Differentiation of embryonic stem cells into oligodendrocyte precursors.
Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing Na(V;)1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.
Jiang
Peng
P
Department of Cell Biology and Human Anatomy Institute for Pediatric Regenerative Medicine, School of Medicine, University of California, Davis, CA, USA.
Selvaraj
Vimal
V
Deng
Wenbin
W
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003584:Cytological Techniques / Q000379:methods*; D055724:Electrophysiological Phenomena; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009836:Oligodendroglia / Q000166:cytology* / Q000187:drug effects; D011687:Purines / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
D009025:Morpholines; D011687:Purines; D014212:Tretinoin; C470893:purmorphamine