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Generation of oligodendrocytes and establishment of an all-human myelinating platform from human pluripotent stem cells
- Source :
- Nature Protocols. 15:3716-3744
- Publication Year :
- 2020
- Publisher :
- Springer Science and Business Media LLC, 2020.
-
Abstract
- 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.
- Subjects :
- 0303 health sciences
biology
Neurogenesis
SOX10
General Biochemistry, Genetics and Molecular Biology
Myelin basic protein
Viral vector
Cell biology
03 medical and health sciences
Myelin
0302 clinical medicine
medicine.anatomical_structure
nervous system
Cell culture
biology.protein
medicine
Progenitor cell
Induced pluripotent stem cell
030217 neurology & neurosurgery
030304 developmental biology
Subjects
Details
- ISSN :
- 17502799 and 17542189
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- Nature Protocols
- Accession number :
- edsair.doi...........f98136f80a20117e9b2504536d46fc80