Name: generating self-assembling human heart organoids derived from pluripotent stem cells
Uploaded: 2021-09-15T15:00:00
Description: Watch this Scientific Journal Video about Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells at JoVE.com

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 direct...

<ol>
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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...

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 ...

This protocol allows us to create a complex 3D structure that closely resembles the developing human heart in a simple cost effective way, that is reproducible across cell lines. It consists of a single protocol with three steps of differentiation, which gives rise to all the cell types of the heart, including chambers and a vascular network. This technique allows us to model a wide range of human cardiovascular diseases and to screen effective pharmacological treatments.Thanks to the high throughput nature of its design. This method is useful for investigating the developing human heart as well as diseases commonly associated with it. One of them being congenital heart defects.This protocol requires basic cell culture skills. We recommend attentive and delicate pipetting as well as gentle handling of the organoids. Begin by creating embryoid bodies or EBs two days before differentiation.Wash sub-confluent HPSCs with DBPS for at least 10 seconds. Then aspirate the DPBS. Add one milliliter of room temperature ACUTA and gently tap the plate approximately five times every minute for three to five minutes to induce detachment, while checking under the microscope.Then add one milliliter of PSC medium and Thias Avin or Thias to stop the reaction. Pipette the media up and down in the well, two to three times to generate a single cell suspension and collect the cells. Then transfer the single cell suspension to a centrifuge tube and spin for five minutes at 300 times G.Aspirate the supernatant and re-spend the cells in one milliliter of PSC medium and Thias.Dilute the cells in DPBS. Count the cells using a cell counter or hemo cystometer and dilute the cells in PSC medium with Thias to a concentration of 100, 000 cells per milliliter. Add 100 microliters of diluted cells suspension to each well of a round bottom ultra low attachment 96 well plate.Centrifuge the plate at 100 times G for three minutes and incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. After 24 hours carefully remove 50 microliters of medium from each well and add 200 core litters of fresh PSC medium, warm to 37 degrees Celsius for a volume of 250 microliters per well. Incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.Remove 166 microliters or two thirds of medium from one side of each well, and add 166 microliters of RPMI 1640 containing insulin free B27 supplement, six micromolar cheer 990211, 0.875 nanograms per milliliter of bone morphogenetic protein four and 1.5 nanograms per milliliter of Activin A.Incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. After 24 hours remove 166 microliters of medium from one side of each well and add 166 microliters of fresh RPMI 1640 with insulin free B27 supplement. Incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide.To induce cardiac mesoderm specification on day two, remove 166 microliters of medium from each well and add 166 microliters of RPMI 1640, containing insulin free B27 supplement and three micromolar Wnt-C59 and continue incubation for 48 hours. On day four, remove 166 microliters of medium from each well and add 166 microliters of fresh RPMI 1640 with insulin free B27 supplement, then incubate another 48 hours. On day six, remove 166 microliters of medium from each well and add 166 microliters of RPMI 1640 with B27 supplement.For this and all subsequent media changes, use B27 supplement with insulin, incubate for another 24 hours. On day seven, the cell culture is ready for pro epicardial differentiation. Remove 166 microliters of medium from each well and add 166 microliters of fresh RPMI 1640 containing B27 supplement and three micromolar cheer 99021.Then in incubate for one hour, after one hour remove the plate from the incubator, remove 166 microliters of medium from one side of each well and add 166 microliters of fresh RPMI 1640 containing B27 supplement continue incubation for another 48 hours. Repeat this process us every 48 hours until collection. Organoids are ready for analyses and experimentation on day 15 To transfer the whole organoids, cut the tip of a P200 pipette, five to 10 millimeters from the opening with a diameter of approximately two to three millimeters.Press the pipette plunger and insert the tip vertically into the round bottom well with the organoid. Take up approximately 100 to 200 microliters of medium enough to collect the organoids and transfer them to the target destination. Before differentiation EBS appear as blacks spherical masses the differentiation process starts on day zero with a Wnt pathway activation and growth factor addition for exactly 24 hours.Further the edit of Wnt C59 on day two results in enlargement of the organoid from approximately 200 micrometers to a maximum of one millimeter. The human heart organoids began beating as early as day six and 100%of the organoids showed visible beating by day 10. High magnification images of day seven organoid revealed that most hand one expressing cells were cardiomyocyte in origin and hand two were non myosite cells from second Heartfield, progenitor cells.RNA transcripts for both hand one and hand two were expressed from day three onwards in RNA sequencing data. The FHF markers were highly expressed during days three and 11 while the SF marker was more highly expressed after day 13. Immunofluorescence stain revealed the presence of various human heart cell type lineages that make up the human heart.Such as myocardial and epicardial tissue, endocardial cells, endothelial cells, and cardiac fibroblast. The markers expressing the above cell lineages were also observed in the RNA SAC gene expression profiles. Live calcium imaging of individual cells in whole organoids was used to measure the electrophysiological function of the organoids.Fluo-4 fluorescence intensity varied over time due to calcium entry exit from the cell revealing regular action potentials. Heat maps showing calcium intensities over a high magnification region of the organoid, show increased intensity due to calcium transients and individual cells. When attempting this protocol, keep in mind that the EBs and subsequent organoids are not attached to the bottom of the plate, and we'll move around with the removal in addition of media requiring attentive media changes.Organoids can be culture for longer periods of time introduced to maturation techniques, or even exposed to external stimuli, such as mechanical or electrical conditioning, and even stressors such as toxins. This technique allow as access to stages of human fetal heart development that are otherwise inaccessible in research, paving the way for new insights into heart development and disease etiology.

Research

JoVE Journal

Biochemistry

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

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Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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

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

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Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

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Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

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Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

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