We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate “spiking” OPCs that can fire action potentials.
Embryonic stem cells (ESCs), isolated from a blastocyst embryo, can differentiate into all cell lineages of the organism 3, 4, providing an in vitro model system for studying early mammalian development, including oligodendrocyte specification. mESCs have been shown to differentiate into oligodendrocyte precursor cells (OPCs) with the treatment of sonic hedgehog (Shh) 5. Moreover, the Shh-induced OPC differentiation from mESCs retains the correct timing observed in embryonic development 6. Hence, the nature of in vitro OPC differentiation from mESCs is considered to be consistent with what has been learned from in vivo development. Here, using the small molecules RA and the Shh agonist Pur, we successfully differentiated the G-Olig2 mESCs into GFP+Olig2+NG2+ OPCs with high efficiency.
OPCs, characterized by the expression of the proteoglycan NG2 7 and the helix-loop-helix transcription factor Olig2 8, generate oligodendrocytes in the developing and mature CNS, where they comprise a significant percentage (~ 5%) of the total cells and are the main proliferating cell type 9. For nearly two decades researchers have demonstrated NaV channels are expressed in a subpopulation of OPCs and can be activated upon depolarization 10, 11. Moreover, a recent striking observation9 was that in in situ rat CNS white matter, OPCs (~ 50%) generated action potentials when depolarized depending upon the expression of voltage-gated sodium (NaV) channels, and thus could be subdivided into spiking and nonspiking subpopulations. However, the functions of these spiking properties are still largely unknown. We found that, electrophysiologically, the mESC-derived OPCs differentiated with the Shh-dependent protocol, were not the same as the in situ brain OPCs. After introducing subunit NaV1.2, these silent mESC-derived OPCs were capable of spiking.
Thus, the spiking/nonspiking mESC-derived OPCs and differentiation protocol described here may facilitate (1) studying the functional differences between spiking and nonspiking OPCs, (2) screening new factors that could promote sodium channel expression in the mESC-derived OPCs, (3) developing and optimizing the differentiation protocol of OPCs from human ESC or induced pluripotent stem cells (iPSCs).
The authors have nothing to disclose.
This work was in part supported by grants to W.D. from National Institutes of Health (RO1 NS059043 and RO1 ES015988), National Multiple Sclerosis Society, Roche Foundation for Anemia Research, Feldstein Medical Foundation, and the Shriners Hospitals for Children.
We would like to thank Dr. David Pleasure and Jennifer Plane for suggestions. We declare no competing interests related to this article.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
MEFs | GlobalStem | GSC-6001G | ||
TrypLE Express | Invitrogen | 12604 | ||
Retinoic acid | Sigma | R-2625 | ||
Purmorphamine | Cayman Chemical | 10009634 | ||
FGF-2 | Millipore | GF003 | ||
BacMam NaV 1.2 | Invitrogen | B10341 |