This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award numbers K01HL135464 and R01HL151505 and by the American Heart Association under award number 19IPLOI34660342. We wish to thank the MSU Advanced Microscopy Core and Dr. William Jackson at the MSU Department of Pharmacology and Toxicology for access to confocal microscopes, the IQ Microscopy Core, and the MSU Genomics Core for sequencing services. We also wish to thank all members of the Aguirre Lab for their valuable comments and advice.

1. hPSC culture and maintenance
NOTE: The human induced PSCs (hiPSCs) or human embryonic stem cells (hESCs) need to be cultured for at least 2 consecutive passages after thawing before being used to generate EBs for differentiation or further cryopreservation. hPSCs are cultured in PSC medium (see the Table of Materials) on basement-membrane-extracellular matrix (BM-ECM)-coated 6-well culture plates. When performing medium changes on hPSCs in 6-well plates, add the medium directly to the inner side of the well rather than directly on top of the cells to prevent unwanted cell detachment or stress. Users should be wary of pre-warming PSC media that should not be warmed at 37 &#176;C; all PSC media used in this protocol were thermostable.
<ol>
	<li>To coat the well-plates with the BM-ECM, thaw one aliquot of the BM-ECM (stored at -20 &#176;C according to the manufacturer&#39;s instructions) on ice and mix 0.5 mg of the BM-ECM with 12 mL of cold Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)/F12 medium (stored at 4 &#176;C). Distribute 2 mL of the DMEM/F12-BM-ECM mixture onto each well of a 6-well plate and incubate at 37 &#176;C for at least 2 h.</li>
	<li>To thaw the cells, first, thaw the hPSC cryovial in a 37 &#176;C bead or water bath for 1-2 min until only a small amount of ice is visible. Transfer the thawed cells to a centrifuge tube and slowly add 8-9 mL of the PSC medium supplemented with 2 &#181;M of the ROCK inhibitor, thiazovivin (Thiaz), and centrifuge at 300 &#215; g for 5 min. Remove the supernatant and resuspend the cell pellet in the PSC medium supplemented with 2 &#181;M of Thiaz. Distribute the cells in the culture medium into 1-2 wells depending on the cryovial cell concentration and culture at 37 &#176;C, 5% CO2 for 24 h before changing the PSC medium.</li>
	<li>Change the medium on the cells at 48 h intervals. Perform washes and medium changes using DMEM/F12 (1 mL/well) and the PSC medium (2 mL/well), respectively. 
	NOTE: Washes help remove cell waste and debris while fresh media changes provide cells with a renewed source of nutrients.</li>
	<li>Passage the cells upon subconfluency (60-80% confluent) by aspirating the medium, then washing each well with 1 mL of 1x Dulbecco&#39;s phosphate-buffered solution (no calcium, no magnesium; DPBS). Aspirate the DPBS and add 1 mL of the dissociation reagent for hPSCs (see the Table of Materials), followed by the aspiration of all but a thin film of the reagent after 10 s.</li>
	<li>Incubate for 2-5 min with the thin film of the dissociation reagent for hPSCs until gaps form between cells. 
	NOTE: The time to stop the dissociation is cell-line-dependent.</li>
	<li>Add 1 mL of the PSC medium supplemented with 2 &#181;M of Thiaz (PSC medium+Thiaz) to the well and gently tap the plate to induce cell detachment. Pipette the detached cells in the medium 1-2 times to break up any large colonies, and resuspend the cells in PSC medium+Thiaz in a 1:6 well ratio (cells from 1 well resuspended in 12 mL of culture medium). Replate the cells on BM-ECM-coated wells.</li>
</ol>
2. Generation of 3D self-assembling human heart organoids
<ol>
	<li>Embryoid body (EB) formation: 
	NOTE: It is imperative to limit observable differentiated cells prior to embryoid body formation. Two to three wells of a 6-well plate at a 60-80% confluency will yield enough cells for a single 96-well plate of organoids. All media should be aliquoted and warmed in a 37 &#176;C bead or water bath before any medium changes to minimize temperature shock to the EBs or organoids (this does not include cell dissociation reagents). See Figure 1A,B.
	<ol>
		<li>Day -2
		<ol>
			<li>To create EBs, on day -2, wash sub-confluent hPSCs (60-80% confluent) with DPBS for at least 10 s to wash any cell debris and aspirate the DPBS.</li>
			<li>To detach the cells and release them into a single-cell state, add 1 mL of room-temperature cell dissociation reagent (see the Table of Materials) to each well for 3-6 min. Gently tap the plate ~5 times every minute to induce detachment while checking under the microscope. Add 1 mL of PSC medium+Thiaz to stop the reaction.</li>
			<li>To collect the cells and break up any remaining aggregates, pipette the media up and down in the well 2-3 times to generate a single-cell suspension. Transfer the single-cell suspension to a centrifuge tube and spin for 5 min at 300 &#215; g.</li>
			<li>To obtain the desired cell concentration, discard the supernatant and resuspend the cells in 1 mL of PSC medium+Thiaz. Count the cells using a cell counter or hemocytometer and dilute the cells in PSC medium+Thiaz to a concentration of 100,000 cells/mL.</li>
			<li>To distribute the cells for EB formation, use a multichannel pipette to add 100 &#181;L (10,000 cells) to each well of a round-bottom ultra-low attachment 96-well plate. Centrifuge the plate at 100 &#215; g for 3 min and incubate for 24 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day -1
		<ol>
			<li>Carefully remove 50 &#181;L of medium from each well and add 200 &#181;L of fresh PSC medium warmed to 37 &#176;C to achieve a final volume of 250 &#181;L per well. Incubate the cells for 24 h at 37 &#176;C, 5% CO2. 
			NOTE: Remove and add medium carefully on the side of the well to avoid disturbing the EBs at the bottom of the well. Due to the delicate nature of the EBs and the suspension culture, it is necessary to leave a small volume of liquid in each well when changing the medium to avoid disturbing the EBs.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Human Heart Organoid (hHO) Differentiation: 
	NOTE: All media should be warmed in a 37 &#176;C bead or water bath prior to any media changes. Remove and add medium carefully on the side of the well to avoid disturbing the developing organoids at the bottom of the well. Washes are not needed between media changes to minimize agitation and allow the gradual removal of inhibitors and growth factors. RPMI with 2% B-27 supplement (Table of Materials) was used throughout the differentiation protocol. B-27 supplement contains insulin unless specified (insulin-free in days 0-5). See Figure 1C.
	<ol>
		<li>Day 0
		<ol>
			<li>To initiate differentiation towards a mesoderm lineage, remove 166 &#181;L of medium from each well (~2/3rd of total well volume) and add 166 &#181;L of RPMI 1640 containing insulin-free B-27 supplement, 6 &#181;M CHIR99021, 1.875 ng/mL bone morphogenetic protein 4 (BMP4), and 1.5 ng/mL Activin A for a final well concentration of 4 &#181;M CHIR99021, 1.25 ng/mL BMP4, and 1 ng/mL Activin A. Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day 1
		<ol>
			<li>Remove 166 &#181;L of medium from each well and add 166 &#181;L of fresh RPMI 1640 with insulin-free B-27 supplement. Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day 2
		<ol>
			<li>To induce cardiac mesoderm specification, remove 166 &#181;L of medium from each well and add 166 &#181;L of RPMI 1640 containing insulin-free B-27 supplement and 3 &#181;M Wnt-C59 for a final well concentration of 2 &#181;M Wnt-C59. Incubate for 48 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day 4
		<ol>
			<li>Remove 166 &#181;L of medium from each well and add 166 &#181;L of fresh RPMI 1640 with insulin-free B-27 supplement. Incubate for 48 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day 6
		<ol>
			<li>Remove 166 &#181;L of medium from each well and add 166 &#181;L of RPMI 1640 with B-27 supplement. Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Day 7
		<ol>
			<li>To induce proepicardial differentiation, remove 166 &#181;L of medium from each well and add 166 &#181;L of fresh RPMI 1640 containing B-27 supplement and 3 &#181;M CHIR99021 for a final well concentration of 2 &#181;M CHIR99021. Incubate for 1 h at 37 &#176;C, 5% CO2.</li>
			<li>Remove 166 &#181;L of medium from each well and add 166 &#181;L of fresh RPMI 1640 containing B-27 supplement. Incubate for 48 h at 37 &#176;C, 5% CO2. 
			NOTE: Extra caution is advised at this second medium change on day 7 as the organoids are more prone to movement because of the media changes.</li>
			<li>From day 7 onwards until collection or transfer for analyses or experimentation, perform medium changes every 48 h by removing 166 &#181;L of medium from each well and add 166 &#181;L of fresh RPMI 1640 containing B-27 supplement. 
			&#8203;NOTE: Organoids are ready for analyses and experimentation at day 15 unless earlier developmental stages are of interest. They can be cultured past day 15 for long-term culture or maturation experiments.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Organoid analysis
<ol>
	<li>Transferring whole organoids (live or fixed) 
	NOTE: For live organoid transfer, ensure that pipette tips used are sterile.
	<ol>
		<li>Cut the tip off a P200 pipette tip 5-10 mm from the tip opening, resulting in a wide opening of ~2-3 mm diameter.</li>
		<li>Insert the tip straight into the round-bottom well containing the organoid so that the pipette is completely vertical (perpendicular to the plate). Ensure that the pipette plunger is already pressed all the way before inserting the tip into the medium.</li>
		<li>Slowly release the pipette plunger, taking up enough medium (100-200 &#181;L) to collect the organoid.</li>
		<li>Transfer the organoid in medium to the target destination (e.g., for fixing, live imaging, electrophysiology recording, new plate culture).</li>
	</ol>
	</li>
	<li>Fixing organoids 
	NOTE: Fixing and staining organoids can be done either in the 96-well culture plate or microcentrifuge tubes. Paraformaldehyde (PFA) should be handled only in a fume hood.
	<ol>
		<li>For fixation in microcentrifuge tubes, transfer live organoids to separate tubes with 1-8 organoids per tube. 
		NOTE: Do not exceed 8 organoids per tube.</li>
		<li>Carefully remove and discard as much medium from the tube as possible without touching the organoids.</li>
		<li>Add 4% PFA to each tube or well (300-400 &#181;L per microcentrifuge tube and 100-200 &#181;L per well of a 96-well plate). Incubate at room temperature for 30-45 min. 
		NOTE: Incubation times over 1 h may require antigen retrieval steps and are not recommended.</li>
		<li>Safely discard the PFA without disturbing the organoids. Perform 3 washes with DPBS supplemented with 1.5 g/L glycine (DPBS/Gly), using the same volume used for the 4% PFA, waiting 5 min between washes. Remove DPBS/Gly and proceed to immunostaining or other analyses or add DPBS and store at 4 &#176;C for future use for up to 2 weeks. 
		NOTE: Storing fixed organoids for longer than 2 weeks may result in tissue degradation and contamination and is not recommended.</li>
	</ol>
	</li>
	<li>Whole-mount immunofluorescent staining
	<ol>
		<li>Add 100 &#181;L of blocking/permeabilization solution (10% normal donkey serum + 0.5% bovine serum albumin (BSA) + 0.5% Triton X-100 in 1x DPBS) to each well or tube containing the fixed organoids. Incubate at room temperature overnight on a shaker. 
		NOTE: Do not exceed 8 organoids per tube.</li>
		<li>Carefully remove and discard as much of the blocking solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.</li>
		<li>Prepare the primary antibody solution (1% normal donkey serum + 0.5% BSA + 0.5% Triton X-100 in 1x DPBS) with the desired primary antibodies at the recommended concentrations. Incubate at 4 &#176;C for 24 h on a shaker.</li>
		<li>Carefully remove and discard as much of the antibody solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.</li>
		<li>Prepare secondary antibody solution (1% normal donkey serum + 0.5% BSA + 0.5% Triton X-100 in 1x DPBS) with the desired secondary antibodies at the recommended concentrations. If the antibodies are fluorescently labeled, incubate at 4 &#176;C in the dark (e.g., covered in aluminum foil) for 24 h on a shaker.</li>
		<li>Carefully remove and discard as much of the antibody solution as possible without touching the organoids. Perform 3 washes with DPBS, waiting 5 min between washes.</li>
		<li>Prepare slides with beads (90-300 &#181;m in diameter) mounted in a mounting medium (see the Table of Materials) near the edges of the slide where the coverslip with the organoids will be placed. 
		NOTE: It is recommended to allow the mounting medium around the beads to dry before proceeding; this will prevent the beads from moving around. See Figure 2.</li>
		<li>Transfer the stained organoids using a cut pipette tip onto the slide, between the beads, ensuring spacing to avoid contact between the organoids once on the slide. Use the corner of a rolled-up laboratory wipe to carefully remove excess liquid around the organoid.</li>
		<li>Cover the organoids with mounting-clearing medium (fructose-glycerol clearing solution is 60% (vol/vol) glycerol and 2.5 M fructose)37 using 120-150 &#181;L of the mounting-clearing medium per slide. 
		NOTE: It is recommended to use a cut pipette tip when working with the mounting-clearing medium as it is very viscous.</li>
		<li>Hover the coverslip over the slide with the organoids covered with mounting-clearing solution and slowly press the coverslip over the slide, ensuring the organoids are between the mounted beads.</li>
		<li>Seal the perimeter of the coverslip on the slide using top coat nail varnish. Allow the slide to dry in the dark at room temperature for 1 h. Store at 4 &#176;C in the dark for long-term storage.</li>
	</ol>
	</li>
	<li>Calcium transient imaging in live heart organoids 
	NOTE: According to the manufacturer&#39;s instructions, Fluo4-AM was reconstituted in dimethyl sulfoxide (DMSO) to a final stock solution concentration of 0.5 mM. Fluo4-AM was added directly to the organoid well in the 96-well plate.
	<ol>
		<li>Perform 2 washes on the organoids using RPMI 1640 medium.
		<ol>
			<li>Remove 166 &#181;L of the spent medium from the well.</li>
			<li>Add 166 &#181;L of warmed RPMI 1640 medium, remove 166 &#181;L of medium, and add 166 &#181;L of fresh RPMI 1640 medium. 
			NOTE: The washes are done to remove waste material and cell debris. Two-thirds of the medium is removed from the wells during the washes to avoid disturbing the organoids at the bottom of the well before the functional assay.</li>
		</ol>
		</li>
		<li>Add Fluo4-AM medium to the organoids.
		<ol>
			<li>Add Fluo4-AM reconstituted in DMSO to RPMI 1640 containing B-27 supplement to prepare a 1.5 &#181;M solution.</li>
			<li>Remove 166 &#181;L of medium from the well.</li>
			<li>Add 166 &#181;L of 1.5 &#181;M Fluo4-AM in RPMI 1640 containing B-27 supplement for a final well concentration of 1 &#181;M. Incubate at 37 &#176;C, 5% CO2 for 30 min.</li>
		</ol>
		</li>
		<li>Perform 2 washes as in step 3.4.1.</li>
		<li>Add 166 &#181;L of RPMI 1640 containing B-27 supplement to the well.</li>
		<li>Using a cut tip of a P200 pipette tip, transfer the organoid to a glass-bottom Petri dish (e.g., 8-well chambered cover glass with #1.5 high-performance coverglass) with 100-200 &#181;L of medium. 
		NOTE: See section 3.1 on transferring whole organoids.</li>
		<li>Image the organoids live under a microscope with a temperature- and CO2-controlled chamber at 37 &#176;C, 5% CO2.</li>
		<li>Record several 10-20 s videos at various locations across the organoid, showing the increase and decrease in fluorescence intensity levels as the calcium enters and exits the cells. 
		NOTE: For high-resolution recordings, it is recommended to record at a speed of 10 fps or faster; 50 fps is recommended.</li>
		<li>Analyze the videos using image analysis software (e.g., ImageJ) by selecting regions of interest and measuring intensity levels over time.</li>
		<li>Normalize the intensity recordings using &#916;F/F0&#160;vs. time in milliseconds and plot.</li>
	</ol>
	</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=Impaired+mitophagy+facilitates+mitochondrial+damage+in+Danon+disease.">Impaired mitophagy facilitates mitochondrial damage in Danon disease.</a> Journal of Molecular and Cellular Cardiology. 108, 86-94 (2017).</li><li>Sun, N., 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=Patient-specific+induced+pluripotent+stem+cells+as+a+model+for+familial+dilated+cardiomyopathy.">Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.</a> Science Translational Medicine. 4 (130), (2012).</li><li>Stroud, M. 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=Luma+is+not+essential+for+murine+cardiac+development+and+function.">Luma is not essential for murine cardiac development and function.</a> Cardiovascular Research. 114 (3), 378-388 (2018).</li><li>Liang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+using+a+library+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+reveals+disease-specific+patterns+of+cardiotoxicity.">Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li><li>Mills, R. 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=Functional+screening+in+human+cardiac+organoids+reveals+a+metabolic+mechanism+for+cardiomyocyte+cell+cycle+arrest.">Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (40), 8372-8381 (2017).</li><li>Braam, S. R., 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=Prediction+of+drug-induced+cardiotoxicity+using+human+embryonic+stem+cell-derived+cardiomyocytes.">Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes.</a> Stem Cell Research. 4 (2), 107-116 (2010).</li><li>Burridge, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cell-derived+cardiomyocytes+recapitulate+the+predilection+of+breast+cancer+patients+to+doxorubicin-induced+cardiotoxicity.">Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity.</a> Nature Medicine. 22 (5), 547-556 (2016).</li><li>Pinto, A. R., 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=Revisiting+cardiac+cellular+composition.">Revisiting cardiac cellular composition.</a> Circulation Research. 118 (3), 400-409 (2017).</li><li>Bertero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+genome+reorganization+during+human+cardiogenesis+reveal+an+RBM20-dependent+splicing+factory.">Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory.</a> Nature Communications. 10 (1), 1538 (2019).</li><li>Gilbert, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+plate+mesoderm:+Heart+and+Circulatory+System.">Lateral plate mesoderm: Heart and Circulatory System.</a> Developmental Biology. 6th edition. , 591-610 (2000).</li><li>Richards, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiac+organoids+for+the+modelling+of+myocardial+infarction+and+drug+cardiotoxicity.">Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.</a> Nature Biomedical Engineering. 4 (4), 446-462 (2020).</li><li>Lewis-Israeli, Y. R., Wasserman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Organoids+and+Engineered+Heart+Tissues:+Novel+Tools+for+Modeling+Human+Cardiac+Biology+and+Disease.">Heart Organoids and Engineered Heart Tissues: Novel Tools for Modeling Human Cardiac Biology and Disease.</a> Biomolecules. 1277, (2021).</li></ol>

The authors have no conflicts of interest to declare.

Recent advances in human stem cell-derived cardiomyocytes and other cells of cardiac origin have been used to model human heart development22,24,25 and disease26,27,28 and as tools to screen therapeutics29,30 and toxic agents31,<sup class="xref...

Congenital heart defects (CHDs) are the most common type of congenital defect in humans and affect approximately 1% of all live births1,2,3. Under most circumstances, the reasons for CHDs remain unknown. The ability to create human heart models in the lab that closely resemble the developing human heart constitutes a significant step forward to directly study the underlying causes of CHDs in humans rather than in surrogate animal models.
The epitome of laboratory-grown tissue models are organoids, 3D cell constructs that resemble an organ of interest in cell composition and physiological function. Organoids are often derived from stem cells or progenitor cells and have been successfully used to model many organs such as the brain4,5, kidney6,7, intestine8,9, lung10,11, liver12,13, and pancreas14,15, just to name a few. Recent studies have emerged demonstrating the feasibility of creating self-assembling heart organoids to study heart development in vitro. These models include using mouse embryonic stem cells (mESCs) to model early heart development16,17 up to atrioventricular specification18 and human pluripotent stem cells (hPSCs) to generate multi-germ layer cardiac-endoderm organoids19 and chambered cardioids20 with highly complex cellular composition.
This paper presents a novel 3-step WNT modulation protocol to generate highly complex hHOs in an efficient and cost-effective manner. Organoids are generated in 96-well plates, resulting in a scalable, high-throughput system that can be easily automated. This method relies on creating hPSC aggregates and triggering developmental steps of cardiogenesis, including mesoderm and cardiac mesoderm formation, first and second heart field specification, proepicardial organ formation, and atrioventricular specification. After 15 days of differentiation, hHOs contain all major cell lineages found in the heart, well-defined internal chambers, atrial and ventricular chambers, and a vascular network throughout the organoid. This highly sophisticated and reproducible heart organoid system is amenable to investigating structural, functional, molecular, and transcriptomic analyses in the study of heart development, and diseases, and pharmacological screening.

<table><tbody><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Donkey anti- mouse</td><td>Invitrogen</td><td>A-21202</td><td>1:200</td></tr><tr><td>Alexa Fluor 488 Donkey anti- rabbit</td><td>Invitrogen</td><td>A-21206</td><td>1:200</td></tr><tr><td>Alexa Fluor 594 Donkey anti- mouse</td><td>Invitrogen</td><td>A-21203</td><td>1:200</td></tr><tr><td>Alexa Fluor 594 Donkey anti- rabbit</td><td>Invitrogen</td><td>A-21207</td><td>1:200</td></tr><tr><td>Alexa Fluor 647 Donkey anti- goat</td><td>Invitrogen</td><td>A32849</td><td>1:200</td></tr><tr><td>HAND1</td><td>Abcam</td><td>ab196622</td><td>Rabbit; 1:200</td></tr><tr><td>HAND2</td><td>Abcam</td><td>ab200040</td><td>Rabbit; 1:200</td></tr><tr><td>NFAT2</td><td>Abcam</td><td>ab25916</td><td>Rabbit; 1:100</td></tr><tr><td>PECAM1</td><td>DSHB</td><td>P2B1</td><td>Rabbit; 1:50</td></tr><tr><td>TNNT2</td><td>Abcam</td><td>ab8295</td><td>Mouse; 1:200</td></tr><tr><td>THY1</td><td>Abcam</td><td>ab133350</td><td>Rabbit; 1:200</td></tr><tr><td>TJP1</td><td>Invitrogen</td><td>PA5-19090</td><td>Goat; 1:250</td></tr><tr><td>VIM</td><td>Abcam</td><td>ab11256</td><td>Goat; 1:250</td></tr><tr><td>WT1</td><td>Abcam</td><td>ab89901</td><td>Rabbit; 1:200</td></tr><tr><td>Media and Reagents</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>NC9464543</td><td>cell dissociation reagent</td></tr><tr><td>Activin A</td><td>R&amp;amp;D Systems</td><td>338AC010</td><td></td></tr><tr><td>B-27 Supplement (Minus Insulin)</td><td>Gibco</td><td>A1895601</td><td>insulin-free cell culture supplement</td></tr><tr><td>B-27 Supplement</td><td>Gibco</td><td>17504-044</td><td>cell culture supplement</td></tr><tr><td>BMP-4</td><td>Gibco</td><td>PHC9534</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Bioworld</td><td>50253966</td><td></td></tr><tr><td>CHIR-99021</td><td>Selleck</td><td>442310</td><td></td></tr><tr><td>D-(-)-Fructose</td><td>Millipore Sigma</td><td>F0127</td><td></td></tr><tr><td>DAPI</td><td>Thermo Scientific</td><td>62248</td><td>1:1000</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Millipore Sigma</td><td>D2650</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>10566016</td><td></td></tr><tr><td>Essential 8 Flex Medium Kit</td><td>Gibco</td><td>A2858501</td><td>pluripotent stem cell (PSC) medium containing 1% penicillin-streptomycin</td></tr><tr><td>Fluo4-AM</td><td>Invitrogen</td><td>F14201</td><td></td></tr><tr><td>Glycerol</td><td>Millipore Sigma</td><td>G5516</td><td></td></tr><tr><td>Glycine</td><td>Millipore Sigma</td><td>410225</td><td></td></tr><tr><td>Matrigel GFR</td><td>Corning</td><td>CB40230</td><td>Basement membrane extracellular matrix (BM-ECM)</td></tr><tr><td>Normal Donkey Serum</td><td>Millipore Sigma</td><td>S30-100mL</td><td></td></tr><tr><td>Paraformaldehyde</td><td>MP Biomedicals</td><td>IC15014601</td><td>Powder dissolved in PBS Buffer &amp;ndash; use at 4%</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffer Solution</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Phosphate Buffer Solution (10x)</td><td>Gibco</td><td>70011044</td><td></td></tr><tr><td>Polybead Microspheres</td><td>Polysciences, Inc.</td><td>73155</td><td>90 &amp;micro;m</td></tr><tr><td>ReLeSR</td><td>Stem Cell Technologies</td><td>NC0729236</td><td>dissociation reagent for hPSCs</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Thiazovivin</td><td>Millipore Sigma</td><td>SML1045</td><td></td></tr><tr><td>Triton X-100</td><td>Millipore Sigma</td><td>T8787</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Gibco</td><td>1525006</td><td></td></tr><tr><td>VECTASHIELD Vibrance Antifade Mounting Medium</td><td>Vector Laboratories</td><td>H170010</td><td></td></tr><tr><td>WNT-C59</td><td>Selleck</td><td>NC0710557</td><td></td></tr><tr><td>Other</td><td></td><td></td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>02682002</td><td></td></tr><tr><td>15 mL Falcon Tubes</td><td>Fisher Scientific</td><td>1495970C</td><td></td></tr><tr><td>2 mL Cryogenic Vials</td><td>Corning</td><td>13-700-500</td><td></td></tr><tr><td>50 mL Reagent Reservoirs</td><td>Fisherbrand</td><td>13681502</td><td></td></tr><tr><td>6-Well Flat Bottom Cell Culture Plates</td><td>Corning</td><td>0720083</td><td></td></tr><tr><td>8 Well chambered cover Glass with #1.5 high performance cover glass</td><td>Cellvis</td><td>C8-1.5H-N</td><td></td></tr><tr><td>96-well Clear Ultra Low Attachment Microplates</td><td>Costar</td><td>07201680</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>Image processing software</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>06-666</td><td>laboratory wipes</td></tr><tr><td>Micro Cover Glass</td><td>VWR</td><td>48393-241</td><td>24 x 50 mm No. 1.5</td></tr><tr><td>Microscope Slides</td><td>Fisherbrand</td><td>1255015</td><td></td></tr><tr><td>Moxi Cell Counter</td><td>Orflo Technologies</td><td>&amp;nbsp;MXZ001</td><td></td></tr><tr><td>Moxi Z Cell Count Cassette &amp;ndash; Type M</td><td>Orflo&amp;nbsp;Technologies</td><td>MXC001</td><td></td></tr><tr><td>Multichannel Pipettes</td><td>Fisherbrand</td><td>FBE1200300</td><td>30-300 &amp;micro;L</td></tr><tr><td>Olympus cellVivo</td><td>Olympus</td><td></td><td>For Caclium Imaging, analysis with Imagej</td></tr><tr><td>Sorvall Legend X1 Centrifuge</td><td>ThermoFisher Scientific</td><td>75004261</td><td></td></tr><tr><td>Thermal Mixer</td><td>ThermoFisher Scientific</td><td>13-687-717</td><td></td></tr><tr><td>Top Coat Nail Varish</td><td>Seche Vite</td><td></td><td>Can purchase from any supermarket</td></tr></tbody></table>

generating self-assembling human heart organoids derived from pluripotent stem cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

To achieve self-organizing hHO in vitro, we modified and combined differentiation protocols previously described for 2D monolayer differentiation of cardiomyocytes21 and epicardial cells22 using Wnt pathway modulators and for 3D precardiac organoids16 using the growth factors BMP4 and Activin A. Using the 96-well plate EB and hHO differentiation protocol described here and shown in Figure 1, the concentrations a...

Watch this Scientific Journal Video about Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells at JoVE.com

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

generating-self-assembling-human-heart-organoids-derived-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.1K Views.  Michigan State University. Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues. 

Video: Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

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 ...

Developmental Biology

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

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.

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. ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Bioengineering

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

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

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

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

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 ...

Biology

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...