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

Journal name:
Nature Protocols
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Published online


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.

At a glance


  1. Timeline of oligodendrocyte differentiation.
    Figure 1: Timeline of oligodendrocyte differentiation.

    (a,b) Timelines show the differences in following the original protocol (Step 32A, a) and the fast protocol (Step 32B, b). Adapted with permission from ref. 16. Colored triangles represent the recommended time points to evaluate the expression of stage-specific markers through immunofluorescence. SB, SB431542; LDN, LDN193189; SAG, smoothened agonist; T3, thriiodothytonine; RA, all-trans retinoic acid; PDGF, platelet-derived growth factor; HGF, hepatocyte growth factor; IGF-I, insulin-like growth factor-1; NT3, neurotrophin 3; AA, ascorbic acid.

  2. Fundamental steps of hPSC differentiation to oligodendrocytes.
    Figure 2: Fundamental steps of hPSC differentiation to oligodendrocytes.

    (a) PAX6+ neural stem cells at day 8 of differentiation (PAX6, green; nuclei are stained with DAPI, blue). (b) Typical morphology of the culture at day 12, depicting 3D structures. (c) Expression of OLIG2 and NKX2.2 at day 12, as shown through immunofluorescence analysis (OLIG2, green; NKX2.2, red; nuclei are stained with DAPI, blue). (d) Sphere selection at Step 30. Arrows indicate the good spheres, which are round-shaped, golden or brown in color, with a darker core and with a diameter between 300 and 800 μm. The exclamation mark (!) indicates a pair of joined spheres that can be broken into single spheres by gentle pipetting. Aggregates that should be avoided are small and transparent (arrowheads) or very large and irregular in shape, usually derived by mechanical digestion (asterisks,*). (e) Immunofluorescence staining of progenitor cells at day 56, coexpressing NKX2.2 (green), SOX10 (red) and OLIG2 (blue). (f) O4 (green) live staining showing the highly ramified morphology of the cells. Reproduced in part from Douvaras et al.16 with permission. (g) MBP+ (red) oligodendrocytes at the end of the differentiation (nuclei are stained with DAPI, blue). The solid box is a magnification of the cells in the dashed box, to highlight the morphology. (h) Morphology of MBP+ (red) oligodendrocytes at higher magnification. MAP2+ (green) and GFAP+ (blue) cells are also present in the culture. (i) Purified O4+ cells 24 h after sorting still retain the typical ramified morphology. Scale bars, 500 μm (a,b,g); 200 μm (c,e,f,i); 1 mm (d); 100 μm (h).

  3. Live imaging and quantification of O4+ OPCs.
    Figure 3: Live imaging and quantification of O4+ OPCs.

    (af) Live O4 (green) imaging at day 53 (a,d), day 63 (b,e) and day 73 (c,f) of differentiation following the fast protocol. (a–c) Representative fields at low magnification showing an increase in the number of O4+ cells with time. (d–f) Higher-magnification images of ac, respectively, to highlight the morphology of the cells. (g) Representative examples of O4+ cell frequencies calculated by flow cytometry at different time points using the fast protocol (days 55, 63 and 75) and the original protocol. Scale bars, 500 μm (ac) and 200 μm (d,e,f).


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

    • Panagiotis Douvaras &
    • Valentina Fossati


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

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The authors declare no competing financial interests.

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