mouse es cell line gfp - olig2 ( g - olig2 ) , is purchased from the american type culture collection ( atcc ) . the mescs are routinely passaged every 3 days onto an irradiated mouse embryonic fibroblast ( mef ) feeder layer . the mef cells are plated into 0.1% gelatin - coated six - well tissue culture plate at least one day before passaging the escs . the mesc culture medium is dulbecco 's modified eagle 's medium ( dmem ) ( gibco ) supplemented with 20% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 1 mm sodium pyruvate , 0.1 mm -mercaptoethanol/ , 1% nonessential amino acids ( neaa ) , and 1,000 u / ml leukemia inhibitory factor . mes colonies are trypsinized ( tryple , 5 min , 37c ) into single cells and suspended in knockout serum replacement ( ksr ) medium , then the cells are transferred to a costar ultra - low attachment six - well plate ( corning ) at a cell density of 50,000 cells / cm . in these conditions , mescs form embryoid bodies ( ebs ) . ksr medium consists of -minimal essential medium ( -mem ) supplemented with 20% ksr , 1 mm sodium pyruvate , 1% neaa , and 0.1 mm -mercaptoethanol/. from day 4 to day 7 , retinoic acid ( ra , 0.2 m ) and purmorphamine ( pur , 1 m ) are included as shown in fig . the n2 medium is -mem containing 1x n2 supplement , 1 mm sodium pyruvate , 1% neaa , and 0.1 mm -mercaptoethanol/. the medium is changed everyday . at day 8 , ebs are disaggregated using tryple ( 5min , 37c ) and plated on 0.01% polyornithine - coated dishes in opc medium that consists of n2 medium and fibroblast growth factor-2 ( fgf-2 , 20 ng / ml ) . the cells are trypsinized ( tryple , 3 min , 37c ) and replated approximately every week when they become confluent . at day 30 , over 80% of the cells show gfp / olig2 expression and are ng2-positive , characteristic of opcs . to generate spiking opcs , the bacmam na channel kit is used to introduce nav1.2 subunit into these mesc - derived opcs . the bacmam reagent ( 1 ml , component a ) is mixed with pbs to a final volume of 5 ml . then , the mixed bacmam reagent is added into mesc - derived opcs culture in a 60 mm culture dish ( at 50 - 70% confluence ) prior to rinsing with pbs . after 2 hr incubation , the mixed bacmam reagent is removed and replaced with opc medium supplemented with 1x enhancer . next , after additional 2-hr incubation the medium with enhancer is removed and replaced with complete opc medium . the cells are assayed 24 hrs later . following the differentiation protocol shown in fig . 1a , g - olig2 mescs were initially suspended in ksr medium and for 4 days to form embryoid bodies ( ebs ) ( fig . the ebs did not show any gfp fluorescence at day 4 and expressed strong gfp fluorescence at d8 ( fig . at this time point , the ebs were trypsinized and plated in n2 medium supplemented with growth factors fgf-2 . after further culture for 22 days ( d30 ) , the percentage of gfp+ cell reached 80.3  0.6 % according to our flow cytometry results . 1c , gfp+ cells overlapped with ng2 staining consistently ( 96.4  1.3% of gfp positive cells were ng2 positive , and 94.8  1.5% of ng2 positive cells were gfp positive ) . thus , these cells express gfp / olig2 and ng2 , defining them as opcs . the mesc - derived opcs ( 76 cells ) failed to fire action potentials upon depolarization ( fig . after transducing with a baculovirus carrying the nav1.2 subunit , these mesc - derived opcs ( 94.1% , 16 of 17 cells ) could be classified as spiking ( fig . ( a ) scheme showing the protocol of the embryoid body ( eb)-based and small molecule - driven differentiation . at d8 , ( c ) immunostaining of ng2 ( red ) was consistent with gfp ( green ) expression at d30 . generation of spiking opcs from nonspiking mesc - derived opcs by virus - mediated nav channel expression . ( a ) the membrane potential was held at -60 mv and mesc - derived opcs failed to fire action potentials , even when the cell was depolarized to 0 mv . ( b ) an example of mesc - derived opc firing action potential ( highlighted in red ) after the viral transduction . mouse es cell line gfp - olig2 ( g - olig2 ) , is purchased from the american type culture collection ( atcc ) . the mescs are routinely passaged every 3 days onto an irradiated mouse embryonic fibroblast ( mef ) feeder layer . the mef cells are plated into 0.1% gelatin - coated six - well tissue culture plate at least one day before passaging the escs . the mesc culture medium is dulbecco 's modified eagle 's medium ( dmem ) ( gibco ) supplemented with 20% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 1 mm sodium pyruvate , 0.1 mm -mercaptoethanol/ , 1% nonessential amino acids ( neaa ) , and 1,000 u / ml leukemia inhibitory factor . mes colonies are trypsinized ( tryple , 5 min , 37c ) into single cells and suspended in knockout serum replacement ( ksr ) medium , then the cells are transferred to a costar ultra - low attachment six - well plate ( corning ) at a cell density of 50,000 cells / cm . in these conditions , mescs form embryoid bodies ( ebs ) . ksr medium consists of -minimal essential medium ( -mem ) supplemented with 20% ksr , 1 mm sodium pyruvate , 1% neaa , and 0.1 mm -mercaptoethanol/. from day 4 to day 7 , retinoic acid ( ra , 0.2 m ) and purmorphamine ( pur , 1 m ) are included as shown in fig . the n2 medium is -mem containing 1x n2 supplement , 1 mm sodium pyruvate , 1% neaa , and 0.1 mm -mercaptoethanol/. the medium is changed everyday . at day 8 , ebs are disaggregated using tryple ( 5min , 37c ) and plated on 0.01% polyornithine - coated dishes in opc medium that consists of n2 medium and fibroblast growth factor-2 ( fgf-2 , 20 ng / ml ) . the cells are trypsinized ( tryple , 3 min , 37c ) and replated approximately every week when they become confluent . at day 30 , over 80% of the cells show gfp / olig2 expression and are ng2-positive , characteristic of opcs . to generate spiking opcs , the bacmam na channel kit is used to introduce nav1.2 subunit into these mesc - derived opcs . the bacmam reagent ( 1 ml , component a ) is mixed with pbs to a final volume of 5 ml . then , the mixed bacmam reagent is added into mesc - derived opcs culture in a 60 mm culture dish ( at 50 - 70% confluence ) prior to rinsing with pbs . after 2 hr incubation , the mixed bacmam reagent is removed and replaced with opc medium supplemented with 1x enhancer . next , after additional 2-hr incubation the medium with enhancer is removed and replaced with complete opc medium . 1a , g - olig2 mescs were initially suspended in ksr medium and for 4 days to form embryoid bodies ( ebs ) ( fig . the ebs did not show any gfp fluorescence at day 4 and expressed strong gfp fluorescence at d8 ( fig . at this time point , the ebs were trypsinized and plated in n2 medium supplemented with growth factors fgf-2 . after further culture for 22 days ( d30 ) , the percentage of gfp+ cell reached 80.3  0.6 % according to our flow cytometry results . 1c , gfp+ cells overlapped with ng2 staining consistently ( 96.4  1.3% of gfp positive cells were ng2 positive , and 94.8  1.5% of ng2 positive cells were gfp positive ) . thus , these cells express gfp / olig2 and ng2 , defining them as opcs . the mesc - derived opcs ( 76 cells ) failed to fire action potentials upon depolarization ( fig . after transducing with a baculovirus carrying the nav1.2 subunit , these mesc - derived opcs ( 94.1% , 16 of 17 cells ) could be classified as spiking ( fig . ( a ) scheme showing the protocol of the embryoid body ( eb)-based and small molecule - driven differentiation . at d8 , ( c ) immunostaining of ng2 ( red ) was consistent with gfp ( green ) expression at d30 . generation of spiking opcs from nonspiking mesc - derived opcs by virus - mediated nav channel expression . ( a ) the membrane potential was held at -60 mv and mesc - derived opcs failed to fire action potentials , even when the cell was depolarized to 0 mv . ( b ) an example of mesc - derived opc firing action potential ( highlighted in red ) after the viral transduction . embryonic stem cells ( escs ) , isolated from a blastocyst embryo , can differentiate into all cell lineages of the organism , 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 ) . moreover , the shh - induced opc differentiation from mescs retains the correct timing observed in embryonic development . 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   and the helix - loop - helix transcription factor olig2 , 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 . for nearly two decades researchers have demonstrated nav channels are expressed in a subpopulation of opcs and can be activated upon depolarization . moreover , a recent striking observation 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 . 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 , 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 ) .
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 nav1.2 subunit . 
 the spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of opcs .