because of their unique ability to self - renew while retaining pluripotency , human embryonic stem cells ( hescs ) are considered an invaluable resource in regenerative medicine . stem cell mediated repopulation of diseased muscles and hesc- or induced pluripotent stem cells ( ipsc)-derived generation of skeletal muscles for in vitro disease modeling are key goals of studies aimed at identifying treatments and elucidating the pathogenesis of many neuromuscular diseases .
however , these studies have been challenged by the resistance of hescs to convert into skeletal muscle cells and by the paucity of information regarding the molecular regulation of hesc - commitment toward skeletal myogenesis .
indeed , previous attempts to generate skeletal muscle progenitors from hescs revealed that only embryoid body ( eb)-derived progenies or mesenchymal cells are competent to activate skeletal myogenesis following ectopic expression of transcriptional activators ( e.g. pax3 or myf5 ) or by exposure to specific culture conditions .  
we have previously shown that the swi / snf component , baf60c ( encoded by smarcd3 ) , is an essential component of the transcriptional machinery that allows myod - mediated activation of muscle - specific loci .
we have recently discovered that the selective absence of baf60c confers onto hescs the resistance to myod - mediated activation of skeletal myogenesis that is otherwise observed in baf60c expressing somatic cells , a process commonly referred to as myogenic conversion .
forced expression of baf60c enables myod to directly activate skeletal myogenesis in hescs , upon specific culture conditions , with baf60c and myod imposing an epigenetic signature that commits hescs towards the myogenic lineage . of note ,
previous proteomic analysis revealed the selective absence of baf60c , among the swi / snf components , in escs .
we used this knowledge to impose an epigenetic commitment of hescs onto the myogenic lineage , leading to the generation of a homogeneous population of skeletal myoblasts that could be aggregated to form three - dimensional contractile structures ( myospheres ) that functionally mimic miniaturized skeletal muscles . indeed , our method of generating skeletal muscle progenitors from hescs relies on the epigenetic commitment of hescs to the myogenic lineage , which is phenotypically latent until cells are exposed to differentiation signals , such as cell aggregation and culture in differentiation medium ( see specific protocol ) .
this strategy permits the expansion of a homogeneous population of hescs that are epigenetically committed to skeletal muscle lineage and suitable to formation of three - dimensional contractile myospheres that recapitulate histological and functional properties of skeletal muscles .
the myospheres provide the first evidence of miniaturized muscles exploitable for a  disease in a dish 
derived ipscs , these myospheres have the potential to elucidate longstanding developmental questions and the pathogenesis of rare diseases , in addition to offering the tremendous potential as a tool for high throughput screening of therapeutic compounds
. we also note that one immediate readout of myosphere analysis could be provided by immunohistochemistry on sections , as described in a jove protocol by gomes et al .
recommendations before starting in order to ensure high efficiency of infection , plate hescs for infection on feeder - free conditions ( figure 2a ) to eliminate cellular competitors during the viral uptake .
indeed , mefs are notoriously easier to infect as compared to hescs and are competent for myod - mediated conversion .
thus , eliminating mefs from hesc cultures increases the infection rate.it is recommended to have high titer lentiviruses to ensure high efficiency of infection .
options to improve efficiency of infection are discussed in the section " checking infection".prepare matrigel - coated plates by adding 1 ml of 1 mg / ml matrigel in each well and leave at least 1 hr at rt ( or o / n sealed at 4 c if prepared the day before ) . 
  in order to ensure high efficiency of infection , plate hescs for infection on feeder - free conditions ( figure 2a ) to eliminate cellular competitors during the viral uptake
indeed , mefs are notoriously easier to infect as compared to hescs and are competent for myod - mediated conversion .
options to improve efficiency of infection are discussed in the section " checking infection " .
prepare matrigel - coated plates by adding 1 ml of 1 mg / ml matrigel in each well and leave at least 1 hr at rt ( or o / n sealed at 4 c if prepared the day before ) .
infection procedure day before the infection add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) . 

day 0 - infection with baf60c note : perform the infection of hescs with baf60c first followed by infection with myod . 

dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c.transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.during the centrifugation , prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm).re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic.add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubatorcollect the cell suspension containing the viruses and divide equally in two matrigel - coated wells .
then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator . 

day 1 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
day2 - infection with myod before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection .
if not , see the section " checking infection".if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding.remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.repeat as described from1.2.2.2 - 1.2.2.6 adding myod virus at 10moi . 

day 3 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement.plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells . 

day 4 remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.remove the supernatant and re - suspend in 6 ml of hesc medium.remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c.remove collagenase iv and add 1 ml of hesc medium.under a dissection microscope scrape the colonies with a 10 ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 . 

 
day before the infection add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) . 
 
add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) .
day 0 - infection with baf60c note : perform the infection of hescs with baf60c first followed by infection with myod . 
 dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c.transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.during the centrifugation ,
prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm).re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic.add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubatorcollect the cell suspension containing the viruses and divide equally in two matrigel - coated wells . then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator . 
 
dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c .
transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm . during the centrifugation ,
prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm ) .
re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic . add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubator collect the cell suspension containing the viruses and divide equally in two matrigel - coated wells . then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator .
day 1 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
cells will start clumping as shown in figure 2b . 
  carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
day2 - infection with myod before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection .
if not , see the section " checking infection".if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding.remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.repeat as described from1.2.2.2 - 1.2.2.6 adding myod virus at 10moi . 
 
before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection . if not , see the section " checking infection " .
if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding .
day 3 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement.plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells . 
 
carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells .
day 4 remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.remove the supernatant and re - suspend in 6 ml of hesc medium.remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c.remove collagenase iv and add 1 ml of hesc medium.under a dissection microscope scrape the colonies with a 10 ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 . 
 
transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm . remove the supernatant and re - suspend in 6 ml of hesc medium . remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c .
remove collagenase iv and add 1 ml of hesc medium . under a dissection microscope scrape the colonies with a 10 ml tip .
collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 .
checking infection while propagating the cells , it is recommended to plate the infected cells in a smaller scale to harvest for rna analysis and perform protein expression analysis by immunofluorescence in order to check for biomarkers that reflect the correct biological status at each stage ( see representative results ) .
for high titer lentiviruses the percentage of expected infected cells should be at least 80%.to ensure high efficiency , the lentiviral vectors can be modified , introducing a fluorescent selection marker and/or an antibiotic resistance .
our lentiviral plasmid expressing baf60c also carries gfp fluorescence.sorting hescs for a fluorescent marker . in the case of low efficiency of infection
is recommended to use fluorescence activated cells sorting ( facs - sorting ) to enrich for cells expressing baf60c virus and then infect the cells with high titer myod virus .
add he s cell cloning & recovery supplement in the medium the day before the sorting.the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs-tubes.at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement . 

  while propagating the cells , it is recommended to plate the infected cells in a smaller scale to harvest for rna analysis and perform protein expression analysis by immunofluorescence in order to check for biomarkers that reflect the correct biological status at each stage ( see representative results ) .
for high titer lentiviruses the percentage of expected infected cells should be at least 80% . to ensure high efficiency , the lentiviral vectors can be modified , introducing a fluorescent selection marker and/or an antibiotic resistance .
our lentiviral plasmid expressing baf60c also carries gfp fluorescence . sorting hescs for a fluorescent marker . in the case of low efficiency of infection
is recommended to use fluorescence activated cells sorting ( facs - sorting ) to enrich for cells expressing baf60c virus and then infect the cells with high titer myod virus .
add he s cell cloning & recovery supplement in the medium the day before the sorting.the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs-tubes.at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement . 
 
add he s cell cloning & recovery supplement in the medium the day before the sorting .
the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs - tubes . at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement .
induction and differentiation procedure day 0 - eb induction make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c).for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
incubate for 5 min at 37 c.remove collagenase and add 1 ml of eb medium.under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate . 

if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows.add 1.5 ml of fresh eb medium in the wells 
 day 2 change the medium by collecting the aggregates from the wells in a 15-ml tube .
let them settle down for 2 min.remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells . 

collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment.remove pbs and add 3 ml of dm medium ; plate on the same wells . 

day 6 - day 20 from d6 to d20 replace the medium with fresh medium every 3 days .
day 0 - eb induction make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c).for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
incubate for 5 min at 37 c.remove collagenase and add 1 ml of eb medium.under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate . 
 
make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c ) .
for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
remove collagenase and add 1 ml of eb medium . under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip .
collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min .
remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate .
if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows.add 1.5 ml of fresh eb medium in the wells 
  check the size of colonies and the cell death before proceeding .
if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows .
add 1.5 ml of fresh eb medium in the wells day 2 change the medium by collecting the aggregates from the wells in a 15-ml tube .
let them settle down for 2 min.remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells . 
 
remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells .
collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment.remove pbs and add 3 ml of dm medium ; plate on the same wells . 
 
collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment . remove pbs and add 3 ml of dm medium ; plate on the same wells .
day 6 - day 20 from d6 to d20 replace the medium with fresh medium every 3 days .
checking myosphere differentiation before proceeding further , it is recommended to check the efficiency of myogenic differentiation within the myospheres by making sections of the myospheres embedded in o.c.t .
perform an immunofluorescence staining for myogenic markers such as myognenin ( clone f5d , dshb ) and myosin heavy chain ( clone mf20 , dshb ) ( see representative results ) . tip ! to be successful with the staining for myogenin on eb sections it is critical to perform antigen retrieval by treatment with sodium dodecyl sulfate ( sds ) 0.5% for 5 min .
additionally , it is possible to use this protocol for the double staining using f5d and mf20 antibodies . 
  before proceeding further
, it is recommended to check the efficiency of myogenic differentiation within the myospheres by making sections of the myospheres embedded in o.c.t .
perform an immunofluorescence staining for myogenic markers such as myognenin ( clone f5d , dshb ) and myosin heavy chain ( clone mf20 , dshb ) ( see representative results ) .
it is critical to perform antigen retrieval by treatment with sodium dodecyl sulfate ( sds ) 0.5% for 5 min .
additionally , it is possible to use this protocol for the double staining using f5d and mf20 antibodies .
esc medium dmem - f12 1 mm l - glutamine 20% knockout serum replacement medium ( kosr ) 0.1 mm nonessential amino acids ( neaa ) 50 u / ml penicillin / streptomycin ( pen / strep ) 0.1 mm beta - mercaptoethanol 8 ng / ml basic fibroblast growth factor ( bfgf ) eb medium dmem f12 1 mm l - glutamine 15% fbs 5% horse serum dm medium dmef f12 1x its liquid media supplement 1x n-2 supplement
recommendations before starting in order to ensure high efficiency of infection , plate hescs for infection on feeder - free conditions ( figure 2a ) to eliminate cellular competitors during the viral uptake .
indeed , mefs are notoriously easier to infect as compared to hescs and are competent for myod - mediated conversion .
thus , eliminating mefs from hesc cultures increases the infection rate.it is recommended to have high titer lentiviruses to ensure high efficiency of infection .
options to improve efficiency of infection are discussed in the section " checking infection".prepare matrigel - coated plates by adding 1 ml of 1 mg / ml matrigel in each well and leave at least 1 hr at rt ( or o / n sealed at 4 c if prepared the day before ) . 
  in order to ensure high efficiency of infection , plate hescs for infection on feeder - free conditions ( figure 2a ) to eliminate cellular competitors during the viral uptake
indeed , mefs are notoriously easier to infect as compared to hescs and are competent for myod - mediated conversion .
options to improve efficiency of infection are discussed in the section " checking infection " .
prepare matrigel - coated plates by adding 1 ml of 1 mg / ml matrigel in each well and leave at least 1 hr at rt ( or o / n sealed at 4 c if prepared the day before ) .
infection procedure day before the infection add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) . 

day 0 - infection with baf60c note : perform the infection of hescs with baf60c first followed by infection with myod . 

dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c.transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.during the centrifugation , prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm).re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic.add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubatorcollect the cell suspension containing the viruses and divide equally in two matrigel - coated wells .
then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator . 

day 1 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
day2 - infection with myod before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection .
if not , see the section " checking infection".if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding.remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.repeat as described from1.2.2.2 - 1.2.2.6 adding myod virus at 10moi . 

day 3 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement.plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells . 

day 4 remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.remove the supernatant and re - suspend in 6 ml of hesc medium.remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c.remove collagenase iv and add 1 ml of hesc medium.under a dissection microscope scrape the colonies with a 10 ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 . 

 
day before the infection add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) . 
 
add he s cell cloning & recovery supplement in the medium at 1,000x dilution ( 2 mm final concentration ) .
day 0 - infection with baf60c note : perform the infection of hescs with baf60c first followed by infection with myod . 
 dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c.transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.during the centrifugation ,
prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm).re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic.add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubatorcollect the cell suspension containing the viruses and divide equally in two matrigel - coated wells . then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator . 
 
dissociate a well of a 6-well plate of hescs grown as colonies in a single - cell suspension by incubation with 1 ml of tryple for 5 min at 37 c .
transfer suspension to a 15-ml tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm . during the centrifugation ,
prepare the infection mix in a clean tube by adding to 1 ml of mtesr1 , polybrene ( 6 mg / ml ) and he s cell cloning & recovery supplement ( 2 mm ) .
re - suspend the cell pellet ( approximately 5 x 10 - 1 x 10 cells from one confluent well of hescs ) with 1 ml of infection mix and plate onto a 6-well low attachment plate , which prevents the cells from adhering to the plastic . add baf60c - ires - gfp virus at 10moi and incubate 3 hr in the incubator collect the cell suspension containing the viruses and divide equally in two matrigel - coated wells . then add 2 ml of mtesr1 + he s cell cloning & recovery supplement to each well and leave o / n in the incubator .
day 1 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
cells will start clumping as shown in figure 2b . 
  carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
day2 - infection with myod before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection .
if not , see the section " checking infection".if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding.remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.repeat as described from1.2.2.2 - 1.2.2.6 adding myod virus at 10moi . 
 
before proceeding check the fluorescence status of the cells under a fluorescence microscope to make sure to have a high efficiency of infection . if not , see the section " checking infection " .
if the cells have reached at least 80% of confluence , proceed as follows ; otherwise wait for an additional day prior to proceeding .
day 3 carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement.plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells . 
 
carefully remove the medium containing the virus and replace with 2 ml of mtesr1and he s cell cloning & recovery supplement .
plate mefs on matrigel coated plates at 2.5 x 10/cmfor the next day to allow propagation of the infected cells .
day 4 remove the medium and add 1 ml of tryple reagent to dissociate the cells .
incubate 5 min at 37 c.transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm.remove the supernatant and re - suspend in 6 ml of hesc medium.remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c.remove collagenase iv and add 1 ml of hesc medium.under a dissection microscope scrape the colonies with a 10 ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 . 
 
transfer the cells in a 15-ml falcon tube , add 9 ml of hesc medium and centrifuge for 5 min at 1,200 rpm . remove the supernatant and re - suspend in 6 ml of hesc medium . remove the medium from the mefs - containing 6-well plate and plate hescs at 1:2 split ratio , dispensing 3 ml for each mefs - containing well .
when the colonies reach confluence , some of the wells of myod / baf60c - infected cells can be frozen in liquid nitrogen as a reserve stock . freezing medium includes 90% of kosr and 10% dmso plus 2 mm he s cell cloning & recovery supplement .
the remaining wells can be then exposed to the differentiation protocol - see below for description .
if more cells are needed to produce more myospheres , infected hescs can be propagated further as follows : remove the medium and incubate with 1 ml of 1 mg / ml collagenase iv for 5 min at 37 c .
remove collagenase iv and add 1 ml of hesc medium . under a dissection microscope scrape the colonies with a 10 ml tip .
collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . then remove the supernatant , resuspend in hesc medium and plate on mefs plates using a ratio 1:4 .
checking infection while propagating the cells , it is recommended to plate the infected cells in a smaller scale to harvest for rna analysis and perform protein expression analysis by immunofluorescence in order to check for biomarkers that reflect the correct biological status at each stage ( see representative results ) .
for high titer lentiviruses the percentage of expected infected cells should be at least 80%.to ensure high efficiency , the lentiviral vectors can be modified , introducing a fluorescent selection marker and/or an antibiotic resistance .
our lentiviral plasmid expressing baf60c also carries gfp fluorescence.sorting hescs for a fluorescent marker . in the case of low efficiency of infection
is recommended to use fluorescence activated cells sorting ( facs - sorting ) to enrich for cells expressing baf60c virus and then infect the cells with high titer myod virus .
add he s cell cloning & recovery supplement in the medium the day before the sorting.the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs-tubes.at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement . 

  while propagating the cells , it is recommended to plate the infected cells in a smaller scale to harvest for rna analysis and perform protein expression analysis by immunofluorescence in order to check for biomarkers that reflect the correct biological status at each stage ( see representative results ) .
for high titer lentiviruses the percentage of expected infected cells should be at least 80% . to ensure high efficiency , the lentiviral vectors can be modified , introducing a fluorescent selection marker and/or an antibiotic resistance .
our lentiviral plasmid expressing baf60c also carries gfp fluorescence . sorting hescs for a fluorescent marker . in the case of low efficiency of infection
is recommended to use fluorescence activated cells sorting ( facs - sorting ) to enrich for cells expressing baf60c virus and then infect the cells with high titer myod virus .
add he s cell cloning & recovery supplement in the medium the day before the sorting.the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs-tubes.at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement . 
 
add he s cell cloning & recovery supplement in the medium the day before the sorting .
the day of the sorting , dissociate the cells by tryple ( as described at day 0 ) and resuspend the cells in ko replacement serum plus he s cell cloning & recovery supplement in facs - tubes . at the end of the sorting , collect the cells in ko replacement serum and plate on mefs plates adding he s cell cloning & recovery supplement .
induction and differentiation procedure day 0 - eb induction make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c).for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
incubate for 5 min at 37 c.remove collagenase and add 1 ml of eb medium.under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate . 

if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows.add 1.5 ml of fresh eb medium in the wells 
 day 2 change the medium by collecting the aggregates from the wells in a 15-ml tube .
let them settle down for 2 min.remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells . 

collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment.remove pbs and add 3 ml of dm medium ; plate on the same wells . 

day 6 - day 20 from d6 to d20 replace the medium with fresh medium every 3 days .
day 0 - eb induction make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c).for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
incubate for 5 min at 37 c.remove collagenase and add 1 ml of eb medium.under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip.collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min . remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate . 
 
make sure , before starting differentiation from baf60c / myod - infected hescs , to have high number of colonies in the well ( figure 2c ) .
for each well remove the medium , wash with pbs and add 1 ml of 1 mg / ml collagenase iv .
remove collagenase and add 1 ml of eb medium . under a dissection microscope quickly mechanically dissociate the colonies into 400 - 800 cell aggregates by scraping with a 10-ml tip .
collect the chunks of colonies in a 15-ml tube and allow to sediment for 3 min .
remove the supernatant , re - suspend in 3 ml of eb medium and transfer the cell suspension to one well of a 6-well low attachment plate .
if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows.add 1.5 ml of fresh eb medium in the wells 
  check the size of colonies and the cell death before proceeding .
if big chunks are present , break them by gently pipetting up and down with a 5-ml pipette , 3 - 4 times .
if the cell death is very high ( many floating cells and debris ) replace the medium by collecting the aggregates in a 15-ml tube by gravity and plate them with 3 ml of fresh eb medium , otherwise proceed as follows .
add 1.5 ml of fresh eb medium in the wells day 2 change the medium by collecting the aggregates from the wells in a 15-ml tube .
let them settle down for 2 min.remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells
. 
  change the medium by collecting the aggregates from the wells in a 15-ml tube .
remove the supernatant , replace with 3 ml of fresh eb medium and redistribute on the same wells .
collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment.remove pbs and add 3 ml of dm medium ; plate on the same wells . 
 
collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of pbs ; allow the aggregated to sediment . remove pbs and add 3 ml of dm medium ; plate on the same wells .
day 6 - day 20 from d6 to d20 replace the medium with fresh medium every 3 days .
checking myosphere differentiation before proceeding further , it is recommended to check the efficiency of myogenic differentiation within the myospheres by making sections of the myospheres embedded in o.c.t .
perform an immunofluorescence staining for myogenic markers such as myognenin ( clone f5d , dshb ) and myosin heavy chain ( clone mf20 , dshb ) ( see representative results ) .
it is critical to perform antigen retrieval by treatment with sodium dodecyl sulfate ( sds ) 0.5% for 5 min .
additionally , it is possible to use this protocol for the double staining using f5d and mf20 antibodies . 
  before proceeding further ,
it is recommended to check the efficiency of myogenic differentiation within the myospheres by making sections of the myospheres embedded in o.c.t .
perform an immunofluorescence staining for myogenic markers such as myognenin ( clone f5d , dshb ) and myosin heavy chain ( clone mf20 , dshb ) ( see representative results ) .
to be successful with the staining for myogenin on eb sections it is critical to perform antigen retrieval by treatment with sodium dodecyl sulfate ( sds ) 0.5% for 5 min .
additionally , it is possible to use this protocol for the double staining using f5d and mf20 antibodies .
esc medium dmem - f12 1 mm l - glutamine 20% knockout serum replacement medium ( kosr ) 0.1 mm nonessential amino acids ( neaa ) 50 u / ml penicillin / streptomycin ( pen / strep ) 0.1 mm beta - mercaptoethanol 8 ng / ml basic fibroblast growth factor ( bfgf ) eb medium dmem f12 1 mm l - glutamine 15% fbs 5% horse serum dm medium dmef f12 1x its liquid media supplement 1x n-2 supplement
the critical steps to control ( indicated by the arrows ) are the efficiency of the infection in hescs and the myogenic conversion within the myospheres . in our experiments
we usually infect the cells with baf60c and facs - sort 72 hr after the infection to enrich for the population expressing baf60c .
when the cells reach around 70 - 80% of confluence , we infect with myod lentivirus . after the first infection it normally takes two or three days for the cells to reach that confluence .
figure 1b shows expression levels of the exogenous genes as fold induction relative to hescs not infected .
figure 1c shows the expression and distribution at the protein level of the factors introduced .
baf60c expression is shown as gfp fluorescence , while myod expression is detected by immunofluorescence with a myod antibody .
we consider day 0 the day we start the differentiation of infected hescs into eb - like aggregates .
after 15 days in dm medium , a portion of the myospheres ( usually one entire well ) is embedded in o.c.t .
figure 1d shows representative staining for myogenin and myosin heavy chain in an optimal myogenic differentiation .
myospheres can have different or unusual shapes , perhaps as a result of fusion with smaller myospheres .
starting from day 10 , it is possible to observe sporadic contraction in the myospheres ( see movie 1 and 2 ) .
b ) mrna levels of the exogenous genes in hescs infected with baf60c and myod ( h9-bm ) monitored by qrt - pcr and expressed as fold induction , as compared to mock infected hescs ( h9-ctr ) .
c ) immunofluorescence images showing myod staining ( red ) and baf60c fluorescence ( green ) due to the ires - gfp element in the vector .
d ) immunofluorescence performed on myosphere sections stained for myogenin ( myog ) , myosin heavy chain ( myhc ) and counterstained for dapi ( see immunofluorescence details ) .
representative pictures of cell appearance at different stages of the protocol from the infection ( part i ) to the differentiation ( part ii ) .
scale bar is 100 m . whereas eb - like structures from d1 to d5 are mostly circular in shape ( d ) , myospheres grown over 15 days according the scheme in figure 1a present an unpredictable shape and are heterogeneous in culture ; see two examples in e and f.
the protocol proposed here describes how to generate three - dimensional clusters of contractile myofibers ( myospheres ) directly from hescs .
the strategy proposed has the unprecedented and extraordinary potential to produce mini muscles in suspension that can be suitable as a " disease in a dish " model for both screening assays and developmental studies .
moreover , the method to generate myospheres from hescs is straightforward and does not require any facs - sorting step during the differentiation , which typically has a negative impact on the yield of cells recovered .
besides , a sorting procedure during the differentiation would interfere with the aggregation since it implies a dissociation of the eb into single cells .
the method proposed is based on the epigenetic reprograming of hescs with specific factors , myod and baaf60c , which are not expressed in pluripotent embryonic stem cells .
myod and baf60c provide the " core " protein complex that marks the genomic loci from which transcription proceeds to activate the skeletal myogenic program .
the two factors are delivered to the cells by means of lentiviral infections and it is therefore critical to obtain a high efficiency of infection of both factors in all cells .
this can be achieved by using high titer viruses or viruses endowed with selection markers .
culture conditions also play an important role in optimizing the extent of the myogenic differentiation .
we propose a serum - based differentiation protocol for the differentiation of myod / baf60c expressing hescs into clusters of myogenic precursors , followed by incubation in serum - free defined medium ( containing insulin transferrin - its ) to achieve conversion of myogenic precursors into skeletal myotubes .
however , it is possible that other defined media can achieve an equal or better myogenic differentiation .
one current limitation of the protocol is that it relies on the use of fetal bovine serum , which , besides containing many animal proteins and substances , shows lot - to - lot variations .
this may result in lower efficiency or heterogeneity of myogenic conversion within myospheres . of note ,
not all the eb - like structures derived from baf60/myod - expressing hescs are myospheres ; namely , some are eb - like aggregates fully composed of myofibers .
the number of myospheres normally derived from hescs expressing baf60c and myod can vary from 30 to 60% as estimated by immunostaining on eb sections performed at the end of the protocol ( see results ) . a potential approach to enrich a culture dish for only myospheres would be to use a myogenic reporter .
this would facilitate discarding the partially or not differentiated eb - like clusters and selecting only the myospheres .
lastly , a better understanding of the mechanisms underlying the temporal requirements of the factors introduced would be useful to improve the method of delivery .
for example , if baf60c and myod are required only for short time to " kick " the cells in the right direction , methods based on modified mrna delivery might be applied . by contrast , if the factors are required for a longer time before the cells exploit their endogenous proteins , an episomal approach would be recommended to eliminate variability and uncontrolled effects due to integration in the genome . finally , we note that it is mandatory to express baf60c prior to or at the same time as myod ( never after ) in order to achieve hesc conversion into skeletal muscles .
the requirement for prior expression of baf60c presumably relies on its role in pre - setting the epigenetic landscape for proper myod chromatin distribution through the genome . as such
, future protocols might be improved by compounds or other manipulations that promote baf60c expression in hescs .
 generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells ( hescs ) is a requirement for cell - based therapies and for a " disease in a dish " model of human neuromuscular diseases . 
 major hurdles , such as low abundance and heterogeneity of the population of interest , as well as a lack of protocols for the formation of three - dimensional contractile structures , have limited the applications of stem cells for neuromuscular disorders . 
 we have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hescs - the muscle determination factor myod and swi / snf chromatin remodeling complex component baf60c - that are able to reprogram hescs into skeletal muscle cells . 
 here we describe the protocol established to generate hesc - derived myoblasts and promote their clustering into tridimensional miniaturized structures ( myospheres ) that functionally mimic miniaturized skeletal muscles7 . 
Introduction
Protocol
1. Infection of hESCs
2. EB-like Induction and Differentiation
3. Media Recipes
Representative Results
Discussion
Disclosures