This study was supported by the American Heart Association (AHA) Career Development Award 18CDA34110293 (M-T.Z.), Additional Ventures AVIF and SVRF awards (M-T.Z.), National Institutes of Health (NIH/NHLBI) grants 1R01HL124245, 1R01HL132520 and R01HL096962 (I.D.). Dr. Ming-Tao Zhao was also supported by startup funds from the Abigail Wexner Research Institute at Nationwide Children&#39;s Hospital.

The experimental protocols and informed consent for human subjects were approved by the Institutional Review Board (IRB) at Nationwide Children&#39;s Hospital.
1. Preparation of cell culture media, solutions, and reagents
<ol>
	<li>Prepare PBMC media
	<ol>
		<li>Mix 20 mL of basal PBMC culture media (1x) and 0.52 mL of supplement. Add 20 &#956;L of SCF and FLT3 each (stock concentration: 100 &#956;g/mL), 4 &#956;L of IL3, IL6 and EPO each (stock concentration: 100 &#956;g/mL) and 200 &#956;L of L-glutamine alternative (100x). Mix them thoroughly. Filter in a sterile hood using a 0.22-&#956;m filter unit. Name this as Complete Blood Media.</li>
		<li>Mix 20 mL of basal PBMC culture media (1x) and 0.52 mL of the Supplement. Add 200 &#956;L of L-glutamine alternative (100x). Mix them thoroughly. Filter using a 0.22-&#956;m filter unit. Name this as Supplement Blood Media.</li>
	</ol>
	</li>
	<li>Prepare complete E8 media
	<ol>
		<li>Mix 500 mL of E8 basal media and 10 mL of E8 supplement (thawed overnight at 4 &#176;C) to make complete E8 media. Equilibrate to room temperature (RT) before use.</li>
	</ol>
	</li>
	<li>Prepare iPSC passaging media
	<ol>
		<li>Add 40 &#956;L of Y-27632 Rock inhibitor (1:5,000 dilution, stock concentration: 10 mM) to 200 mL of complete E8 media. Mix it thoroughly. Equilibrate to RT before use.</li>
	</ol>
	</li>
	<li>Prepare cardiomyocyte differentiation media
	<ol>
		<li>Media I: Mix 500 mL of RPMI1640 with 10 mL of B27 minus insulin supplement (50x).</li>
		<li>Media II: Add an appropriate volume of CHIR99021 (GSK3 inhibitor) stock to Media I (CHIR99021 final concentration of 6 &#956;M). Mix thoroughly.</li>
		<li>Media III: Add an appropriate volume of IWR-1 (Wnt inhibitor) stock to Media I (IWR-1&#160;final concentration of 5 &#956;M). Mix thoroughly.</li>
		<li>Media IV: Mix 500 mL of RPMI1640 with 10 mL of B27 supplement (50x). Mix thoroughly.</li>
		<li>Media V: Mix 500 mL of RPMI1640 (no glucose) with 10 mL of B27 supplement (50x). Mix thoroughly.</li>
		<li>Media VI: Add an appropriate volume of CHIR99021 stock to Media IV (CHIR99021 final concentration of 2 &#956;M). Mix thoroughly.</li>
	</ol>
	</li>
	<li>Prepare iPSC-CM passaging media
	<ol>
		<li>Add 10 mL of Knockout Serum Replacement (KSR) to 90 mL of Media IV (KSR final concentration: 10%). Mix well.</li>
	</ol>
	</li>
	<li>Prepare iPSC-CM freezing media
	<ol>
		<li>Add 1 mL of DMSO to 9 mL of KSR (final concentrations: 10% DMSO/90% KSR) and mix well.</li>
	</ol>
	</li>
	<li>Prepare basement membrane matrix medium-coated plates
	<ol>
		<li>Thaw basement membrane matrix medium at 4 &#176;C overnight and aliquot in 1.5 mL tubes. Add 1 mL of this medium to 250 mL of DMEM/F12 media (1:250 dilution) and mix them thoroughly. Apply 2 mL of the diluted solution per well in a 6-well plate and incubate in 5% CO2 at 37 &#176;C for 30 min before use.</li>
	</ol>
	</li>
</ol>
2. iPSC reprogramming of PBMCs
<ol>
	<li>Separate PBMCs from blood samples.
	<ol>
		<li>Collect patient blood samples (~5 mL) and transfer into blood cell separation tubes (see Table of Materials). Mix by inverting 10x.</li>
		<li>Centrifuge at 1,500 x g for 30 min at room temperature.</li>
		<li>Take the tubes out carefully and spray with 70% ethanol. Under a biosafety hood, remove the caps without disturbing the mononuclear cells (buffy layer). PBMCs will be in a whitish layer just under the plasma layer (Figure 1A). Collect the whole buffy layer using a 1000 &#956;L pipette and transfer to a 15 mL conical tube.</li>
		<li>Count the cell number using an automated cell counter. Spin the tube at 300 x g for 25 min at RT.</li>
		<li>Discard the supernatant. Wash with 10 mL of DPBS (Ca2+/Mg2+ free).</li>
		<li>Spin the tube at 300 x g for 15 min at RT.</li>
		<li>Remove the supernatant. Resuspend cell pellets in 1 mL of the freezing media (KSR plus 10% DMSO). Adjust cell density to make 1 x 106 cells per vial.</li>
		<li>Place PBMC cryovials in a cell freezing container and keep at -80 &#176;C overnight. Transfer to a liquid nitrogen tank for long-time storage the next day.</li>
	</ol>
	</li>
	<li>iPSC reprogramming.
	<ol>
		<li>Add 3 mL of Supplement Blood Media in a 15 mL conical tube. Thaw PBMCs in 37 &#176;C water bath and transfer them to a conical tube. Spin at 300 x g for 7 min at RT.</li>
		<li>Discard the supernatant. Resuspend PBMCs with Complete Blood Media. Seed them into two wells of a 24 well tissue culture plates (no basement membrane matrix).</li>
		<li>Incubate in 5% CO2 at 37 &#730;C overnight. The next day gently remove half of the old media (0.5 ml) and add 0.5 mL of fresh Complete Blood Media.</li>
		<li>Change media every other day by refreshing half of the old media.</li>
		<li>After a week, aggressively wash the well with 1 mL of Supplement Blood Media and transfer cells into a 15 mL centrifuge tube.</li>
		<li>Count the cell number. Take 2 x 105 cells and centrifuge at 300 x g for 7 min.</li>
		<li>Discard the supernatant. Resuspend cells with 300 &#956;L of Complete Blood Media. Perform transfection by adding appropriate volume of Sendai virus reprogramming vectors according to the manufacturer&#39;s instructions. Transfer them into one well of a 24-well plate (no basement membrane matrix). Incubate in 5% CO2 at 37 &#176;C overnight.</li>
		<li>The next day spin at 300 x g for 7 min. Remove the supernatant and resuspend in 2 mL of Complete Blood Media. Transfer into one well of a 6 well plate pre-coated with basement membrane matrix. This is Day 1 (D1).</li>
		<li>Don&#39;t touch the plate the next day.</li>
		<li>On D3, remove 1 mL of the old media. Add 1 mL of Supplement Blood Media.</li>
		<li>Repeat step 2.2.10 on D5.</li>
		<li>On D7, remove 1 mL of the old media. Add 1 mL of complete E8 media.</li>
		<li>On D8, Repeat step 2.2.12.</li>
		<li>On D9, remove the old media. Add 2 mL of complete E8 media. Completely reprogrammed cells are expected to attach and start forming colonies.</li>
		<li>Refresh with 2 mL of complete E8 media every day.</li>
		<li>Around 2 weeks after Sendai virus transduction, large iPSC colonies will appear and be ready for picking.</li>
		<li>Cut iPSC colonies under a stereomicroscope in the hood and transfer individual colonies to a basement membrane matrix-coated 24-well plate pre-loaded with 0.5 mL of iPSC passaging media.</li>
		<li>Refresh with 0.5 mL of complete E8 media every day until iPSC colonies grow large enough for passaging into a new basement membrane matrix-coated 6-well plate.</li>
	</ol>
	</li>
</ol>
3. Human iPSC maintenance and passaging
<ol>
	<li>When human iPSCs reach over 90% confluency, remove old media. Rinse with 3 mL of DPBS once.</li>
	<li>Add 1 mL of 0.5 mM EDTA in DPBS solution. Incubate in 5% CO2 at 37 &#176;C for 5-8 min.</li>
	<li>Remove EDTA by aspiration. Add 1 mL of iPSC passaging media. Manually dislodge iPSCs.</li>
	<li>Take 600-900 &#956;L of single cell suspension and re-plate them onto a basement membrane matrix-coated 6-well plate (dilution: 1:6 to 1:10). Incubate in 5% CO2 at 37 &#176;C overnight.</li>
	<li>Refresh with 2 mL of complete E8 media every day. The iPSC cultures usually reach confluency after 3-4 days.</li>
</ol>
4. Chemically defined cardiomyocyte differentiation
<ol>
	<li>Culture human iPSCs in complete E8 media until 95% confluent (3-4 days).</li>
	<li>Remove the old media. Add 2 mL of CM differentiation Media II (6 &#956;M CHIR in RPMI1640 plus B27 minus insulin supplement) to each well of a 6-well plate. This is D0. Do not touch on D1.</li>
	<li>On D2, replace with 2 mL of CM differentiation Media I (RPMI1640 plus B27 minus insulin supplement).</li>
	<li>On D3, replace with 2 mL of CM differentiation Media III (5 &#956;M IWR-1 in RPMI1640 plus B27 minus insulin supplement). Do not touch on D4.</li>
	<li>On D5, replace with 2 mL of CM differentiation Media I.</li>
	<li>On D7, replace with 2 mL of CM differentiation Media IV (RPMI1640 plus B27 supplement). Thereafter, refresh the media every other day.</li>
	<li>On D11 when contracting cells are observed, replace with 2 mL of CM differentiation Media V (no glucose).</li>
	<li>On D13, replace with 2 mL of CM differentiation Media V.</li>
	<li>On D15, replace with 2 mL of CM differentiation Media IV.</li>
	<li>On D17-D21, replace with 2 mL of CM differentiation Media IV every other day.</li>
</ol>
5. Passage human iPSC-CMs
<ol>
	<li>Remove old media and rinse cells with 3 mL of DPBS once.</li>
	<li>Apply 1 mL of CM dissociation solution (see Table of Materials) to each well of a 6-well plate. Incubate in 5% CO2 at 37 &#176;C for 5-8 min.</li>
	<li>Mechanically dissociate iPSC-CMs into single cells by vigorous pipetting.</li>
	<li>Transfer cells into a 15 mL conical tube. Add 2 mL of CM passaging media (10% KSR in RMPI1640 plus B27 supplement) to neutralize CM dissociation solution.</li>
	<li>Spin at 300 x g for 5 min at RT.</li>
	<li>Discard the supernatant. Resuspend cells with a desired volume of CM passaging media. Seed them into a basement membrane matrix-coated plate/dish. Human iPSC-CMs resume beating 1-3 days after passaging.</li>
</ol>
6. Expansion of human iPSC-CMs
<ol>
	<li>Rinse D10-12 beating iPSC-CMs with 3 mL of DPBS for each well of a 6-well plate once. Add 1 mL of CM dissociation solution (Step 5.2). Incubate in 5% CO2 at 37 &#176;C for 7-10 min.</li>
	<li>Mechanically dissociate iPSC-CMs into single cells by vigorous pipetting.</li>
	<li>Transfer cells into a 15 mL conical tube. Add 2 mL of CM passaging media to neutralize CM dissociation solution.</li>
	<li>Spin at 300 x g for 5 min at RT.</li>
	<li>Discard the supernatant. Resuspend cells with an appropriate volume of CM passaging media. Pipette up and down to make single cell suspension. Seed one million of iPSC-CMs into a basement membrane matrix-coated 10 cm dish.</li>
	<li>The next day remove old media. Add 10 mL of cardiomyocyte proliferation media (Media VI: 2 &#956;M CHIR99021). Change the media every other day.</li>
	<li>When iPSC-CMs become confluent after 7-9 days&#39; culture, repeat the passaging step for further expansion of iPSC-CMs.</li>
</ol>
7. Immunofluorescence
<ol>
	<li>Before immunofluorescence staining, seed iPSC-CMs onto basement membrane matrix-coated coverslips that are placed in a 24 well plate (seeding density: 0.5-1 x 106 cells/mL). Maintain iPSC-CMs in culture for at least 4 days.</li>
	<li>Wash cells using 1 mL of DPBS once.</li>
	<li>Add 0.5 mL of 4% paraformaldehyde (PFA) and incubate for 15 min at RT.</li>
	<li>Wash cells using 1 mL of DPBS. Repeat once.</li>
	<li>Add 0.5 mL of 0.1% Triton X-100 and incubate for 20 min at RT.</li>
	<li>Wash with 1 mL of DPBS twice.</li>
	<li>Add 0.5 mL of 0.2% BSA in DPBS (blocking solution). Incubate at RT for 1 h.</li>
	<li>Add 200 &#956;L of primary antibody diluted with blocking solution (dilution: 1:400-1:1000). Incubate at 4 &#176;C overnight.</li>
	<li>Wash cells using 0.5 mL of blocking solution for 3 min with shaking. Repeat twice.</li>
	<li>Add 200 &#956;L of secondary antibody diluted in the blocking solution. Incubate at RT for 1 h.</li>
	<li>Rinse cells three times with 0.5 mL of DPBS, each for 3 min with shaking.</li>
	<li>Counterstain nuclei with DAPI (1:2000 dilution) and incubate for 5 min at RT.</li>
	<li>Rinse cells three times with 0.5 mL of DPBS.</li>
	<li>Mount cells on the coverslips onto a microscope slide using 5 &#956;l of mounting media. Store at 4 &#176;C and protect from light.</li>
</ol>
8. Flow cytometry sample preparation
<ol>
	<li>Wash human iPSC-CMs with 3 mL of DPBS once.</li>
	<li>Add 1 mL of CM dissociation solution and incubate in 5% CO2 at 37 &#176;C for 7-10 min.</li>
	<li>Dislodge cells using a 1,000-&#956;L pipette. Transfer cell suspension to a round FACS tube through a strainer cap. The FACS tube is pre-filled with 1 mL of iPSC-CM passaging media (10% KSR) to neutralize the enzyme activity.</li>
	<li>Spin at 300 x g for 5 min.</li>
	<li>Remove supernatant without disturbing cell pellet. Add 250 &#956;L of Fixation/Permeabilization solution (see Table of Materials). Incubate for 20 min at 4 &#176;C.</li>
	<li>Add 1 mL of Perm/Wash buffer. Vortex briefly and spin at 300 x g for 4 min.</li>
	<li>Discard the supernatant. Add 100 &#956;L of diluted primary antibodies (1:200-1:500) in 1x Perm/Wash buffer. Vortex briefly and incubate overnight at 4 &#176;C.</li>
	<li>Wash cells by adding 1 mL of Perm/Wash buffer. Vortex briefly and spin at 300 x g for 4 min.</li>
	<li>Discard the supernatant. Add 100 &#956;L of diluted secondary antibodies (1:500-1:1,000). Vortex briefly and incubate at RT for 1 h. Protect from light if secondary antibodies are conjugated with light-sensitive fluorescence.</li>
	<li>Wash cells by adding 1 mL of Perm/wash buffer. Vortex briefly and spin at 300g for 4 min.</li>
	<li>Discard the supernatant. Resuspend cells with 400 &#956;L of FACS staining buffer (PBS/4% FBS). Store at 4 &#176;C until loading to a FACS instrument.</li>
</ol>
9. Real time qPCR
<ol>
	<li>Remove old media in human iPSC-CM culture. Add 500-700 &#956;L of lysis buffer to lysate cells. Incubate for 3 min at RT. Scape cell lysate and transfer to a 1.5-ml RNase-free tube. Proceed to total RNA extraction immediately or store at -80 &#176;C.</li>
	<li>Isolate total RNA using an RNA extraction kit following the manufacturer&#39;s instruction.</li>
	<li>Measure the RNA concentration and assess the quality of total RNA by a spectrophotometer.</li>
	<li>Perform reverse transcription reaction using a cDNA synthesis kit. Total volume of RT reaction is 20 &#956;L including 4 &#956;L of reaction mix (5x), 1 &#956;L of reverse transcriptase,1 &#956;g of total RNA and RNase-free water.</li>
	<li>Incubate the complete RT reaction mix in a thermal cycler using the following protocol: 25 &#176;C for 5 min; 46 &#176;C for 20 min; 95 &#176;C for 1 min; hold at 4 &#176;C.</li>
	<li>Dilute cDNA by 1:10 using nuclease-free water. Set up real time qPCR reaction by mixing 1 &#956;L of cDNA template, 1 &#956;L of primers/probe, 10 &#956;L of qPCR master mix and 8 &#956;L of nuclease-free water.</li>
	<li>Run in a real-time PCR system. The cycling protocol is 50 &#176;C 2 min (hold), 95 &#730;C 10 min (hold), 95 &#176;C 15 sec, 60 &#176;C 1 min, repeat for 40 cycles.</li>
	<li>Collect CT values for each gene in each sample. Relative mRNA abundance is calculated by subtracting the CT value of target gene from the CT value of a housekeeping gene. Relative gene expression is analyzed by the 2-&#916;&#916;CT method.</li>
</ol>
10. Whole-cell patch clamp recording
<ol>
	<li>Dissociate iPSC-CMs into single cells using CM dissociation solution as previously described.</li>
	<li>Seed cells at a low density on basement membrane matrix-coated coverslips. Culture them for 3-4 days in Media IV.</li>
	<li>Pull pipettes (resistance 0.9-1.5 M&#937;) from borosilicate glass capillaries using a horizontal microelectrode puller.</li>
	<li>Incubate cells in Tyrode&#39;s solution (pH=7.35).</li>
	<li>Fill pipettes with electrode solution (pH=7.3) composed of the following chemicals: 120 mM aspartic acid, 20 mM KCl, 2 mM MgCl2, 5 mM HEPES, 10 mM NaCl, 5 mM EGTA, 0.3 mM Na-GTP, 14 mM phosphocreatine, 4 mM K-ATP and 2mM creatine phosphokinase.</li>
	<li>Place cells in the current clamp mode using a 1.5-2 diastolic threshold 5 ms current pulse at 1 Hz.</li>
	<li>Record action potentials (APs) using a microelectrode amplifier and a software-driven acquisition board.</li>
</ol>

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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=Sinoatrial+node+cardiomyocytes+derived+from+human+pluripotent+cells+function+as+a+biological+pacemaker.">Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker.</a> Nature Biotechnology. 35 (1), 56-68 (2017).</li><li>Ren, 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=Canonical+Wnt5b+signaling+directs+outlying+Nkx2.5++mesoderm+into+pacemaker+cardiomyocytes.">Canonical Wnt5b signaling directs outlying Nkx2.5+ mesoderm into pacemaker cardiomyocytes.</a> Developmental Cell. 50 (6), 729-743 (2019).</li><li>Zhang, Q., 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=Direct+differentiation+of+atrial+and+ventricular+myocytes+from+human+embryonic+stem+cells+by+alternating+retinoid+signals.">Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals.</a> Cell Research. 21 (4), 579-587 (2011).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proceedings of the Japan Academy, Seriers B, Physical and Biological Sciences. 85 (8), 348-362 (2009).</li><li>Stacey, G. N., Crook, J. M., Hei, D., Ludwig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banking+human+induced+pluripotent+stem+cells:+lessons+learned+from+embryonic+stem+cells.">Banking human induced pluripotent stem cells: lessons learned from embryonic stem cells.</a> Cell Stem Cell. 13 (4), 385-388 (2013).</li><li>Karbassi, 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=Cardiomyocyte+maturation:+advances+in+knowledge+and+implications+for+regenerative+medicine.">Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine.</a> Nature Reviews Cardiology. 17 (6), 341-359 (2020).</li><li>Zhao, L., Ben-Yair, R., Burns, C. E., Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+notch+signaling+promotes+cardiomyocyte+proliferation+in+the+regenerating+zebrafish+heart+through+Wnt+pathway+antagonism.">Endocardial notch signaling promotes cardiomyocyte proliferation in the regenerating zebrafish heart through Wnt pathway antagonism.</a> Cell Reports. 26 (3), 546-554 (2019).</li><li>Heallen, T. R., Kadow, Z. A., Wang, J., Martin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+cardiac+growth+and+size.">Determinants of cardiac growth and size.</a> Cold Spring Harbor Perspectives in Biology. 12 (3), 037150 (2020).</li><li>Campa, V. 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=Notch+activates+cell+cycle+reentry+and+progression+in+quiescent+cardiomyocytes.">Notch activates cell cycle reentry and progression in quiescent cardiomyocytes.</a> Journal of Cell Biology. 183 (1), 129-141 (2008).</li><li>Collesi, C., Zentilin, L., Sinagra, G., Giacca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+signaling+stimulates+proliferation+of+immature+cardiomyocytes.">Notch1 signaling stimulates proliferation of immature cardiomyocytes.</a> Journal of Cell Biology. 183 (1), 117-128 (2008).</li><li>Heallen, 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=Hippo+pathway+inhibits+Wnt+signaling+to+restrain+cardiomyocyte+proliferation+and+heart+size.">Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size.</a> Science. 332 (6028), 458-461 (2011).</li></ol>

The authors declare no competing financial interests.

During iPSC reprogramming, it is critical to culture PBMCs for 1 week until they are enlarged with clear nuclei and cytoplasm. Because PBMCs do not proliferate, an appropriate cell number for viral transduction is important for successful iPSC reprogramming. Cell number of PBMCs, multiplicity of infection (MOI) and titer of virus should be considered and adjusted to reach the optimal transduction outcomes. For cardiac differentiation, initial seeding density is critical for iPSCs to reach over 90% confluent on the day when CHIR99021 is administered. On one hand, if iPSCs are less confluent at the time of cardiac differentiation, CHIR99021 will be toxic and lead to substantial cell death. On the other hand, if iPSCs are over confluent, they will undergo spontaneous differentiation which will compromise the efficiency of directed cardiac differentiation. For the expansion of early iPSC-CMs, the timing and cardiomyocyte quality should be taken into account. Early iPSC-CMs can robustly multiply only when the purity of cardiomyocytes is high enough. Existing non-cardiomyocytes in the culture may also proliferate in response to CHIR99021 treatment, which will negatively affect the proliferation of early iPSC-CMs. In addition, it is crucial to stimulate cardiomyocyte expansion by day 20 of differentiation. Once iPSC-CMs pass over day 30, it will be difficult for them to resume robust dividing.
Human iPSCs were initially derived from dermal and lung fibroblasts via retrovirus-mediated transfection1,2. There are two major issues with these reprogramming methods that prevent the progress in clinical translation of patient iPSCs: 1) the retrovirus integrates into the host genome thus introducing potential genetic mutations; 2) patient-derived fibroblasts require skin biopsies which many patients may decline. In this protocol, we describe a protocol that utilizes commercial non-integration Sendai virus23 and PBMCs to robustly derive patient iPSCs. These iPSCs are free of exogenous reprogramming vectors and can be maintained with self-renewal and pluripotency indefinitely. In addition, patient blood samples are easily collected in clinical laboratories. Our protocol is versatile and can be used for mass production of patient- and disease-specific iPSCs for large-scale repository and clinical translations24.
Robust cardiomyocyte differentiation is achieved by sequential modulation of specific signaling pathways during cardiac differentiation from human iPSCs. Key pathways involved in cardiac specification and proliferation include Wnt, BMP, Activin, NOTCH, VEGF and retinoic acid (RA) 10,12. Here we present an efficient cardiac differentiation protocol by sequential modulation of Wnt signaling by small chemicals: first activation by CHIR99021 and then inhibition by IWR-113,14. Small chemicals are stable and give consistent differentiation outcomes compared to those using growth factors. Most iPSC-CMs generated by this protocol are ventricular-like cardiomyocytes, mixed with atrial- and nodal-like cells. Precision generation of subtype-specific cardiomyocytes is achieved through fine-tuning later differentiation steps10,12. For example, addition of RA immediately after IWR-1 treatment yields a high percentage of atrial-like cardiomyocytes whereas RA inhibition promotes generation of ventricular-like iPSC-CMs18,22. Wnt signaling activation at a later stage of differentiation promotes the induction of cardiac progenitor cells to nodal-like cardiomyocytes19,21, which is promising for the generation of patient-specific biological pacemaker cells.
Human iPSC-CMs are immature and have limited proliferation ability25. During embryonic cardiac development, the maturation proceeds while the proliferation diminishes. There is a narrow window when iPSC-CMs can be stimulated for robust proliferation, which is reflective of embryonic cardiomyocyte expansion. Here we use a Wnt activator CHIR99021 to promote the proliferation of early iPSC-CMs for a limited period, which is consistent with a recent report17. It is speculated that the Wnt signaling pathway affects cardiomyocyte proliferation possibly through the crosstalk with multiple upstream pathways such as NOTCH and Hippo26,27. NOTCH signaling promotes cardiomyocyte proliferation whereas the Hippo pathway restricts cardiac growth and heart size28,29,30. It is still unknown how the interaction between NOTCH and Hippo determines downstream Wnt activity and fine-tunes an appropriate degree of cardiac proliferation. Our protocol has provided an interesting model for cardiomyocyte proliferation to study disease mechanisms of congenital heart defects that are caused by the hypoplasia of ventricular cardiomyocytes, such as hypoplastic left heart syndrome (HLHS) and pulmonary atresia with intact ventricular septum (PA-IVS).

The discovery of human induced pluripotent stem cells has revolutionized modern cardiovascular medicine1,2. Human iPSCs are capable of self-renewing and generating all cell types in the heart, including cardiomyocytes, endothelial cells, smooth muscle cells and cardiac fibroblasts. Patient iPSC-derived cardiomyocytes (iPSC-CMs) can serve as indefinite resources for modeling genetically inheritable cardiovascular diseases (CVDs) and testing cardiac safety for new drugs3. In particular, patient iPSC-CMs are well poised to investigate genetic and molecular etiologies of CVDs that are derived from defects in cardiomyocytes, such as long QT syndrome4 and dilated cardiomyopathy (DCM)5. Combined with CRISPR/Cas9-mediated genome editing, patient iPSC-CMs have opened an unprecedented avenue to understand the complex genetic basis of CVDs including congenital heart defects (CHDs)6,7,8. Human iPSC-CMs have also exhibited potentials to serve as autologous cell sources for replenishing the damaged myocardium during a heart attack9. In recent years, it has become paramount to generate high-quality human iPSC-CMs with defined subtypes (atrial, ventricular and nodal) for cardiac regeneration and drug testing10.
Cardiac differentiation from human iPSCs has been greatly advanced in the past decade. Differentiation methods have gone from embryoid body (EB)-based spontaneous differentiation to chemically defined and directed cardiac differentiation11. Key signaling molecules essential for embryonic heart development, such as Wnt, BMP, Nodal, and FGF are manipulated to enhance cardiomyocyte differentiation from human iPSCs10,12. Significant advances include sequential modulation of Wnt signaling (activation followed by inhibition) for robust generation of cardiomyocytes from human iPSCs13,14. Chemically defined cardiac differentiation recipes have been explored to facilitate large-scale production of beating cardiomyocytes15,16, which have the potential to be upgraded to industrial and clinical level production. Moreover, robust expansion of early human iPSC-CMs is achieved by exposure to constitutive Wnt activation using a small chemical (CHIR99021)17. Most recently, subtype-specific cardiomyocytes are generated through manipulation of retinoic acid (RA) and Wnt signaling pathways at specific differentiation windows during cardiomyocyte lineage commitment from human iPSCs18,19,20,21,22.
In this protocol, we detail a working procedure for robust generation and proliferation of human CMs originating from patient peripheral blood mononuclear cells. We present protocols for 1) reprogramming human PBMCs to iPSCs, 2) robust generation of beating cardiomyocytes from human iPSCs, 3) rapid expansion of early iPSC-CMs, 4) molecular characterization of human iPSC-CMs, and 5) electrophysiological measurement of human iPSC-CMs at the single-cell level by patch clamp. This protocol covers the detailed experimental procedures on converting patient blood cells into beating cardiomyocytes.

<table><tbody><tr><td>ABI 7300 Fast Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Axon Axopatch 200B Microelectrode Amplifier</td><td>Molecular Devices</td><td></td><td>Microelectrode Amplifier</td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>B27 supplement minus insulin</td><td>Thermo Fisher Scientific</td><td>A1895601</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td><td>BD Biosciences</td><td>554714</td><td>Fixation/Permeabilization solution, Perm/Wash buffer</td></tr><tr><td>BD Vacutainer CPT tube</td><td>BD Biosciences</td><td>362753</td><td>Blood cell separation tube</td></tr><tr><td>CHIR99021</td><td>Selleck Chemicals</td><td>S2924</td><td></td></tr><tr><td>CytoTune-iPS 2.0 Sendai Reprogramming Kit</td><td>Thermo Fisher Scientific</td><td>A16517</td><td>Sendai virus reprogramming kit</td></tr><tr><td>Digidata 1200B</td><td>Axon Instruments</td><td></td><td>Acquisition board</td></tr><tr><td>Direct-zol RNA Miniprep kit</td><td>Zymo Research</td><td>R2050</td><td>RNA extraction kit</td></tr><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>11330057</td><td></td></tr><tr><td>Essential 8 medium</td><td>Thermo Fisher Scientific</td><td>A1517001</td><td>E8 media for iPSC culture</td></tr><tr><td>GlutaMAX supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>L-glutamine alternative</td></tr><tr><td>Growth factor reduced Matrigel</td><td>Corning</td><td>356231</td><td>Basement membrane matrix</td></tr><tr><td>iScript cDNA Snythesis Kit</td><td>Bio-Rad</td><td>1708891</td><td>cDNA synthesis</td></tr><tr><td>IWR-1-endo</td><td>Selleck Chemicals</td><td>S7086</td><td></td></tr><tr><td>KnockOut Serum Replacement (KSR)</td><td>Thermo Fisher Scientific</td><td>10828028</td><td></td></tr><tr><td>pCLAMP 7.0</td><td>Molecular Devices</td><td></td><td>Electrophysiology data acquisition &amp;amp; analysis software</td></tr><tr><td>Recombinant human EPO</td><td>Thermo Fisher Scientific</td><td>PHC9631</td><td></td></tr><tr><td>Recombinant human FLT3</td><td>Thermo Fisher Scientific</td><td>PHC9414</td><td></td></tr><tr><td>Recombinant human IL3</td><td>Peprotech</td><td>200-03</td><td></td></tr><tr><td>Recombinant human IL6</td><td>Thermo Fisher Scientific</td><td>PHC0065</td><td></td></tr><tr><td>Recombinant human SCF</td><td>Peprotech</td><td>300-07</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Thermo Fisher Scientific</td><td>11875093</td><td></td></tr><tr><td>RPMI 1640 medium, no glucose</td><td>Thermo Fisher Scientific</td><td>11879020</td><td></td></tr><tr><td>SlowFade Gold Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>S36936</td><td>Mounting media</td></tr><tr><td>StemPro-34 SFM</td><td>Thermo Fisher Scientific</td><td>10639011</td><td>PBMC culture media</td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444964</td><td>qPCR master mix</td></tr><tr><td>TrypLE Select Enzyme 10x, no phenol red</td><td>Thermo Fisher Scientific</td><td>A1217703</td><td>CM dissociation solution</td></tr><tr><td>UltraPure 0.5 M EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>iPSC dissociation solution</td></tr><tr><td>Y-27632 2HCl</td><td>Selleck Chemicals</td><td>S1049</td><td></td></tr></tbody></table>

generation and expansion of human cardiomyocytes from patient peripheral blood mononuclear cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Human iPSC reprogramming from PBMCs 
After pre-culture with Complete Blood Media for 7 days, PBMCs become large with visible nuclei and cytoplasm (Figure 1B), indicating that they are ready for virus transfection. After transfection with the Sendai virus reprogramming factors, PBMCs will undergo an epigenetic reprogramming process for another week. Typically, we get 30-50 iPSC colonies from the transfection of 1 x 105 PBMCs and the reprogramming efficiency is 0.03%-0.05%. Completely reprogrammed cells will attach and start forming colonies when they are introduced to the complete E8 media (Figure 1C). These early iPSC colonies are expanded for another 7 days and then mechanically cut and picked up individually. Each iPSC colony is transferred to one well of a 6-well plate to establish individual iPSC lines. After 4-5 passages, iPSC colonies will become pure with very few differentiated cells around (Figure 1D). At this stage, most of the cells in iPSC colonies are OCT4 and NANOG positive (Figure 1E), demonstrating their pluripotency. Stable iPSC lines are established by the fifth passage.
Cardiac differentiation 
The cardiac differentiation protocol is depicted in Figure 1F. Cardiac differentiation is initiated when iPSCs are maintained for at least 10 passages. The degree of iPSC confluency is critical when CHIR99021 is applied. The cell density is more than 90% confluent but not over confluent. If iPSC colonies become too crowded, they will start spontaneous differentiation which will negatively affect the directed cardiomyocyte differentiation efficiency. Beating cardiomyocytes are usually observed after day 12 of differentiation (Video 1). The date in which the onset of beating occurs varies and is dependent on the iPSC lines in use. After glucose starvation and replating, iPSC-CMs show spontaneous beating (Video 2) and aligned sarcomere structure with intercalated cardiac troponin T (TNNT2) and &#945;-actinin (Figure 1G-H). In addition, the purity of iPSC-CMs is high, with more than 93% of cells being TNNT2+ as shown by FACS analysis (Figure 1I).
Although iPSC-CMs are relatively immature compared to adult cardiomyocytes, they show ventricular- and atrial-like action potentials measured by whole-cell patch clamp (Figure 2A,B). In a typical cardiac differentiation, day 30 iPSC-CMs are a mixture of ventricular-, atrial-, and nodal-like subtypes, with ventricular CMs accounting for the majority (60%, Figure 2C) using the abovementioned differentiation protocol (Figure 1F). Different differentiation protocols yield varying percentages of cardiomyocyte subtypes due to distinct signaling pathways activation during cell lineage determination10. Ventricular CMs are labeled with MYL2 (MLC2v, Figure 2D) whereas atrial iPSC-CMs are marked by NR2F2 (COUP-TFII, Figure 2E). These markers are highly expressed in more mature iPSC-CMs (&#62;D30) rather than those in early stage.
Expansion of iPSC-CMs by Wnt activation 
In mammals, adult cardiomyocytes do not actively divide for self-renewal. This phenomenon also takes place for human iPSC-CMs. Once mature beyond D30, cell division of iPSC-CMs is a rare event, thus limiting their ability for clinical- and industrial-level mass production. To mimic the developmental environment during embryonic cardiomyocyte proliferation, we activate the Wnt pathway by CHIR99021 to stimulate the multiplication of early iPSC-CMs. D12-14 iPSC-CMs (after purification by glucose deprivation) are seeded at a low density in the presence of 2 &#956;M CHIR99021. Wnt activation stimulates cell division of iPSC-CMs and promotes the expression of cell cycle regulators such as Cyclin D1 (Figure 3A) which can push the cell cycle to advance through the G1 phase. Interestingly, CHIR99021 enables robust proliferation of early iPSC-CMs for 2 passages compared to the controls (Figure 3B). However, the proliferation ability of iPSC-CMs diminishes with extensive passage (Figure 3B), which is consistent with the limited and well-controlled cardiac proliferation during embryonic heart development. In addition, it does not appear that CHIR99021 is able to stimulate the expansion of more mature iPSC-CMs when they reach over 30 days of differentiation and develop stable sarcomere structures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62206/62206fig01.jpg" /> 
Figure 1: Human iPSC reprogramming and cardiomyocyte differentiation. (A) A schematic diagram showing the PBMC layer after separation of patient blood samples. (B) Enlarged PBMCs are ready for transfection. (C) Early human iPSC colonies. (D) An established iPSC line at passage 5. (E) Human iPSCs are positive for the pluripotency markers OCT4 (green) and NANOG (red). Nuclei are counterstained by DAPI (blue). (F) Overview of a cardiomyocyte differentiation protocol. (G-H) Sarcomere structure of iPSC-CMs is revealed by immunofluorescence staining using antibodies against TNNT2 (green) and &#945;-actinin (red). Nuclei are counterstained by DAPI (blue). (I) FACS analysis of iPSC-CMs using an antibody against TNNT2. Scale bars: 200 &#956;m (B-D), 50 &#956;m (E and G) and 20 &#956;m (H). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62206/62206fig01large.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/62206/62206fig02.jpg" /> 
Figure 2: Cardiomyocyte subtypes in human iPSC-CMs. (A-B) Representative action potential durations for ventricular-like (A) and atrial-like (B) iPSC-CMs. (C) Representative percentages of ventricular-, atrial- and nodal-like subtypes in human iPSC-CMs. (D-E) D30 iPSC-CMs are stained with antibodies against ventricular cardiomyocyte marker MYL2 (D) and atrial marker NR2F2 (E). Cells are simultaneously stained with a TNNT2 antibody. Nuclei are counterstained by DAPI (blue). Scale bars: 50 &#956;m (D-E). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62206/62206fig02large.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/62206/62206fig03.jpg" /> 
Figure 3: Expansion of human iPSC-CMs by Wnt activation. (A) Percentage of Cyclin D1 positive iPSC-CMs is increased in the presence of CHIR99021. Cells are double stained with antibodies against TNNT2 (red) and Cyclin D1 (green). Nuclei are counterstained by DAPI (blue). (B) Cell number fold changes during the expansion of human iPSC-CMs with or without CHIR99021 in the first 3 passages. Y-axis shows the cell number fold changes. CHIR99021 stimulates the robust proliferation of early iPSC-CMs. Scale bars: 50 &#956;m (A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62206/62206fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1. Beating human iPSC-CMs at day 18 of differentiation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62206/Video 1.avi" target="_blank">Please click here to download&#160;this video.</a>
Video 2. Beating human iPSC-CMs at day 25 after metabolic purification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62206/Video 2.avi" target="_blank">Please click here to download&#160;this video.</a>

Watch this Scientific Journal Video about Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells at JoVE.com

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

generation-expansion-human-cardiomyocytes-from-patient-peripheral

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

4.0K Views.  Nationwide Children’s Hospital. Dit zijn uitgebreide protocolfaciliteiten, ruwe stimuleert de generatie en het gebruik van pouncing van patiëntspecifieke culturen uit perifere mononucleaire bloedcellen. Deze tactiek is reproduceerbaar en gemakkelijk te volgen, speciaal voor beginners. En niet nodig, speciaal expertiseren in stamcel ballads, of cardiovasculaire geneeskunde.Ons protocol kan worden gebruikt om patiëntspecifieke cardiomyocyten te genereren uit een enkele bloedafname die kan worden gebruikt voor het mod...

Video: Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

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 ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetics

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

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 ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biology

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Bioengineering

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

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