PLP is an Associate Investigator of Sanford Children&#39;s Health Research Center. This work has been supported by the following grants to PLP: R01AR056712, R01AR052779 and P30 AR061303 from the National Institute of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), MDA and Sanford Children Health Research award. This work has partly benefited from research funding from the European Community's Seventh Framework Programme in the project FP7-Health - 2009 ENDOSTEM 241440 (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue). SA was supported by CIRM fellowship.

1. Infection of hESCs
This protocol requires high titer lentiviruses encoding BAF60C and MyoD13. These constructs are available upon request.
<ol>
	<li>Recommendations before starting
		<ol>
			<li>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.</li>
			<li>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".</li>
			<li>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 &#176;C if prepared the day before).</li>
		</ol>
	</li>
	<li>Infection procedure
		<ol>
			<li>Day before the infection
				<ol>
					<li>Add hES Cell Cloning &#38; Recovery Supplement in the medium at 1,000X dilution (2 mM final concentration).</li>
				</ol>
			</li>
			<li>Day 0 - Infection with BAF60C 
				Note: Perform the infection of hESCs with BAF60C first followed by infection with MyoD.
				<ol>
					<li>Dissociate a well of a 6-well plate of hESCs grown as colonies14 in a single-cell suspension by incubation with 1 ml of TrypLE for 5 min at 37 &#176;C.</li>
					<li>Transfer suspension to a 15-ml tube, add 9 ml of hESC medium and centrifuge for 5 min at 1,200 rpm.</li>
					<li>During the centrifugation, prepare the infection mix in a clean tube by adding to 1 ml of mTeSR1, polybrene (6 mg/ml) and hES Cell Cloning &#38; Recovery Supplement (2 mM).</li>
					<li>Re-suspend the cell pellet (approximately 5 x 105-1 x 106 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.</li>
					<li>Add BAF60C-iRES-GFP virus at 108 MOI and incubate 3 hr in the incubator</li>
					<li>Collect the cell suspension containing the viruses and divide equally in two matrigel-coated wells. Then add 2 ml of mTeSR1+ hES Cell Cloning &#38; Recovery Supplement to each well and leave O/N in the incubator.</li>
				</ol>
			</li>
			<li>Day 1
				<ol>
					<li>Carefully remove the medium containing the virus and replace with 2 ml of mTeSR1and hES Cell Cloning &#38; Recovery Supplement. Cells will start clumping as shown in Figure 2B.</li>
				</ol>
			</li>
			<li>Day2 - Infection with MyoD 
				<ol>
					<li>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".</li>
					<li>If the cells have reached at least 80% of confluence, proceed as follows; otherwise wait for an additional day prior to proceeding.</li>
					<li>Remove the medium and add 1 ml of TrypLE reagent to dissociate the cells. Incubate 5 min at 37 &#176;C.</li>
					<li>Repeat as described from1.2.2.2 - 1.2.2.6 adding MyoD virus at 108 MOI.</li>
				</ol>
			</li>
			<li>Day 3
				<ol>
					<li>Carefully remove the medium containing the virus and replace with 2 ml of mTeSR1and hES Cell Cloning &#38; Recovery Supplement.</li>
					<li>Plate MEFs on matrigel coated plates at 2.5 x 105/cm2 for the next day to allow propagation of the infected cells.</li>
				</ol>
			</li>
			<li>Day 4
				<ol>
					<li>Remove the medium and add 1 ml of TrypLE reagent to dissociate the cells. Incubate 5 min at 37 &#176;C.</li>
					<li>Transfer the cells in a 15-ml falcon tube, add 9 ml of hESC medium and centrifuge for 5 min at 1,200 rpm.</li>
					<li>Remove the supernatant and re-suspend in 6 ml of hESC medium.</li>
					<li>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. 
						TIP! 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 hES Cell Cloning &#38; 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:</li>
					<li>Remove the medium and incubate with 1 ml of 1 mg/ml collagenase IV for 5 min at 37 &#176;C.</li>
					<li>Remove collagenase IV and add 1 ml of hESC medium.</li>
					<li>Under a dissection microscope scrape the colonies with a 10 ml tip.</li>
					<li>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.</li>
				</ol>
			</li>
		</ol>
	</li>
	<li>Checking infection
		<ol>
			<li>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%.</li>
			<li>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.</li>
			<li>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.
				<ol>
					<li>Add hES Cell Cloning &#38; Recovery Supplement in the medium the day before the sorting.</li>
					<li>The day of the sorting, dissociate the cells by TrypLE (as described at day 0) and resuspend the cells in KO replacement serum plus hES Cell Cloning &#38; Recovery Supplement in FACS-tubes.</li>
					<li>At the end of the sorting, collect the cells in KO replacement serum and plate on MEFs plates adding hES Cell Cloning &#38; Recovery Supplement.</li>
				</ol>
			</li>
		</ol>
	</li>
</ol>
2. EB-like Induction and Differentiation
<ol>
	<li>Induction and differentiation procedure
		<ol>
			<li>Day 0 - EB induction
				<ol>
					<li>Make sure, before starting differentiation from BAF60C/Myod-infected hESCs, to have high number of colonies in the well (Figure 2C).</li>
					<li>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 &#176;C.</li>
					<li>Remove collagenase and add 1 ml of EB medium.</li>
					<li>Under a dissection microscope quickly mechanically dissociate the colonies into 400-800 cell aggregates by scraping with a 10-ml tip.</li>
					<li>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.</li>
				</ol>
			</li>
			<li>Day 1
				<ol>
					<li>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.</li>
					<li>Add 1.5 ml of fresh EB medium in the wells</li>
				</ol>
			</li>
			<li>Day 2
				<ol>
					<li>Change the medium by collecting the aggregates from the wells in a 15-ml tube. Let them settle down for 2 min.</li>
					<li>Remove the supernatant, replace with 3 ml of fresh EB medium and redistribute on the same wells.</li>
				</ol>
			</li>
			<li>Day 3
				<ol>
					<li>Add 1.5 ml of fresh EB medium in the wells.</li>
				</ol>
			</li>
			<li>Day 4
				<ol>
					<li>Proceed as described in "Day 2".</li>
				</ol>
			</li>
			<li>Day 5 - Myogenic differentiation of EB-like clusters (Figure 2D).
				<ol>
					<li>Collect the aggregates as described in 2.1.3.1 and wash once with 2 ml of PBS; allow the aggregated to sediment.</li>
					<li>Remove PBS and add 3 ml of DM medium; plate on the same wells.</li>
				</ol>
			</li>
			<li>Day 6 - Day 20
				<ol>
					<li>From d6 to d20 replace the medium with fresh medium every 3 days. Representative myospheres are shown in Figure 2E.</li>
				</ol>
			</li>
		</ol>
	</li>
	<li>Checking myosphere differentiation
		<ol>
			<li>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. compound, as described in a previous JoVE protocol12. 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.</li>
		</ol>
	</li>
</ol>
3. Media Recipes
<ol>
	<li>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)</li>
	<li>EB medium 
		DMEM F12 
		1 mM L-glutamine 
		15% FBS 
		5% Horse Serum</li>
	<li>DM medium 
		DMEF F12 
		1X ITS Liquid Media Supplement 
		1X N-2 Supplement</li>
</ol>

<ol>
	<li>Darabi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+skeletal+muscle+regeneration+from+differentiating+embryonic+stem+cells.">Functional skeletal muscle regeneration from differentiating embryonic stem cells.</a> Nat Med. 14, 134-143 (2008).</li><li>Darabi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ES-+and+iPS-derived+myogenic+progenitors+restore+DYSTROPHIN+and+improve+contractility+upon+transplantation+in+dystrophic+mice.">Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice.</a> Cell Stem Cell. 10, 610-619 (2012).</li><li>Iacovino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+cassette+exchange:+a+rapid+and+efficient+system+enabling+conditional+gene+expression+in+embryonic+stem+and+primary+cells.">Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.</a> Stem Cells. 29, 1580-1588 (2011).</li><li>Barberi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+engraftable+skeletal+myoblasts+from+human+embryonic+stem+cells.">Derivation of engraftable skeletal myoblasts from human embryonic stem cells.</a> Nat Med. 13, 642-648 (2007).</li><li>Goudenege, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblasts+derived+from+normal+hESCs+and+dystrophic+hiPSCs+efficiently+fuse+with+existing+muscle+fibers+following+transplantation.">Myoblasts derived from normal hESCs and dystrophic hiPSCs efficiently fuse with existing muscle fibers following transplantation.</a> Mol Ther. 11, 2153-2167 (2012).</li><li>Forcales, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal-dependent+incorporation+of+MyoD-BAF60c+into+Brg1-based+SWI/SNF+chromatin-remodelling+complex.">Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex.</a> Embo J. 31, 301-316 (2012).</li><li>Albini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+of+human+embryonic+stem+cells+into+skeletal+muscle+cells+and+generation+of+contractile+myospheres.">Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres.</a> Cell Rep. 3, 661-670 (2013).</li><li>Weintraub, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+muscle-specific+genes+in+pigment,+nerve,+fat,+liver,+and+fibroblast+cell+lines+by+forced+expression+of+MyoD.">Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.</a> Proc Natl Acad Sci U S A. 86, 5434-5438 (1989).</li><li>Tapscott, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD1:+a+nuclear+phosphoprotein+requiring+a+Myc+homology+region+to+convert+fibroblasts+to+myoblasts.">MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts.</a> Science. 242, 405-411 (1988).</li><li>Davis, R. L., Weintraub, H., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+single+transfected+cDNA+converts+fibroblasts+to+myoblasts.">Expression of a single transfected cDNA converts fibroblasts to myoblasts.</a> Cell. 51, 987-1000 (1987).</li><li>Ho, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+embryonic+stem+cell+chromatin+remodeling+complex,+esBAF,+is+essential+for+embryonic+stem+cell+self-renewal+and+pluripotency.">An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency.</a> Proc Natl Acad Sci U S A. 106, 5181-5186 (2009).</li><li>Gomes, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pluripotent+stem+cells+by+using+cryosections+of+embryoid+bodies.">Analysis of pluripotent stem cells by using cryosections of embryoid bodies.</a> J Vis Exp. (46), (2010).</li><li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+lentiviral+vectors.">Production and purification of lentiviral vectors.</a> Nat Protoc. 1, 241-245 (2006).</li><li>Barcova, M., Campa, V. M., Mercola, M., Loring, J. F., Wesselschmidt, R. L., Schwartz, P. 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We have nothing to disclose.

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 genome6,15. As such, future protocols might be improved by compounds or other manipulations that promote BAF60C expression in hESCs.

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)1-3 or by exposure to specific culture conditions4-5. 

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 loci6. We have recently discovered that the selective absence of BAF60C confers onto hESCs the resistance to MyoD-mediated activation of skeletal myogenesis7 that is otherwise observed in BAF60C expressing somatic cells8-10, 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 lineage7. Of note, previous proteomic analysis revealed the selective absence of BAF60C, among the SWI/SNF components, in ESCs11. 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 muscles7. 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 &#8220;disease in a dish&#8221; model of muscular diseases. When generated from patient 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.12

<table><tbody><tr><td>6-well plate</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>6-well low attachment plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>GFR Matrigel</td><td>BD Biosciencies</td><td>356231</td><td></td></tr><tr><td>MEFs (growth arrested)&lt;sup&gt;14&lt;/sup&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td></td></tr><tr><td>DMEM-F12</td><td>Invitrogen</td><td>15-090-CV</td><td></td></tr><tr><td>KOSR</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>1 mM L-glutamine (100X)</td><td>Invitrogen</td><td>35050-61</td><td></td></tr><tr><td>Pen/Strep (100X)</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Invitrogen</td><td>21985-023</td><td></td></tr><tr><td>NEAA (100X)</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td>ITS Liquid Media Supplement</td><td>Sigma</td><td>I3146</td><td></td></tr><tr><td>N2-supplement&amp;nbsp;</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>TrypLE express</td><td>Invitrogen</td><td>12605-010</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SH30396.03</td><td></td></tr><tr><td>HS</td><td>Invitrogen</td><td>16050114</td><td></td></tr><tr><td>&lt;a href=&quot;https://www.stemgent.com/products/193&quot;&gt;hES Cell Cloning &amp;amp; Recovery Supplement&lt;/a&gt;</td><td>Stemgent</td><td>01-0014-100</td><td></td></tr><tr><td>bfgf</td><td>Sigma</td><td>4114-TC</td><td></td></tr><tr><td>mTeSR1</td><td>Stemcell Technologies</td><td>5850</td><td></td></tr><tr><td>Anti-MyoD</td><td>BD Biosciences</td><td>554130</td><td></td></tr><tr><td>Anti-MyHC</td><td>DSHB</td><td>MF20</td><td></td></tr><tr><td>Anti-myogenin</td><td>DSHB</td><td>F5D</td><td></td></tr></tbody></table>

generation of myospheres from hescs by epigenetic reprogramming

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.

Figure 1A shows a schematic of the protocol proposed to generate myospheres from hESCs. 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 take advantage of a lentiviral vector encoding BAF60C that carries GFP fluorescence. 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. compound12 and sectioned to perform immunofluorescence for myogenic markers to check myogenic conversion efficiency. 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).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51243/51243fig1highres.jpg" src="/files/ftp_upload/51243/51243fig1.jpg" /> 
Figure 1. A) Schematic of the protocol used for the generation of myospheres from hESCs. 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). Scale bar is 100 &#181;m.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51243/51243fig2highres.jpg" src="/files/ftp_upload/51243/51243fig2.jpg" /> 
Figure 2. 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 &#181;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.

Watch this Scientific Journal Video about Generation of Myospheres From hESCs by Epigenetic Reprogramming at JoVE.com

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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7.8K Views.  Sanford-Burnham Institute for Medical Research. Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Video: Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Developmental Biology

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAIN&#945;/&#946;), (&#946;III TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

Generation of Induced Pluripotent Stem Cells from Turner Syndrome 45XO Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

This protocol describes the generation of integration free iPSCs from fetal tissue fibroblasts through delivery of episomal plasmids by nucleofection followed by description of methods used for iPSC characterization and neuronal differentiation.

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
The video demonstrates a method to generate astrospheres from progenitors from human pluripotent stem cells. It involves the growth and proliferation of stem cells with nutrient media containing growth factors and kinase inhibitors. These cells then aggregate and differentiate into astrocytes, forming astrospheres.

1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		Dilute extracellular matrix (ECM) coating solution with DMEM/F12 ...