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


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.

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


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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.

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

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Correspondence to Valentina Fossati.

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The authors declare no competing financial interests.

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Douvaras, P., Fossati, V. Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Nat Protoc 10, 1143–1154 (2015).

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