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 with medium, seed 1 x 105 MyoD-ER transduced IDEMs in the 3.5 cm tissue culture dish and incubate at 37 &#176;C in growth medium.</li>
		<li>Expect that cells reach confluence in one or two days and then add 1 &#956;M 4OH-tamoxifen into the growth medium (1st dose).</li>
		<li>After 24 hr, replace the growth medium with differentiation medium supplemented with 1 &#956;M 4OH-tamoxifen (2nd and last dose).</li>
		<li>Replace half of the medium with fresh differentiation medium every other day.</li>
		<li>Examine daily the cultures for myotube formation.</li>
		<li>After one week (5 days in differentiation medium), wash the plates gently with PBS and fix with 4 % paraformaldehyde for 5 min at RT.</li>
		<li>Perform an immunofluorescence staining with antibodies against myosin heavy chain (MyHC) to confirm the presence of myotubes. Counterstain with a nuclear dye (e.g.&#160;Hoechst).</li>
	</ol>
	</li>
</ol>
The efficiency of differentiation is evaluated as the percentage of nuclei inside MyHC-positive cells: proceed to the next step if the efficiency is &#62;50%.
<ol start="2">
	<li>In vivo: engraftment in a model of acute muscle regeneration 
	To evaluate the in vivo contribution of MyoD-ER IDEMs to muscle regeneration, the cells are previously labeled with a vector encoding for the green fluorescent protein (GFP) that will enable to trace them inside the tissue. MyoD-ER GFP IDEMs are then injected into adult murine muscles, previously injured with a myotoxin (e.g. cardiotoxin). To avoid immune rejection against xenogeneic (such as HIDEMs) or genetically manipulated/corrected cells it is required to use either immunodeficient or immunosuppressed mice.
	<ol>
		<li>Pretreat the animals with an intra-peritoneal injection of 3.5 &#956;l/g of 10 mg/ml tamoxifen (liposoluble form) 24 hr before transplantation and pretreat cells adding 1 &#956;M 4OH-tamoxifen (aqueous form) into the growth medium overnight before the transplantation day.</li>
		<li>Administer anesthesia and analgesia to the mouse following the specific guidelines that regulate surgical procedures in the animal facility.</li>
		<li>Inject 25 &#956;l of 100 &#956;M cardiotoxin (CTX; from Naja mossambica mossambica; CAUTION: potentially harmful substance) in the tibialis anterior (TA) muscles.</li>
		<li>24 hr after treating the animals with tamoxifen and CTX, detach the cells by trypsinization and count.</li>
		<li>Centrifuge the cells at 232 x g for 5 min.</li>
		<li>Wash the cell pellet in Ca2+- and Mg2+-free PBS, centrifuge and then gently resuspend the cell pellet in Ca2+- and Mg2+-free PBS to a final concentration of 106 cells/30 &#956;l, which will be the final volume of each injection.</li>
		<li>Inject 30 &#956;l of cell suspension into the previously injured muscles using a syringe with 29 or 30 G needle. Pay particular attention while removing the needle from the muscle. Do it slowly, avoiding spilling the cell suspension through the needle&#39;s track. Do not transplant the contralateral TA and use it as control, injecting 30 &#956;l of Ca2+- and&#160;Mg2+-free PBS to replicate the conditions of the transplanted one.</li>
		<li>Treat the animals with tamoxifen for six additional days and administer analgesia (e.g.&#160;Carprofen) for two additional days.</li>
		<li>Explant the muscles from 14 days after transplantation onwards. Process and analyze the samples as detailed in Protocols 4 and 5.</li>
	</ol>
	</li>
</ol>
2. Transplantation in Mouse Models of Muscular Dystrophy
This transplantation assay allows evaluating the extent of engraftment of IDEMs in mouse models of muscular dystrophy. The animals, treated as follows, can also be assessed for functional amelioration of the disease phenotype. Functional tests can be performed starting from two weeks after transplantation. In order to enhance engraftment consider performing pretransplantation treadmill exercise (as described in Protocol 3) and/or serial cell injections every three weeks for 3x (i.e.&#160;for a total of 3 injections/muscle).
<ol>
	<li>Intramuscular transplantation
	<ol>
		<li>Pretreat the animals with an intra-peritoneal injection of 3.5 &#956;l/g of 10 mg/ml tamoxifen (liposoluble formulation) 24 hr before transplantation and pretreat cells adding 1 &#956;M 4OH-tamoxifen (aqueous formulation) into the growth medium overnight before the transplantation day.</li>
		<li>Detach by trypsinization, count and centrifuge the cells at 232 x g for 5 min.</li>
		<li>Wash the cell pellet in Ca2+- and Mg2+-free PBS, centrifuge and then resuspend the pellet in Ca2+- and Mg2+-free PBS to a concentration of 106 cells/30 &#956;l.</li>
		<li>Administer analgesia and disinfect the skin of the animal (optional) with a povidone iodine- or chlorexidine-based disinfectant. This will also help to localize the tibialis anterior (TA), gastrocnemius (GC), and quadriceps femoris (QC; specifically the vastus intermedius) muscles.</li>
		<li>Inject 30 &#956;l of cell suspension into the muscles using a syringe with 29 or 30 G needle. For the TA, insert 5 mm of the needle 2 mm below the insertion of the proximal tendon (craniocaudal direction) with a 15&#176; inclination relative to the tibia and slowly inject the cell suspension while retracting the needle (empty the syringe with 2 mm of the needle still inside the muscle). For the GC and QC, repeat the same procedure as detailed for the TA, with the main difference being the caudocranial insertion of the needle 2 mm above the myotendinous junction of the Achilles tendon for the GC and 2 mm above the distal tendon for the QC (15&#176; inclination with respect to the femur). Pay attention while removing the needle from the muscle in order to avoid spilling the cell suspension through the needle&#39;s track. 
		TROUBLESHOOTING: In case of low engraftment consider injecting juvenile (1-2 weeks old) mice7 with 3 x 105 cells/10 &#956;l (note that in this case tamoxifen needs to be administered subcutaneously).</li>
	</ol>
	</li>
	<li>Intra-arterial transplantation 
	This part of the protocol allows the evaluation of the ability of MIDEMs and HIDEMs to be delivered in the arterial circulation, cross the vessel wall and contribute to skeletal muscle regeneration in the muscles downstream to the injection site.
	<ol>
		<li>Pretreat animals and cells as described above in step 2.1.1.</li>
		<li>Detach by trypsinization and filter with a 40 &#956;m cell strainer (to remove clusters from the cell suspension in the unlikely event that overnight exposure to tamoxifen could drive fusion and formation of myotubes from adjacent cells) count and centrifuge the cells at 232 x g for 5 min</li>
		<li>Wash the cell pellet in Ca2+- and Mg2+-free PBS, centrifuge and then resuspend the pellet in Ca2+- and Mg2+-free PBS with 0.2 International 
		Units of sodium heparin (optional) to a final cell concentration of 106 cells/50 &#956;l. Add 10% Patent blue dye (final concentration: 1.25 mg/ml in normal saline or Ca2+- and Mg2+-free PBS) to the solution in order to visualize the distribution of the cell suspension.</li>
		<li>Administer anesthesia and analgesia to the mouse according to the guidelines regulating surgical procedures in the specific institutional animal facility.</li>
		<li>Shave the inguinal region (also know as femoral or Scarpa&#39;s triangle) and disinfect the skin with a povidone iodine- or chlorhexidine-based disinfectant.</li>
		<li>Make a 5-7 mm incision and localize the femoral bundle: vein, artery and nerve (the nerve lays laterally to the artery and the vein medially).</li>
		<li>Gently remove the connective fascia that covers the bundle with forceps.</li>
		<li>Separate the femoral vein and nerve from the artery by gently introducing the tip of a forceps (or a 30 G needle) in between them and by progressively enlarging the hole. 
		TROUBLESHOOTING: The femoral vein is fragile: pay attention not to pinch it with the forceps during its detachment from the femoral artery. In case of hemorrhage, drain the blood with sterile gauze and use a micro cauterizer to facilitate hemostasis.</li>
		<li>Lift the artery with one tip of the forceps and clamp the artery with the other tip.</li>
		<li>Puncture the artery with a syringe equipped with a 30 G needle. Inject 50 &#956;l of cell suspension downstream of the clamped area, at an infusion speed of approximately 5 &#956;l/sec. 
		TROUBLESHOOTING: Carefully resuspend the cells in the syringe before the injection: it is vital to avoid precipitation of cells and air bubble formation inside the syringe. The diameter of the femoral artery is slightly smaller than a 30 G needle: be careful not to truncate the artery while inserting the needle. Patent blue dye allows recognizing the effectiveness of the injection: if correctly injected, the whole limb will rapidly turn light blue.</li>
		<li>Slowly remove the needle and the forceps from the artery to restore bloodstream in the limb.</li>
		<li>Apply pressure with sterile gauze to avoid bleeding and/or cauterize as required.</li>
		<li>Suture the wound and monitor the animals until recovery from anesthesia.</li>
		<li>Administer analgesia for 3 days and inspect the wound daily (in case of wound infection discuss this with the animal facility personnel and administer antibiotics as required).</li>
	</ol>
	</li>
</ol>
3. Outcome Measures on Transplanted Dystrophic Animals: Treadmill Test
Starting from two weeks after cell transplantation, it is possible to evaluate functional amelioration of the motor capacity of treated mice with the treadmill tests. This test allows the evaluation of exercise tolerance/endurance of treated mice. A mouse is considered fatigued when lays in the resting area for more than 5 sec, without attempting to reengage the treadmill after a series of 3 consecutive mechanical stimuli (one every 5 sec). Baseline measurements start approximately one month before treatment and are used to evaluate the improvement of each single tested animal. This test can be followed by additional assays to monitor fiber fragility, force improvement, engraftment, differentiation of transplanted cells, and morphological amelioration of the transplanted muscles (see Discussion).
<ol>
	<li>Acclimatize the animals (usually three groups: treated, untreated,&#160;and wild type/non dystrophic controls) to the exercise before the first measurement: set the treadmill at a speed of 6 m/min&#160;for 10 min. Repeat the procedure every other day for one week. 
	TROUBLESHOOTING: Record all the measurements at the same hour of the day to avoid biases owing to the circadian cycles.</li>
	<li>Place the animals into the treadmill, which has been previously set up with an inclination of 10&#176;.</li>
	<li>Turn on the treadmill, with a starting speed of 6 m/min (provide a gentle mechanical stimulus in case the animals are not willing to start the exercise).</li>
	<li>Start the timer and increase the speed 2 m/min&#160;every 2 min.</li>
	<li>As soon as an animal lies in the resting area for more than 5 sec without attempting to reengage the treadmill, gently touch it with a stick to stimulate the restart of the exercise (see above).</li>
	<li>Record the performance (time and or distance) of each animal.</li>
	<li>Repeat the measurements weekly or every 10 days for at least 3x.</li>
	<li>Repeat the same procedure after transplantation.</li>
	<li>Analyze performance data comparing the measurement of each animal to its baseline performance. We suggest to plot the values in a graph as a percentage of the average motor capacity relative to baseline and to analyze them by a one- or two-way ANOVA test followed by appropriate post-test to compare the groups.</li>
</ol>
TROUBLESHOOTING: use a minimum of 5 age-, genotype-, and sex-matched animals/group and repeat the measurements for at least 3x after transplantation.
4. Evaluation of Cell Engraftment and Differentiation in Transplanted Muscles
Transplanted and control muscles are harvested at the appropriate time point (&#60;2 days for short-term engraftment analysis, 2-3 weeks for mid-term and &#62;1 month for long-term analysis). If the transplanted cells are labeled with GFP, the engraftment in freshly isolated muscles can be assessed by direct fluorescence under an UV-equipped stereomicroscope.
<ol>
	<li>Lay and orient along the vertical axis freshly isolated muscles in tragachanth gum (6% w/v)</li>
	<li>Dehydrate the samples in prechilled isopentane for one minute, freeze them in liquid nitrogen for at least 2 min and place them immediately at -80 &#176;C for storage.</li>
	<li>Process the samples with a cryostat to obtain 7 &#956;m thick sections on polarized slides. Sample the majority of the muscle on the slides, collecting approximately 8-10 slides with 30-40 sections/slide. It is advisable to collect a series of sections into a 1.5 ml tube to perform molecular biology/biochemistry assays.</li>
	<li>Evaluate cell engraftment with different immunofluorescent staining, depending on the experimental setup. For example, in case of HIDEM transplantation in Sgca-null/scid/bg mice, stain sections with: a) an antibody against Lamin A/C to detect grafted human cell nuclei; b) an antibody against Laminin to visualize the overall structure of the muscle; and c) an antibody against Sgca to detect donor-derived restoration of the protein absent in the dystrophic animal.</li>
	<li>Quantify, using a fluorescence microscope, the number of donor nuclei per muscle section inside and outside muscle fibers and the number of donor-derived skeletal myofibers per section.</li>
</ol>
5. Muscle Histopathology
Histopathological analyses allow the evaluation of the morphological structure of the transplanted muscle. Architectural improvement in the tissue structure is expected as an outcome of the cell therapy approach.
<ol>
	<li>Fix the freshly isolated muscles with 4% paraformaldehyde for 1 hr at 4 &#176;C.</li>
	<li>Dehydrate the samples with an ascending sucrose gradient (e.g. 7.5-15-30% w/v).</li>
	<li>Leave the muscles overnight in the highest sucrose solution.</li>
	<li>Embed the samples in Tissue Tek OCT, place them in prechilled isopentane until the OCT becomes solid (avoiding complete immersion of the samples), freeze them in liquid nitrogen for at least 2 min and place them immediately at -80 &#176;C for storage.</li>
	<li>Process the samples with a cryostat to obtain 7 &#956;m thick sections as described above.</li>
	<li>Stain the sections with hematoxylin and eosin or Masson&#39;s trichrome according to standard manufacturer&#39;s protocols.</li>
</ol>
Hematoxylin and eosin staining enables calculating hallmarks of regenerating muscle, such as: a) the number of myofibers; b) cross sectional area; c) the number of myofibers containing a central nucleus. Masson&#39;s trichrome is used to calculate the fibrotic index, done by subtracting the total area occupied by the skeletal myofibers from the total area of the image: the resulting area mainly reflects the connective and fat infiltrate of the muscle. All the analyses on the images could be performed using ImageJ software (NIH) with the measurement tool and cell counter plugin.

<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>

F.S.T. has filed an international patent application (no. PCT/GB2013/050112) including part of the strategy described in this article. He received funding by Promimetic and DWC ltd. for his research.

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

9.4K vues.  University College London. Les progéniteurs myogéniques dérivés de cellules souches pluripotentes induites (csps) sont des candidats prometteurs pour les stratégies de thérapie cellulaire visant à traiter les dystrophies musculaires. Ce protocole décrit la transplantation et les mesures fonctionnelles exigées pour évaluer l’engraftment et la différentiation des mesoangioblasts iPSC-dérivés (un type de progéniteurs de muscle) dans des modèles murins de régénération aiguë et chro...

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...