The authors thank Giulio Cossu, Martina Ragazzi and the whole laboratory for helpful discussion and support, and Jeff Chamberlain for kindly providing MyoD-ER vector. All the experiments involving live animals were completed in accordance and compliance with all relevant regulatory and institutional agencies, regulations and guidelines. Work in the authors laboratory is supported by UK Medical Research Council, European Community 7th Framework projects Optistem and Biodesign and the Italian Duchenne Parent Project.

1. Assessment of Myogenic and Engraftment Potential
<ol>
	<li>In vitro: MyoD-induced differentiation
	<ol>
		<li>Generate a stable cell line of IDEMs transduced with the tamoxifen-inducible MyoD-ER lentiviral vector, titrating the multiplicity of infection (MOI; e.g.&#160;1, 5 and 50) using the staining described in 1.1.9 (MyHC) as an outcome of the efficiency of the procedure.</li>
		<li>Coat a 3.5 cm dish with 1 ml of 1% Matrigel and incubate for 30 min at 37 &#176;C.</li>
		<li>Wash the plate twice...

<ol>
	<li>Dellavalle, A., 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=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat. Cell Biol. 9, 255-267 (2007).</li><li>Minasi, M. G., 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=The+meso-angioblast:+a+multipotent,+self-renewing+cell+that+originates+from+the+dorsal+aorta+and+differentiates+into+most+mesodermal+tissues.">The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues.</a> Development. 129, 2773-2783 (2002).</li><li>Dellavalle, A., 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=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat. Commun. 2, 499 (2011).</li><li>Tedesco, F. S., Dellavalle, A., Diaz-Manera, J., Messina, G., Cossu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repairing+skeletal+muscle:+regenerative+potential+of+skeletal+muscle+stem+cells.">Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells.</a> J. Clin. Invest. 120, 11-19 (2010).</li><li>Sampaolesi, 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=Cell+therapy+of+alpha-sarcoglycan+null+dystrophic+mice+through+intra-arterial+delivery+of+mesoangioblasts.">Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts.</a> Science. 301, 487-492 (2003).</li><li>Sampaolesi, 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=Mesoangioblast+stem+cells+ameliorate+muscle+function+in+dystrophic+dogs.">Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.</a> Nature. 444, 574-579 (2006).</li><li>Tedesco, F. 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=Transplantation+of+Genetically+Corrected+Human+iPSC-Derived+Progenitors+in+Mice+with+Limb-Girdle+Muscular+Dystrophy.">Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy.</a> Sci. Transl. Med. 4, (2012).</li><li>Tedesco, F. 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=Stem+cell-mediated+transfer+of+a+human+artificial+chromosome+ameliorates+muscular+dystrophy.">Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.</a> Sci. Transl. Med. 3, (2011).</li><li>Lattanzi, 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=High+efficiency+myogenic+conversion+of+human+fibroblasts+by+adenoviral+vector-mediated+MyoD+gene+transfer.+An+alternative+strategy+for+ex+vivo+gene+therapy+of+primary+myopathies.">High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.</a> J. Clin. Invest. 101, 2119-2128 (1998).</li><li>Chaouch, 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=Immortalized+skin+fibroblasts+expressing+conditional+MyoD+as+a+renewable+and+reliable+source+of+converted+human+muscle+cells+to+assess+therapeutic+strategies+for+muscular+dystrophies:+validation+of+an+exon-skipping+approach+to+restore+dystrophin+in+Duchenne+muscular+dystrophy+cells.">Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: validation of an exon-skipping approach to restore dystrophin in Duchenne muscular dystrophy cells.</a> Hum. Gene Ther. 20, 784-790 (2009).</li><li>Kimura, E., 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=Cell-lineage+regulated+myogenesis+for+dystrophin+replacement:+a+novel+therapeutic+approach+for+treatment+of+muscular+dystrophy.">Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy.</a> Hum. Mol. Genet. 17, 2507-2517 (2008).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell Stem Cell. 10, 678-684 (2012).</li><li>Wu, S. M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+potential+of+induced+pluripotent+stem+cells+for+regenerative+medicine.">Harnessing the potential of induced pluripotent stem cells for regenerative medicine.</a> Nat. Cell Biol. 13, 497-505 (2011).</li></ol>

iPSCs can be expanded indefinitely while preserving their self-renewal potential and thus be directed to differentiate into a broad range of cell lineages12. For this and other reasons iPSC-derived stem/progenitor cells are considered a promising source for autologous gene and cell therapy approaches13. A previously published work from the Authors reported the generation of mouse and human myogenic progenitors from iPSCs with a phenotype similar to that of pericyte-derived MABs (IDEMs) and their eff...

Mesoangioblasts (MABs) are vessel-associated muscle progenitors derived from a subset of pericytes1-3. The main advantage of MABs over canonical muscle progenitors such as satellite cells4 resides in their ability to cross the vessel wall when delivered intra-arterial and hence contribute to skeletal muscle regeneration in cell therapy protocols. This feature has been evaluated and confirmed in both murine and canine models of muscular dystrophy1,5,6. These preclinical studies have built the foundations for a first-in-man phase I/II clinical trial based upon intra-arterial transplantation of donor HLA-identical MABs in children with Duchenne muscular dystrophy (EudraCT no. 2011-000176-33; currently on going at San Raffaele Hospital of Milan, Italy). One of the main hurdles of cell therapy approaches, where billions of cells are needed to treat the muscle of an entire body, is the limited proliferative potential of the &#34;medicinal product&#34; (the cells). Moreover it has been recently demonstrated that it is not possible to obtain MABs from patients affected by some forms of muscular dystrophies, as it occurs in limb-girdle muscular dystrophy 2D (LGMD2D; OMIM #608099)7.
To overcome these limitations, a protocol for deriving MAB-like stem/progenitor cells from human and murine iPSCs has been recently established7. This procedure generates easily expandable cell populations with a vascular gene signature and immunophenotype highly similar to adult muscle-derived MABs. Upon expression of the myogenic regulatory factor MyoD, both murine and human IDEMs (respectively MIDEMs and HIDEMs) undergo terminal skeletal muscle differentiation. To achieve this aim IDEMs can be transduced with vectors able to drive MyoD expression, either constitutively or in an inducible fashion8-10. A lentiviral vector containing MyoD cDNA fused with the estrogen receptor (MyoD-ER) is used, thus allowing its nuclear translocation upon tamoxifen (an estrogen analogue) administration11. This strategy results in the formation of hypertrophic multinucleated myotubes, with high efficiency7. Notably, IDEMs are nontumorigenic, can engraft and differentiate inside host muscles upon transplantation and can be genetically corrected using different vectors (e.g. lentiviruses or human artificial chromosomes), paving the way for future autologous therapeutic strategies. The derivation, characterization and transplantation of IDEMs have been described in the above publication7. This protocol paper details the assays performed to evaluate the in vitro myogenic differentiation of IDEMs and the subsequent outcome measurements to test the efficacy of their transplantation in mouse models of muscle regeneration.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>MegaCell DMEM</td>
			<td>Sigma</td>
			<td>M3942</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Sigma</td>
			<td>I3390</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Euroclone</td>
			<td>ECS0090L</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Lonza</td>
			<td>DE14801F</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Calcium/Magnesium free</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutammine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilline/Streptomicin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS (Insulin-Transferrin-Selenium)</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acid solution</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Fer-In-Sol</td>
			<td>Mead Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ferlixit</td>
			<td>Aventis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic Acid</td>
			<td>Sigma</td>
			<td>01257-10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>Sigma</td>
			<td>L5900-10 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Human bFGF</td>
			<td>Gibco</td>
			<td>AA 10-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Grow factors-reduced Matrigel</td>
			<td>Becton Dickinson</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium heparin</td>
			<td>Mayne Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Patent blue dye</td>
			<td>Sigma</td>
			<td>19, 821-8</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>TAAB</td>
			<td>P001</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>4-OH Tamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td></td>
		</tr>
		<tr>
			<td>pLv-CMV-MyoD-ER(T)</td>
			<td>Addgene</td>
			<td>26809</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma</td>
			<td>C9759</td>
			<td>HARMFUL</td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tragachant gum</td>
			<td>MP biomedicals</td>
			<td>104792</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopenthane</td>
			<td>VWR</td>
			<td>24,872,323</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>27,480,294</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarized glass slides</td>
			<td>Thermo</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4382</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Bio-Optica</td>
			<td>04-010802</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti Myosin Heavy Chain antibody</td>
			<td>DSHB</td>
			<td>MF20</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti Lamin A/C antibody</td>
			<td>Novocastra</td>
			<td>NLC-LAM-A/C</td>
			<td></td>
		</tr>
		<tr>
			<td>4/11/13</td>
			<td>Cappel</td>
			<td>559762</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma fluka</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti Laminin antibody</td>
			<td>Sigma</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>MATERIALS AND EQUIPMENT</td>
		</tr>
		<tr>
			<td>Adsorbable antibacteric suture 4-0</td>
			<td>Ethicon</td>
			<td>vcp310h</td>
			<td></td>
		</tr>
		<tr>
			<td>30G needle syringe</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>Columbus instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steromicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane unit</td>
			<td>Harvad Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optics</td>
			<td>Euromecs (Holland)</td>
			<td>EK1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Vet Tech</td>
			<td>C17A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Swann-Morton</td>
			<td>11REF050</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Fine Scientific Tools</td>
			<td></td>
			<td>5/45</td>
		</tr>
		<tr>
			<td>High temperature cauteriser</td>
			<td>Bovie Medical</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>MEDIA COMPOSITION</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Media composition is detailed below. 
			HIDEMs growth medium:
			<ul>
				<li>MegaCell Dulbecco&#39;s Modified Eagle Medium (MegaCell DMEM)</li>
				<li>5% fetal bovine serum (FBS)</li>
				<li>2 mM glutamine</li>
				<li>0.1 mM &#946;-mercaptoethanol</li>
				<li>1% non essential amino acids</li>
				<li>5 ng / ml human basic fibroblast growth factor (bFGF)</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
			</ul>
			Alternatively, if some of the above reagents are not available, HIDEMs can be grown in:
			<ul>
				<li>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</li>
				<li>10 % FBS</li>
				<li>2 mM glutamine</li>
				<li>0.1 mM &#946;-mercaptoethanol</li>
				<li>1% Non essential amino acids (NEAA)</li>
				<li>1% ITS (insulin / transferrin / selenium)</li>
				<li>5 ng / ml human bFGF</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
				<li>0.5 &#956;M oleic and linoleic acids</li>
				<li>1.5 &#956;M Fe2+ (Fer-In-Sol)</li>
				<li>0.12 &#956;M Fe3+ (Ferlixit)</li>
			</ul>
			MIDEMs growth medium:
			<ul>
				<li>DMEM</li>
				<li>20 % FBS</li>
				<li>2 mM glutamine</li>
				<li>5 ng / ml human bFGF</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
			</ul>
			Differentiation medium:
			<ul>
				<li>DMEM</li>
				<li>2 % Horse Serum (HS)</li>
				<li>100 IU ml penicillin</li>
				<li>100 mg / ml streptomycin</li>
				<li>2 mM glutamine</li>
			</ul>
			 
			 
			</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. List of Reagents, Materials, Equipment, and Media.

transplantation of induced pluripotent stem cell-derived mesoangioblast-like myogenic progenitors in mouse models of muscle regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

The reported representative results follow the main in vitro/in vivo assays depicted in the workflow in Figure 1. 48 hr after 4OH-tamoxifen administration MyoD-positive nuclei are identifiable within MyoD-ER transduced IDEMs in culture (Figure 2A). The cells then fuse and differentiate into multinucleated myotubes (Figure 2B). When transplanted intramuscularly into a murine model of acute muscle injury, IDEMs contribute to tissue regeneration (Figure 3</...

Watch this Scientific Journal Video about Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration at JoVE.com

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

transplantation-induced-pluripotent-stem-cell-derived-mesoangioblast

Research

JoVE Journal

Bioengineering

9.3K visualizações.  University College London. Progenitores miogênicos derivados de células-tronco pluripotentes induzidas (iPSC) são candidatos promissores para estratégias de terapia celular para tratar distrofias musculares. Este protocolo descreve transplantes e medidas funcionais necessárias para avaliar o enenxerto e diferenciação dos mesoangioblastos derivados do iPSC (um tipo de progenitores musculares) em modelos de camundongos de regeneração muscular aguda e crônica.