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Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells


Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in >60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4+ OPCs can be purified and either cryopreserved or used for functional studies. As an example of the type of functional study for which the derived cells could be used, O4+ cells can be co-cultured with maturing hPSC-derived neurons in 96/384-well-format plates, allowing the screening of pro-myelinating compounds.

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Fig. 1
Fig. 2: Timeline of experimental procedures.
Fig. 3: Representative images of the main stages of the protocol for the generation of OLs using both the lentiviral-based and gene-editing-based methodologies.
Fig. 4: O4 marker expression of the generated OLs from the eight different hPSC lines tested after 8–10 d of SOX10 induction using the lentiviral-based approach, including those derived from MS and familial amyotrophic lateral sclerosis (fALS) patients.
Fig. 5: SOX10-induced OLs myelinate hPSC-derived cortical neurons.
Fig. 6: SOX10-induced OPCs/OLs engraft and myelinate the shiverer Rag2−/− mouse forebrain.

Data availability

Plasmids used and derived during this procedure are available through Addgene: and The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request or available as source data files associated with individual figures. Source data are provided with this paper.


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We acknowledge technical colleagues at SCIL (KU Leuven) for their technical support. J.A.G.-L has been supported by a Fellowship from Alfonso Martín Escudero Foundation (Madrid, Spain), by a contract of doctor reincorporation plan from the I Plan Propio of the University of Malaga (Spain) and by CIBERNED. The work was supported by an IWT-iPSCAF grant (no. 150031) and KUL-PF Stem Cells (no. PFO3) to C.M.V, by Instituto de Salud Carlos III (ISCiii) of Spain, cofinanced by FEDER funds from the European Union (grant PI18/01557 to A.G.), by Junta de Andalucia grants UMA18-FEDERJA-211 and PY18-RT-2233 (to A.G.), co-financed by Programa Operativo FEDER 2014-2020, by Consejeria de Salud of Junta de Andalucia (grant PI-0276-2018 to J.A.G.-L.) and by CIBERNED (grant CB06/05/1116 to A.G.). In vivo studies were supported by the Progressive MS Alliance (PMSA, collaborative research network PA-1604-08492 (BRAVEinMS)), INSERM and ICM grants to A.B.V.-E. and Junta de Andalucía and the European Commission under the Seventh Framework Program of the European Union (agreement no. 291730, contract TAHUB-II-107) to B.G-D. The authors thank the ICM animal, genomic, culture and cellular imaging core facilities.

Author information




J.A.G.-L. and C.M.V. conceived the study and generated the protocol. J.A.G.-L., K.E., L.C.-P., K.N. and R.M.d.C. performed the experiments and analysis of the data. B.G.D. performed the experiments of the shiverer mouse. J.C.D. assisted with electron microscopy. A.B.V.-E. provided scientific support and guidance on the in vivo experiments. A.G. provided scientific guidance and support. J.A.G.-L. and C.M.V. wrote the manuscript with input from B.G.D. and A.B.V.-E. C.M.V. provided scientific guidance and support.

Corresponding author

Correspondence to Juan Antonio García-León.

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Key reference using this protocol

García-León, J. A. et al. Stem Cell Reports 10, 655–672 (2018):

Key data used in this protocol

García-León, J. A. et al. Stem Cell Reports 10, 655–672 (2018):

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Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Methods.

Reporting Summary

Source data

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 6

Statistical source data.

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García-León, J.A., García-Díaz, B., Eggermont, K. et al. Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells. Nat Protoc 15, 3716–3744 (2020).

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