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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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 &#8211; 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 &#181;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>&#160;MXZ001</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxi Z Cell Count Cassette &#8211; Type M</td>
			<td>Orflo&#160;Technologies</td>
			<td>MXC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel Pipettes</td>
			<td>Fisherbrand</td>
			<td>FBE1200300</td>
			<td>30-300 &#181;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>Thermoshaker</td>
			<td>ThermoFisher Scientific</td>
			<td>13-687-711PM</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...

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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 ...

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Biochemistry

9.0K 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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...