valentina Fossati

Authors
  1. Panagiotis Douvaras
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Valentina Fossati
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.D. performed the experiments. P.D. and V.F. designed the protocol, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Valentina Fossati.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Douvaras, P., Fossati, V. Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Nat Protoc 10, 1143–1154 (2015). https://doi.org/10.1038/nprot.2015.075

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.075

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