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 wh...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 an...

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. ...

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Developmental Biology

3.9K Views.  Nationwide Children’s Hospital. Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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 ...