Name: generation of myospheres from hescs by epigenetic reprogramming
Uploaded: 2014-06-21T14:00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Generation of Myospheres From hESCs by Epigenetic Reprogramming at JoVE.com

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. 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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 border="0" cellpadding="0" cellspacing="0" width="841">
	<colgroup>
		<col span="3" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">6 well plate</td>
			<td style="width:259px;">Corning</td>
			<td style="width:259px;">3506</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">6 well low attachment plate</td>
			<td style="width:259px;">Corning</td>
			<td style="width:259px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFR Matrigel</td>
			<td style="width:259px;">BD Biosciencies</td>
			<td style="width:259px;">356231</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:259px;">MEFs (growth arrested)14</td>
			<td style="width:259px;"></td>
			<td style="width:259px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Collagenase IV</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">DMEM-F12</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">15-090-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">KOSR</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">1mM L-glutamine (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">35050-61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Pen/Strep (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">2-Mercaptoethanol</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NEAA (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Polybrene</td>
			<td style="width:259px;">Millipore</td>
			<td style="width:259px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">ITS Liquid Media Supplement</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:259px;">I3146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">N2-supplement&#160;</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">TrypLE express</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">FBS</td>
			<td style="width:259px;">HyClone</td>
			<td style="width:259px;">SH30396.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">HS</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">16050114</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:259px;"><a href="https://www.stemgent.com/products/193">hES Cell Cloning &#38; Recovery Supplement</a></td>
			<td style="width:259px;">Stemgent</td>
			<td style="width:259px;">01-0014-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">bfgf</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:259px;">4114-TC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">mTeSR1</td>
			<td style="width:259px;">Stemcell Technologies</td>
			<td style="width:259px;">5850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-MyoD</td>
			<td style="width:259px;">BD Biosciences</td>
			<td style="width:259px;">554130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-MyHC</td>
			<td style="width:259px;">DSHB</td>
			<td style="width:259px;">MF20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-myogenin</td>
			<td style="width:259px;">DSHB</td>
			<td style="width:259px;">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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Research

JoVE Journal

Biology

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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 Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.