This work was funded by Italian Ministry of Health, Ricerca Corrente to Centro Cardiologico Monzino-IRCCS.

This approach complies with the Declaration of Helsinki and the collection of right ventricular samples was approved (07/06/2012) by &#34;Centro Cardiologico Monzino IRCCS&#34; Ethics Committee.
1. Solutions
<ol>
	<li>Make TMES medium for C-MSC culture with Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), supplemented with 20% Fetal Bovine Serum (FBS), 10 ng/mL basic fibroblast growth factor, 10,000 U/mL Penicillin, 10,000 &#181;g/mL Streptomycin, and 0.02 M L-Glutamine. Filter the TMES medium using a 0.22 &#181;m sterile filter unit, and store at 4 &#176;C.</li>
	<li>To prepare collagenase solution, resuspend the collagenase NB4 mix in IMDM medium to obtain a final solution with a concentration of 3 mg/mL. Vortex gently to dissolve the powder, and filter the collagenase solution using a 0.2 &#181;m syringe filter. Aliquot 1 mL volume into 2 mL sterile tubes and store at -20 &#176;C until needed for use.</li>
	<li>To prepare T. ADIPO medium, make adipogenic medium with IMDM medium supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (pre-diluted in dimethyl sulfoxide (DMSO) at a concentration of 0.5 M and kept at -20 &#176;C until use), 1 &#181;M hydrocortisone (pre-diluted in DMSO at 100 mM and further diluted to 10 mM in sterile distilled water and kept at -20 &#176;C until use) and 0.1 mM indomethacin (pre-diluted in DMSO at a concentration of 0.5 M and kept at -20 &#176;C until use). Filter the T. ADIPO medium using a 0.22 &#181;m sterile filter unit and store at 4 &#176;C.</li>
	<li>Prepare washing buffer composed of phosphate-buffered saline 0.0067 M PO4 (PBS), ethylenediamine tetra acetic acid (EDTA) 5 mM, and 5% bovine serum albumin, and store at 4 &#176;C.</li>
	<li>Prepare oil red O (ORO) working solution by dissolving 1% ORO powder in 60% isopropanol, mixing for 2 h, and filtering using a 0.22 &#181;m filter unit to remove precipitates. Store the working solution at room temperature (RT).</li>
</ol>
2. Isolation of Cardiac Mesenchymal Stromal Cells
<ol>
	<li>Cardiac biopsy collection and processing 
	Note: Before starting, make sure to have the collagenase solution and the TMES medium ready.
	<ol>
		<li>Put the ACM endomyocardial biopsy (usually about 5 mg of tissue) in a sterile tube filled with TMES and transport it to the laboratory. 
		Note: ACM biopsies are collected in the electrophysiology operating room, according to previous reports10. Briefly, the integration of electro-anatomical mapping and intracardiac echocardiography in the right ventricle (see Video 1) is used to guide the endomyocardial biopsy (see Video 2, fluoroscopy) in correspondence to the border zone of the diseased myocardium. Healthy Control (HC) samples are supplied by a tissue bio-bank, obtained from the right ventricular free wall of cadaveric donors within 24 h of death (accidental death, healthy subjects). During transport and prior to dissection, store the biopsy in TMES at 4 &#176;C. Limit the time between biopsy collection and tissue processing (max 24 h).</li>
		<li>Set up the biological safety cabinet with required solutions to carry out the procedure up to the enzymatic digestion.</li>
		<li>Put the sterilizer under the hood and turn on the instrument 15 min before use until the temperature rises 200 - 250 &#176;C.</li>
		<li>Sterilize scissors and tweezers for 10 s. Make sure the sterilized instruments are cold before use. Prepare disposable sterile scalpels.</li>
		<li>Place the tubes with the biopsy in the sterile hood. Open the tube and transfer the biopsy into a sterile plate.</li>
		<li>Wash the right ventricle biopsy twice with 3 mL of sterile PBS. If you want take a picture of the biopsy at the microscope.</li>
		<li>Transfer the right ventricle biopsy, using sterile tweezers, into a sterile 2 mL tube containing 1 mL of the collagenase solution.</li>
		<li>Cut the sample into 0.5 - 1 mm3 pieces with sterile scissors.</li>
		<li>Incubate for 1.5 h at 37 &#176;C under continuous rotating agitation.</li>
		<li>Centrifuge the digested solution at 400 x g for 10 min at RT.</li>
		<li>Remove the supernatant and add 1 mL of sterile PBS to wash the pellet.</li>
		<li>Centrifuge at 400 x g for 10 min at RT.</li>
		<li>Remove the supernatant and resuspend the pellet in 1 mL of TMES medium.</li>
		<li>Plate the obtained suspension onto sterile 60-mm tissue culture treated plate and add TMES medium to the final volume of 3 mL. Incubate the plate in a cell culture incubator at 37 &#176;C with 5% CO2. 
		Note: A higher volume of medium may prevent the correct adhesion of a digested biopsy fragments.</li>
		<li>After 24 h, discard the medium to remove non-adherent cells and debris. 
		Note: Single dissociated mesenchymal cells can attach to the plastic surface and give rise to clones; also, undigested small biopsy fragments can attach to the plastic dish and allow cell sprouting.</li>
		<li>Wash the plate twice with 5 mL of sterile PBS, and add 3 mL of fresh TMES medium.</li>
		<li>Replace the TMES medium three times a week until the cells are 80% confluent. 
		Note: The number of attached cells depends on the quality, amount of tissue, and on the efficiency of digestion.</li>
	</ol>
	</li>
</ol>
3. Cell Expansion
Note: Before starting make sure to have TMES medium ready.
<ol>
	<li>When the cells are 80% confluent, transfer the digested small bioptic samples into a new sterile 60-mm tissue culture treated plate, and add 3 mL of fresh TMES medium. 
	Note: The bioptic samples can be re-used for further C-MSC isolation. It is possible to repeat this procedure until there is a significant number of isolated cells.</li>
	<li>Wash the attached cells twice with 3 mL of sterile PBS. Add trypsin-EDTA solution (0.5 mL for a 60-mm dish), and incubate at 37 &#176;C for 3 - 5 min to allow cell detachment.</li>
	<li>When the cells are detached from the surface of the plate, add 0.5 mL of sterile FBS to inactivate the trypsin and collect cells into a new 50 mL sterile tube.</li>
	<li>Wash the plate twice with 5 mL of sterile PBS to add the remaining cells to the same tube.</li>
	<li>Centrifuge the cells at 400 x g for 10 min at RT.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of TMES medium.</li>
	<li>Load 10 &#181;L of the cell suspension in the cell counting chamber. 
	Note: If the cells are too concentrated, dilute the initial suspension 1:10 in PBS and load 10 &#181;L of the diluted cells in the cell counting chamber.</li>
	<li>Under the microscope, count cells in 3 large squares and on the lines of two of their sides and calculate the average number of cells. 
	Note: To obtain the number of cells/mL, the average of counted cells is multiplied by 104, the dilution factor of the cell counting chamber. If the cells have been further diluted, multiply for the dilution factor. The ratio between the cell number to seed and the cell concentration (number of cells/mL) corresponds to the volume (in mL) of initial suspension to be plated for the next step (point 3.9).</li>
	<li>Plate the resuspension onto sterile 100-mm tissue culture treated plates at a final concentration of 5,000 - 10,000 cells/cm2 and add TMES medium to the final volume of 8 mL. Incubate the plate in a cell culture incubator. 
	Note: Repeat the cell expansion procedure when the cells are 70 - 80% confluent. At every passage (P), it is recommended to cryopreserve part of the cells and plate the remaining amount. Expand the cells until P3 or P4 and use for fluorescent-activated cell sorter (FACS) analysis (section 4) and adipogenic differentiation (section 5).</li>
	<li>For the cryopreservation after detachment, centrifuge the cells at 400 x g for 10 min at RT.</li>
	<li>Count cells as described (steps 3.7 - 3.8) and resuspend 1 x 106 cells in 900 &#181;L of sterile FBS. Transfer to a sterile cryo-vial and add 100 &#181;L of sterile DMSO and mix.</li>
	<li>Quickly store the cryo-vials into a freezing container at -80 &#176;C for at least 2 days and then transfer the vials into liquid nitrogen.</li>
</ol>
4. Characterization of Cardiac Mesenchymal Stromal Cells by Flow Cytometry
Note: Before starting, make sure to have the cell dissociation reagent, washing buffer, and the specific antibodies prepared.
<ol>
	<li>When the cell number reaches at least 3 x 106 (count the cells as described in 3.7 - 3.8), wash the 100-mm plate twice with 10 mL of sterile PBS.</li>
	<li>Add 5 mL of a cell dissociation reagent and wait 7 - 10 min at RT to allow the cell detachment.</li>
	<li>When the cells are detached from the surface of the plate, add 15 mL of sterile washing buffer and collect the suspension into a new 50 mL sterile tube.</li>
	<li>Wash the plate twice with 10 mL of washing buffer and add the remaining cells to the same tube.</li>
	<li>Centrifuge the cells at 400 x g for 10 min at RT and resuspend the pellet in 1 mL of washing buffer.</li>
	<li>Count the cells as described (steps 3.7 - 3.8), and transfer 3 x 106 cells into a new sterile tube and add washing buffer to the final volume of 1.5 mL. 
	Note: The total cell number and the total volume of washing buffer depends on the number of selected markers used for the analysis (3 x 105 cells in 100 &#181;L for each FACS polystyrene tube).</li>
	<li>Distribute 100 &#181;L of cellular suspension in 12 different FACS polystyrene tubes, and add the specific antibody at the concentration indicated in the product datasheet. 
	Note: Antibodies used for C-MSC characterization are CD34, CD105, CD45, CD29, CD90, CD44, CD31, CD14, and HLA-DR (Table 1). It is important to prepare a control sample composed of 100 &#181;L of resuspended cells stained with the isotype control to check the specificity of the signal.</li>
	<li>Incubate the samples for 15 min in the dark.</li>
	<li>Add 1 mL of sterile washing buffer to each tube to stop the reaction.</li>
	<li>Centrifuge the cells at 400 x g for 10 min at RT.</li>
	<li>Remove the supernatant and resuspend the pellet in 250 &#181;L of washing buffer.</li>
	<li>Proceed to C-MSC characterization with flow cytometry.</li>
</ol>
5. Adipogenic Differentiation of Cardiac Mesenchymal Stromal Cells
Note: Before starting, make sure to have prepared T. ADIPO medium and ORO working solution.
<ol>
	<li>Culture in T. ADIPO medium 
	<ol>
		<li>Detach and count the cells as described in section 3.</li>
		<li>Plate 3 x 105 cells onto each well of a sterile 6-well tissue culture treated plate in 2 mL of T. ADIPO medium.</li>
		<li>Let C-MSC differentiate in T. ADIPO medium for 72 h or 1 week, changing medium every 2 - 3 days.</li>
	</ol>
	</li>
	<li>Oil Red O staining 
	Note: Use Oil Red O (ORO) staining to test the lipid accumulation in C-MSC.
	<ol>
		<li>Place the 6-well tissue culture plateunder the fume hood.</li>
		<li>Remove the adipogenic medium and wash the cells twice with 2 mL of PBS.</li>
		<li>Add the enough volume of 4% paraformaldehyde (PFA) to cover each well. Fix the cells for 5 min at RT.</li>
		<li>Discard PFA and wash the cells twice with 2 mL of PBS.</li>
		<li>Incubate the fixed cells with ORO working solution for 1 h at RT, using enough volume to cover each well. 
		Note: Do not keep the 6-well tissue culture plate under the fume hood during ORO incubation to avoid the evaporation of ORO working solution.</li>
		<li>Remove all ORO working solution and wash with 2 mL of PBS 3 - 5 times until the plate is completely cleaned; discard washes. Stop the washes when the PBS used is clear and when you don&#8217;t see, under the microscope, unspecific staining out of the cells.</li>
		<li>Capture 20 images at 20X magnification for each well using the inverted tissue culture phase-contrast microscope.</li>
		<li>Open the pictures with the image processing program to quantify the cell ORO accumulation.</li>
		<li>Separate the different color channels through the &#34;split channels&#34; function in order to quantify only the luminance in the 255-red channel.</li>
		<li>Count the number of cells for each picture by counting the nuclei. Normalize the ORO signal intensity on the cell number.</li>
		<li>Calculate the average of the results obtained for each picture of the same sample.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Brooke, 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=Therapeutic+applications+of+mesenchymal+stromal+cells.">Therapeutic applications of mesenchymal stromal cells.</a> Semin Cell Dev Biol. 18 (6), 846-858 (2007).</li><li>Rossini, 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=Human+cardiac+and+bone+marrow+stromal+cells+exhibit+distinctive+properties+related+to+their+origin.">Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin.</a> Cardiovasc Res. 89 (3), 650-660 (2010).</li><li>Dominici, 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Doppler, S. 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=Cardiac+fibroblasts:+more+than+mechanical+support.">Cardiac fibroblasts: more than mechanical support.</a> J Thorac Dis. 9, 36-51 (2017).</li><li>Moore-Morris, T., Guimaraes-Camboa, N., Yutzey, K. E., Puceat, M., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts:+from+development+to+heart+failure.">Cardiac fibroblasts: from development to heart failure.</a> J Mol Med (Berl). 93 (8), 823-830 (2015).</li><li>Jugdutt, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+remodeling+after+infarction+and+the+extracellular+collagen+matrix:+when+is+enough+enough.">Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough.</a> Circulation. 108 (11), 1395-1403 (2003).</li><li>Brown, R. D., Ambler, S. K., Mitchell, M. D., Long, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardiac+fibroblast:+therapeutic+target+in+myocardial+remodeling+and+failure.">The cardiac fibroblast: therapeutic target in myocardial remodeling and failure.</a> Annu Rev Pharmacol Toxicol. 45, 657-687 (2005).</li><li>Sommariva, 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=Cardiac+mesenchymal+stromal+cells+are+a+source+of+adipocytes+in+arrhythmogenic+cardiomyopathy.">Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy.</a> Eur Heart J. 37 (23), 1835-1846 (2016).</li><li>Sommariva, E., Stadiotti, I., Perrucci, G. L., Tondo, C., Pompilio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+models+of+arrhythmogenic+cardiomyopathy:+advances+and+opportunities.">Cell models of arrhythmogenic cardiomyopathy: advances and opportunities.</a> Dis Model Mech. 10 (7), 823-835 (2017).</li><li>Casella, 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=Electroanatomical+mapping+systems+and+intracardiac+echo+integration+for+guided+endomyocardial+biopsy.">Electroanatomical mapping systems and intracardiac echo integration for guided endomyocardial biopsy.</a> Expert Rev Med Devices. 14 (8), 609-619 (2017).</li><li>Beltrami, A. P., 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=Multipotent+cells+can+be+generated+in+vitro+from+several+adult+human+organs+(heart,+liver,+and+bone+marrow).">Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).</a> Blood. 110 (9), 3438-3446 (2007).</li><li>da Silva Meirelles, L., Chagastelles, P. C., Nardi, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+reside+in+virtually+all+post-natal+organs+and+tissues.">Mesenchymal stem cells reside in virtually all post-natal organs and tissues.</a> J Cell Sci. 119, 2204-2213 (2006).</li><li>Cencioni, 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=The+double+life+of+cardiac+mesenchymal+cells:+Epimetabolic+sensors+and+therapeutic+assets+for+heart+regeneration.">The double life of cardiac mesenchymal cells: Epimetabolic sensors and therapeutic assets for heart regeneration.</a> Pharmacol Ther. 171, 43-55 (2017).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> J Orthop Res. 9 (5), 641-650 (1991).</li><li>Germani, A., Foglio, E., Capogrossi, M. C., Russo, M. A., Limana, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cardiac+progenitor+cells+through+epicardial+to+mesenchymal+transition.">Generation of cardiac progenitor cells through epicardial to mesenchymal transition.</a> J Mol Med (Berl). 93 (7), 735-748 (2015).</li><li>Souders, C. A., Bowers, S. L., Baudino, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast:+the+renaissance+cell.">Cardiac fibroblast: the renaissance cell.</a> Circ Res. 105 (12), 1164-1176 (2009).</li><li>Di Maggio, 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=Non-oxidizable+HMGB1+induces+cardiac+fibroblasts+migration+via+CXCR4+in+a+CXCL12-independent+manner+and+worsens+tissue+remodeling+after+myocardial+infarction.">Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction.</a> Biochim Biophys Acta. , (2017).</li><li>Czapla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Cardiac+Mesenchymal+Stromal+Cells+with+CD105+CD34-+Phenotype+Enhance+the+Function+of+Post-Infarction+Heart+in+Mice.">Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.</a> PLoS One. 11 (7), 0158745 (2016).</li><li>Gambini, 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=C-kit++cardiac+progenitors+exhibit+mesenchymal+markers+and+preferential+cardiovascular+commitment.">C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment.</a> Cardiovasc Res. 89 (2), 362-373 (2010).</li><li>Gourdie, R. G., Dimmeler, S., Kohl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+strategies+targeting+fibroblasts+and+fibrosis+in+heart+disease.">Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.</a> Nat Rev Drug Discov. 15 (9), 620-638 (2016).</li><li>Le Blanc, K., Tammik, C., Rosendahl, K., Zetterberg, E., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+expression+and+immunologic+properties+of+differentiated+and+undifferentiated+mesenchymal+stem+cells.">HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.</a> Exp Hematol. 31 (10), 890-896 (2003).</li><li>Detert, 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=The+Atrial+Appendage+as+a+Suitable+Source+to+Generate+Cardiac-derived+Adherent+Proliferating+Cells+for+Regenerative+Cell-based+Therapies.">The Atrial Appendage as a Suitable Source to Generate Cardiac-derived Adherent Proliferating Cells for Regenerative Cell-based Therapies.</a> J Tissue Eng Regen Med. , (2017).</li><li>Miteva, K., 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+cardiac-derived+adherent+proliferating+cells+reduce+murine+acute+Coxsackievirus+B3-induced+myocarditis.">Human cardiac-derived adherent proliferating cells reduce murine acute Coxsackievirus B3-induced myocarditis.</a> PLoS One. 6 (12), 28513 (2011).</li><li>Bassetti, B., Capogrossi, M. C., Pompilio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power+Is+Nothing+Without+Control:+The+Enduring+Search+for+the+Best+Cell+in+Cardiac+Cell+Therapy+at+a+Crossroads.">Power Is Nothing Without Control: The Enduring Search for the Best Cell in Cardiac Cell Therapy at a Crossroads.</a> Circ Res. 119 (9), 988-991 (2016).</li><li>Nigro, P., 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+for+heart+disease+after+15+years:+Unmet+expectations.">Cell therapy for heart disease after 15 years: Unmet expectations.</a> Pharmacol Res. , (2017).</li><li>Ikebe, C., Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+regenerative+therapy:+optimization+of+cell+preparation+protocols.">Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols.</a> Biomed Res Int. 2014, 951512 (2014).</li><li>Marcus, F. I., 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=Diagnosis+of+arrhythmogenic+right+ventricular+cardiomyopathy/dysplasia:+proposed+modification+of+the+task+force+criteria.">Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria.</a> Circulation. 121 (13), 1533-1541 (2010).</li><li>Pieroni, 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=High+prevalence+of+myocarditis+mimicking+arrhythmogenic+right+ventricular+cardiomyopathy+differential+diagnosis+by+electroanatomic+mapping-guided+endomyocardial+biopsy.">High prevalence of myocarditis mimicking arrhythmogenic right ventricular cardiomyopathy differential diagnosis by electroanatomic mapping-guided endomyocardial biopsy.</a> J Am Coll Cardiol. 53 (8), 681-689 (2009).</li><li>Casella, 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=Feasibility+of+combined+unipolar+and+bipolar+voltage+maps+to+improve+sensitivity+of+endomyocardial+biopsy.">Feasibility of combined unipolar and bipolar voltage maps to improve sensitivity of endomyocardial biopsy.</a> Circ Arrhythm Electrophysiol. 8 (3), 625-632 (2015).</li><li>Cooper, L. 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=The+role+of+endomyocardial+biopsy+in+the+management+of+cardiovascular+disease:+a+scientific+statement+from+the+American+Heart+Association,+the+American+College+of+Cardiology,+and+the+European+Society+of+Cardiology+Endorsed+by+the+Heart+Failure+Society+of+America+and+the+Heart+Failure+Association+of+the+European+Society+of+Cardiology.">The role of endomyocardial biopsy in the management of cardiovascular disease: a scientific statement from the American Heart Association, the American College of Cardiology, and the European Society of Cardiology Endorsed by the Heart Failure Society of America and the Heart Failure Association of the European Society of Cardiology.</a> Eur Heart J. 28 (24), 3076-3093 (2007).</li><li>Leone, O., 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=2011+consensus+statement+on+endomyocardial+biopsy+from+the+Association+for+European+Cardiovascular+Pathology+and+the+Society+for+Cardiovascular+Pathology.">2011 consensus statement on endomyocardial biopsy from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology.</a> Cardiovasc Pathol. 21 (4), 245-274 (2011).</li><li>Monsanto, M. 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=Concurrent+Isolation+of+3+Distinct+Cardiac+Stem+Cell+Populations+From+a+Single+Human+Heart+Biopsy.">Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.</a> Circ Res. 121 (2), 113-124 (2017).</li><li>From, A. M., Maleszewski, J. J., Rihal, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+endomyocardial+biopsy.">Current status of endomyocardial biopsy.</a> Mayo Clin Proc. 86 (11), 1095-1102 (2011).</li></ol>

The authors have nothing to disclose.

MSC and C-MSC: MSC are multipotent cells resident in the stromal fraction of different adult tissues, such as bone marrow, adipose tissue, cartilage, brain, skin, fetal annexes, and heart12. Different studies have been performed to isolate and characterize them for potential applications in basic and translational research12,13.
In healthy conditions, MSC are quiescent, self-renewing at low rates14. Since they are exposed to environmental pathological changes, they react fostering tissue remodeling through either direct transdifferentiation, matrix deposition, or the paracrine effect14.
The cardiac MSC (C-MSC) represent a large non-myocyte cell population of the heart4. They originate from the epicardium and migrate into the myocardium undergoing the process of epithelial-to-mesenchymal transition15. They contribute to the mechanical and electrical integrity of the cardiac structure, both in physiological and in pathological states, through interactions with cardiomyocytes and extracellular matrix homeostasis7,16. However, the broad range of C-MSC functions is still not completely understood. A deeper knowledge of their role both in physiological and pathological conditions can be facilitated by in vitro studies performed after their isolation.
C-MSC have been obtained from different districts of the human heart, such as the atrial appendage2,17 and right ventricle18.
Recently, C-MSC from human right ventricular endomyocardial bioptic samples have been obtained8, demonstrating that the source tissue could be as little as 3-5 mg.
Possible applications: The method outlined in this manuscript allows obtaining cells with few simple passages, such as digestion and selection for plastic adherence, from very small heart specimens.
C-MSC can be considered a cell model, since they are easy to amplify and maintain in vitro, and are able to differentiate into cells of mesenchymal lineage (endothelium, osteocytes, and adipocytes). Moreover, the possibility of obtaining cells directly from patients constitutes a great in vitro tool for mechanistic studies in the context of personalized/precision medicine. Indeed, these cells carry the genetic background and eventually specific mutations of the donors, and are influenced by the specific patients&#39; characteristics, such as clinical conditions, age, sex, lifestyle, and medications. Moreover, the possibility of sorting them for different markers may allow the study of specific C-MSC subsets19.
C-MSC are known to be active players in different cardiovascular diseases, mostly characterized by adverse remodeling of the heart. Therefore, they represent candidate targets for novel therapeutic strategies to counteract heart diseases8,20.
C-MSC stem-like properties and their lack of significant immunogenicity suggests their potential application in cell-therapy for cardiac regenerative medicine. Indeed, like MSC from bone marrow or other sources, C-MSC could be potentially used both in autologous and in allogenic settings, without the need for matching between donor and recipient21.
Moreover, C-MSC, being isolated directly from heart tissue, have the advantage of being preconditioned by the cardiac micro-environment and epigenetic profile. In the context of cardiac regenerative medicine, this could be particularly important to obtain successful results.
To date, preclinical studies of regenerative medicine identified useful therapeutic potential in the C-MSC and their paracrine activity18,22,23. Importantly, clinical trials in which the cell source is the heart are underway either with cardiosfere-derived cells or with subpopulations of C-MSC13,24,25. However, as for bone-marrow-derived MSC, different protocols may be necessary to obtain clinical grade C-MSC26.
C-MSC in ACM: The presented protocol is mostly suitable for the study of pathologies for which an endocardial biopsy is indicated. ACM patients undergo bioptic procedures for diagnostic purposes27. Their myocardium is gradually substituted by scar-tissue, an electrically inert tissue composed of adipocytes and fibrosis. In order to guide the bioptic sampling to the scar area, where the diagnostic yield is maximal, endomyocardial mapping is used10,28,29. The samples used in this protocol are taken in the border zone of the diseased myocardium.
Sommariva et al. has recently defined a pivotal role of C-MSC in the pathogenesis of ACM8, demonstrating that C-MSC are active players in ACM heart adipogenesis, since preadipocytes in those hearts are of mesenchymal origin. Moreover, C-MSC isolated with the present protocol from ACM patients&#39; biopsies showed more propensity to both lipid accumulation and adipogenesis than controls. For this reason, these cells could be used to confirm some of the molecular mechanisms of ACM, proving their suitability as a cell model for mechanistic studies9.
Limitations and critical steps: Despite the advantages of obtaining C-MSC directly from patients (see the paragraph &#34;Possible applications&#34;), this protocol is subjected to different limitations.
First of all, the cardiac bioptic procedure is invasive and often avoided if not strictly necessary. Indeed, sampling cardiac tissue is both ethically and technically problematic. Reasons for performing a cardiac biopsy may be the achievement of a definite diagnosis in the context of cardiomyopathies in differential diagnosis, monitoring the status of cardiac transplants, or ascertaining the presence of a heart tumor30. Therefore, only patients for which an endomyocardial biopsy is indicated by consensus statement31 can be enrolled for research on C-MSC. Moreover, the cardiac bioptic procedure can have clinical complications, above all in cardiomyopathic hearts. Therefore, electrophysiologist&#39;s samplings are always cautious and bioptic samples could be very small, compromising the isolation of cells. Future experiments could overcome this issue by tuning collagenase concentration or timing of digestion.
C-MSC, as all primary human cells, show a high variability among different subjects in all phenotypes. Indeed, cells from different subjects are not only genetically different, but also subjected to variable environmental conditioning. Specifically, within this experiment, a high variability in cell isolation, growth, and adipogenic differentiation is observed.
Critical steps of the present protocol have to be acknowledged. If the bioptic sample includes capillaries, they must be removed to avoid the parallel isolation of endothelial cells, which may contaminate the C-MSC culture, and can be evidenced by the FACS analysis (positivity for CD31). To obtain an efficient adipogenic differentiation, cells must be in an active growth phase. The degree of confluence may also influence lipid accumulation.
Significance of the method: With respect to previous methods of isolation of mesenchymal stromal cells, this is the first time where the description of C-MSC obtainment directly from human ventricular bioptic samples is proposed in detail. Although this method is suggested for the processing of ACM patient samples, it is potentially applicable to all the patients for which a cardiac biopsy is indicated.
This protocol represents a useful implementation of previous methods for the obtainment of cells that required bigger cardiac samples32, which are often difficult to collect.
Moreover, the sample source constitutes an interesting innovation. While the ventricular biopsy is usually performed on the septum33, this protocol takes into account samples obtained from the right ventricular free wall. Cells derived from the diseased right ventricular district may be more representative of the pathologic status of diseases involving RV.
In addition, some of the reagents used in the present protocol are different with respect to other C-MSC isolation and differentiation methods32. For example, the type of collagenase proposed in this manuscript is a mix of class I and class II collagenases with a balanced ratio of proteolytic activities. Moreover, the digestion solution is composed of the collagenase mix dissolved in the same basal medium (IMDM) used for the preparation of C-MSC culture medium, allowing isolated C-MSC to adapt to future growth conditions.
In addition, though sorting procedures could standardize the cell batch, using the whole C-MSC population, isolated only through the plastic adherence property of these cells, constitutes a simplification without altering the immunophenotypic characteristics of C-MSC. The composition of T. ADIPO proposed in this manuscript is able to lead to adipogenic differentiation, avoiding the metabolic dysregulation induced by other components such as insulin.
Moreover, the proposed method of lipid accumulation quantification, which is based on the evaluation of the ORO colorimetric intensity, provides more information about the quantity of the accumulated lipids, if compared with methods based only on the percentage of cells positive to the ORO staining. Often lipid accumulation is quantified by extracting the ORO incorporated by cells with isopropanol and measuring its absorbance. However, this method requires more passages and is subjected to variability due to isopropanol evaporation.

Mesenchymal Stromal Cells (MSC) are adult cells with an important supportive function in many tissues1. Bone marrow represents the historical source of MSC, but they can be isolated from different tissues including placenta, adipose tissue, cord blood, liver, and heart1,2.
In 2006, the International Society for Cellular Therapy (ISCT) specified, for the first time, the minimal criteria to define human MSC3. In particular, MSC must have the ability to adhere to plastic. They express specific surface antigens: the positivity for CD44, CD105, CD29, and CD90 mesenchymal markers, and the negativity for CD14, CD45, CD34, CD31 hematopoietic and endothelial markers characterize MSC. Due to the lack expression of HLA-DR, MSC are unable to trigger alloreactivity. Moreover, they are multipotent cells with the potential to differentiate toward adipogenic, chondrocyte, and osteoblast lineages1,3.
Focusing on cardiac cellular composition, cardiac mesenchymal stromal cells (C-MSC) are abundant in a normal adult heart4,5. They play a critical role both in the normal cardiac function and pathological conditions. While, physiologically, C-MSC provide a microenvironment that supports the structural and functional integrity of the myocardium, in heart diseases they are activated in response to heart injury participating in the wound healing and fibrotic remodeling6,7.
Recently, the involvement of C-MSC in arrhythmogenic cardiomyopathy (ACM) adipose substitution has been demonstrated8. In particular, ACM is a genetic disorder mainly caused by mutations in desmosomal genes that lead to myocardial fibro-fatty replacement, mainly in the right ventricle9. This substitution, extending from epicardium to endocardium, creates a non-conductive substrate that elicits progressive heart failure and worsens ventricular arrhythmias, which can lead, in severe cases, to sudden death. Sommariva et al. demonstrated the mesenchymal origin of the pre-adipocytes present in ACM patients&#39; explanted heart sections. Furthermore, C-MSC isolated from endomyocardial biopsies of ACM hearts express desmosomal genes and therefore can be affected by their mutations. In particular, under adipogenic conditions, ACM C-MSC accumulate more lipids than those from control hearts. This evidence leads to the conclusion that C-MSC both have an active role in the disease pathogenesis and represent a valid cell model to study ACM.
To facilitate future research in this context or in other heart diseases for which a cardiac biopsy is indicated, a detailed protocol is presented for the isolation of C-MSC from small fragments of endomyocardial tissue, their expansion, their immuno-phenotypical characterization, and adipogenic differentiation.

<table><tbody><tr><td>IMDM</td><td>Gibco by Life Technologies&amp;nbsp;</td><td>12440053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>PENICILLIN STREPTOMYCIN</td><td>Life Technologies Italia</td><td>15140122</td><td></td></tr><tr><td>Collagenase NB4</td><td>Serva</td><td>17454.02</td><td></td></tr><tr><td>Basic fibroblast growth factor</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>L-glutammine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Tryple select 1X (cell dissociation reagent)</td><td>Gibco</td><td>12563029</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>Trypsin-EDTA solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6689</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2058</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H4001</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>Paraformaldehyde (PFA) Solution 4% in PBS</td><td>D.b.a. Italia S.r.l.</td><td>sc-281692</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Antibody CD14-FITC (M&amp;phi;P9)</td><td>Becton Dickinson</td><td>345784</td><td></td></tr><tr><td>Antibody Integrin &amp;beta;1 (CD29)-PE (clone MAR4)</td><td>Becton Dickinson</td><td>561795</td><td></td></tr><tr><td>Antibody PECAM-1 (CD31)- APC&amp;nbsp; (clone 9G11)</td><td>R&amp;amp;D Systems</td><td>FAB3567A</td><td></td></tr><tr><td>Antibody Hematopoietic progenitor cell antigen (CD34)-APC (clone 581)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>CD34-581-05</td><td></td></tr><tr><td>Antibody H-CAM (CD44)-PE&amp;nbsp; (clone G44-26)</td><td>Becton Dickinson</td><td>561858</td><td></td></tr><tr><td>Antibody L-CA (CD45)-APC (clone HI30)</td><td>Thermo Fisher Scientific</td><td>MHCD4505-4</td><td></td></tr><tr><td>Antibody THY1 (CD90)-FITC (clone 5E10)</td><td>Becton Dickinson</td><td>561969</td><td></td></tr><tr><td>Antibody Endoglin (CD105)-APC (clone 266)</td><td>Becton Dickinson</td><td>562408</td><td></td></tr><tr><td>Antibody HLA-DR-FITC (clone L243)</td><td>Becton Dickinson</td><td>&lt;br/&gt; 347400</td><td></td></tr><tr><td>Gima Quick Plus sterilizer</td><td>Gima&amp;nbsp;</td><td>35642</td><td></td></tr><tr><td>Bench centrifuge Sigma 3-16K</td><td>Sigma Centrifuges</td><td>10330</td><td></td></tr><tr><td>Mr. Frosty Freezing Container</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>5100-0001</td><td></td></tr><tr><td>AxioVert 200M microscope&amp;nbsp;</td><td>Zeiss</td><td>B 40-080 e 03/01</td><td></td></tr><tr><td>FACS Gallios</td><td>Beckman Coulter&amp;nbsp;</td><td>773231AF</td><td></td></tr><tr><td>ImageJ (image processing program)</td><td>NIH</td><td></td><td></td></tr><tr><td>Stericup filter units</td><td>Merck S.P.A.&amp;nbsp;</td><td>SCGPU05RE</td><td></td></tr><tr><td>Conical Tubes, 50 mL</td><td>Eppendorf</td><td>30122178</td><td></td></tr><tr><td>Conical Tubes, 15 mL</td><td>Eppendorf</td><td>30122151</td><td></td></tr><tr><td>Safe-Lock Tubes, 2 mL</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>60 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>6 well TC-Treated Multiple Well Plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Polystyrene Round-Bottom Tubes, 5 mL</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>BRAND&amp;nbsp;counting chamber BLAUBRAND B&amp;uuml;rker-T&amp;uuml;rk</td><td>Sigma-Aldrich</td><td>BR719520</td><td></td></tr></tbody></table>

isolation and characterization of cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

Cardiac stromal cells isolation: The C-MSC isolation from endomyocardial biopsies procedure is summarized in Figure 1.
Cardiac stromal cells mesenchymal characterization: As established by the International Society for Cellular Therapy (ISCT), the minimal criteria for defining multipotent mesenchymal stromal cells includes their immuno-phenotypic characterization3. In particular, to confirm their mesenchymal lineage, cells are incubated with appropriate FITC/PE/APC-conjugated antibodies and analyzed by flow cytometry. All of the antibodies used in the C-MSC characterization are listed in Table of Materials.
As illustrated in Figure 2, C-MSC obtained from biopsies are positive for the specific mesenchymal surface antigens CD29, CD44, and CD105. The percentage of CD90 positive cells is variable, as reported previously11. The endothelial (CD31, CD34), monocyte/macrophage (CD14), hematopoietic (CD45) markers, and major histocompatibility complex (HLA-DR) are not expressed (Figure 2).
Cardiac mesenchymal stromal cells adipogenic differentiation: To prompt their adipogenic differentiation, C-MSC obtained from patients affected by ACM and HC have to be cultured in T. ADIPO medium (see Solutions section). The cells are maintained in culture for 72 h or 1 week, replacing the medium twice a week.
The accumulation of intracellular lipid droplets is evidenced by ORO staining (Figure 3). Differences in the ability, as well as in the degree of differentiation, can be observed between the cells obtained from ACM and HC. As shown in the representative images, ACM C-MSC accumulates more lipid droplets than HC C-MSC after 72 h of culture in adipogenic medium (Figure 3). These differences are maintained also when the cells are exposed to adipogenic differentiation conditions for a longer period (1 week) (Figure 3).
<img alt="Video 1" src="/files/ftp_upload/57263/57263video1.jpg" /> 
Video 1:&#160;Integration of electroanatomical map and intracardiac echocardiography. Endocardial unipolar electroanatomical maps of the right ventricle (left anterior oblique and right anterior oblique views) in a patient who undergoes endomyocardial biopsy (lower panels). Limited areas of low voltage (red/green) are visible at the apex. Endomyocardial bioptic samples (tagged as circle) are collected in correspondence to the diseased myocardium and the interventricular septum. Real-time intracardiac echocardiography allows checking the correct position of the bioptome at the target area (upper panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57263/Video_1.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 2" src="/files/ftp_upload/57263/57263video2.jpg" /> 
Video 2: Fluoroscopy video in right anterior oblique view. The bioptome is inserted through a long-deflectable sheath and advanced into the ventricle. After a careful check of the bioptome contact with the myocardium, the jaws are opened and then firmly closed to collect the sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57263/Video_2.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57263/57263fig1.jpg" /> 
Figure 1:&#160;Procedure draft. The endomyocardial bioptic sample is collected using a bioptome catheter, a small pincer-shaped cutting instrument. The weight of the obtained biopsy is approximately 5 mg (A). The endomyocardial bioptic sample is minced into 0.5 - 1 mm3 fragments, with sterile scissors (B), and collagenase solution is added. The sample is placed in a 37 &#176;C incubator on a rotating platform mixer for 1.5 h of digestion (C). The digested solution is centrifuged and plated in TMES in a plastic dish (D). C-MSC are obtained thanks to their plastic adherence properties either in single cells or clones, or sprouting from small undigested bioptic fragments (E). C-MSC are then characterized by flow cytometry (F). C-MSC are plated in adipogenic medium and lipid droplet accumulation is tested by Oil Red O staining (G). Scale bars indicate 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57263/57263fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57263/57263fig2.jpg" /> 
Figure 2:&#160;Representative cyto-fluorimetric profile of C-MSC. Each histogram shows the cell count vs. the intensity of fluorescence in the indicated channel (PE: phycoerythrin; APC: allophycocyanin; FITC: fluorescein-isothyocyanate). In each graph the isotype control (white) and a sample conjugated with the specific cell surface marker antibody (red) are shown. C-MSC are positive for the mesenchymal surface antigens CD29, CD44, CD105 and, partially, for CD90, whereas they do not express CD31, CD34, CD14, CD45, and HLA-DR. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57263/57263fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57263/57263fig3.jpg" /> 
Figure 3:&#160;Adipogenic differentiation of C-MSC. Representative images of ACM and HC C-MSC, cultured in adipogenic medium for 72 h and 1 week, stained with ORO (left pictures; n = 3). In the right graphs, quantification of the luminance of the 255-red channel staining is reported: intensity is expressed in arbitrary units (A.U.). ACM C-MSC accumulate more lipid droplets than controls. Student&#39;s T-test was used, *: p&#160;&#60; 0.05. Scale bars indicate 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57263/57263fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients at JoVE.com

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

isolation-characterization-cardiac-mesenchymal-stromal-cells-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K Views.  Centro Cardiologico Monzino-IRCCS. In dit artikel wordt een methode om te isoleren van de cardiale mesenchymale stromale cellen uit de bioptic monsters endomyocardial van arrhythmogenic cardiomyopathie patiënten verstrekt. Hun karakterisering en het protocol bij het stimuleren van hun adipogenic differentiatie worden beschreven.

Video: Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Medicine

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Immunology and Infection

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Biology

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

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