We thank Dr. Peter Zandstra, in whose laboratory this protocol was developed, and Drs. Mark Gagliardi and Gordon Keller who provided assistance in establishing the initial methods on which this process was based. Protocol development was supported by an Ontario Graduate Scholarship in Science and Technology to C.B. and a grant from the Heart and Stroke Foundation of Ontario to Peter Zandstra.

1. Preparation of Medium Components
<ol>
	<li>Prepare Wash Medium. Per 100 ml of DMEM/F12, add 1 ml of 100x Penicillin/Streptomycin (Pen/Strep), 1 ml of 100x L-glutamine, and 5 ml of serum replacement.</li>
	<li>Prepare the chemically defined Basal Cardiac Induction Medium according to the manufacturer&#39;s instructions.</li>
	<li>Prepare the following stock reagents for media that will be used in this protocol.
	<ol>
		<li>L-Ascorbic Acid: Prepare a stock solution of 5 mg per ml in 4 &#176;C, sterile ultra-pure distilled water in a conical tube.
		<ol>
			<li>Leave this solution on ice and vortex the tube periodically until the solute is completely dissolved. Filter-sterilize the Ascorbic Acid solution using a 0.22 &#956;m syringe filter.</li>
			<li>Prepare 1 ml aliquots of the ascorbic acid solution and store these aliquots at -20 &#176;C. Use a freshly thawed aliquot each time medium is prepared.</li>
		</ol>
		</li>
		<li>On the day of medium preparation, dilute 13 &#956;l of monothioglycerol (MTG) in 1 ml of Basal Cardiac Induction Medium. Discard unused, diluted MTG.</li>
		<li>Prepare 1 ml aliquots of Transferrin (30 mg/ml) to store at -20 &#176;C. Store thawed aliquots at 4 &#176;C for up to 3 months.</li>
		<li>Prepare 4 mM hydrochloric acid (HCl) containing 0.1% Bovine Serum Albumin (BSA). In a fume hood, add 30 &#956;l of 6.0 N HCl solution to 50 ml of ultra-pure distilled water. Filter-sterilize the solution using a 0.22 &#956;m syringe filter. Add 2 ml of 25% BSA solution to the 50 ml HCl solution.</li>
		<li>Prepare Phosphate Buffered Saline (PBS) containing 0.1% BSA. Add 20 &#956;l of 25% BSA solution per ml PBS.</li>
		<li>Prepare Human Bone Morphogenetic Protein 4 (BMP-4) stock solution (10 ng/&#181;l). Dissolve 10 &#181;g lyophilized BMP-4 in 1 ml of 4 mM HCl buffer solution containing 0.1% BSA. Prepare 50 &#181;l aliquots to store at -20 &#176;C.</li>
		<li>Prepare Human Fibroblast Growth Factor 2 (bFGF) stock solution (10 ng/&#181;l). Dissolve 10 &#181;g lyophilized bFGF in 1 ml of Phosphate Buffered Saline (PBS) containing 0.1% BSA. Prepare 50 &#181;l aliquots to store at -20 &#176;C.</li>
		<li>Prepare Human Vascular Endothelial Growth Factor (VEGF) stock solution (5 ng/&#181;l). Dissolve 5 &#181;g lyophilized VEGF in 1 ml of PBS containing 0.1% BSA. Prepare 50 &#181;l aliquots to store at -20 &#176;C.</li>
		<li>Prepare Activin A stock solution (10 ng/&#181;l). Dissolve 10 &#181;g lyophilized Activin A in 1 ml of PBS containing 0.1% Bovine Serum Albumin (BSA). Prepare 50 &#181;l aliquots to store at -20 &#176;C.</li>
		<li>Prepare Inhibitor of Wnt Production-2 (IWP-2) stock solution (10 mM). Dissolve 2 mg IWP-2 in 429 &#181;l Dimethyl Sulfoxide (DMSO). Prepare 10 &#181;l aliquots to store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare Complete Cardiac Induction Medium. To 100 ml of Basal Cardiac Induction Medium, add 1 ml of 100x Pen/Strep, 1 ml of 100x L-glutamine, 500 &#181;l of Transferrin, 1 ml of freshly thawed ascorbic acid, and 300 &#181;l of MTG. Discard unused medium.</li>
</ol>
2. Preparation of the Microwell Plate
NOTE: All steps should be performed in a biological safety cabinet.
<ol>
	<li>Add 0.5 ml Rinsing Solution (kit component) to each well that will be used on the plate. To ensure that the solution contacts the entire surface inside each microwell, centrifuge the plate at 840 x g for 2 min.</li>
	<li>Incubate the plate for 30 to 60 min at RT.</li>
	<li>Aspirate the Rinsing Solution from the wells. Wash each well twice as follows: Add 1 ml of PBS to each well, centrifuge the plate at 840 x g for 2 min, and then aspirate the PBS.</li>
</ol>
3. Formation of hPSC Aggregates in the Microwell Plate
NOTE: All steps should be performed in a biological safety cabinet.
<ol>
	<li>Place Wash Medium in a 37 &#176;C water bath. 
	NOTE: The volume required = 1 ml x number of wells of hPSC that will be dissociated.</li>
	<li>Dissociate hPSCs to single cells 
	NOTE: These steps describe the dissociation of cells cultured on 6 well plates &#8212; the reagent volumes can be scaled proportionally for different culture formats.
	<ol>
		<li>Aspirate the culture medium from each hPSC culture well for dissociation. Rinse each well with 1 ml of dissociation enzyme, and then immediately aspirate the dissociation enzyme from each well. 
		NOTE: Residual dissociation enzyme should be sufficient to dissociate the cells.</li>
		<li>Incubate the plate at 37 &#176;C for 3 min.</li>
		<li>Add 1 ml of Wash Medium to each well and mechanically dissociate the cells from the tissue culture surface by pipetting the Wash Medium with a P1000 micropipettor over the tissue culture surface. If the dissociation appears to be incomplete and cell clumps remain, pass the suspension through a strainer at this point.</li>
		<li>Transfer the cell suspension to a 15 ml conical tube and store the tube in the incubator while the cell count is performed in the next step.</li>
		<li>Perform a cell count:
		<ol>
			<li>Take a 10 &#181;l sample of the cell suspension collected in the previous step. Add 30 &#181;l of Trypan Blue and mix the suspension well by pipetting.</li>
			<li>Transfer 10 &#181;l of Trypan Blue-stained cells to each chamber of a hemocytometer and visualize under the inverted microscope with the 10X objective. Count the cells.</li>
		</ol>
		</li>
		<li>From the cell count results, calculate how many wells can be seeded at 1.2 x 106 cells per well of the microwell plate. Calculate Aggregation Medium required to seed the cells at 1 ml per well.</li>
		<li>Prepare aggregation medium. Per 10 ml of Complete Cardiac Induction Medium, add 0.5 &#181;l of BMP4 stock solution (final concentration = 0.5 ng/ml) and Y-27632 ROCK Inhibitor (final concentration = 10 &#181;M).</li>
		<li>Remove the conical tube containing the hPSCs from the incubator and centrifuge the tube at 200 x g for 5 min. Aspirate the wash medium and resuspend the cells in aggregation medium at a density of 1.2 x 106 cells per ml.</li>
		<li>Aspirate the PBS wash solution from each well of the microwell plate. Using a P1000 micropipettor, evenly distribute and seed 1 ml of the cell suspension to each well on the microwell plate. To seed a large number of wells, periodically vortex the cell suspension to prevent settling.</li>
		<li>Centrifuge the plate at 200 x g for 5 min. Observe the plate under the microscope to confirm cells have spun to the bottom of each microwell.</li>
		<li>Incubate the plate for 24 hr at 37 &#176;C in a 5% CO2, 5% O2 (hypoxic) incubator.</li>
	</ol>
	</li>
</ol>
4. Cardiac Induction Stage 1
<ol>
	<li>One day following aggregation (day 1), prepare the required volume of Stage 1 Induction medium (for a 24 well plate, volume = 1 ml x number of wells). For 1 ml of complete cardiac induction medium, add 1 &#181;l of BMP4 stock solution (final concentration = 10 ng/ml), 0.5 &#181;l of bFGF stock solution (final concentration = 5 ng/ml), and 0.6 &#181;l of Activin A (final concentration = 6 ng/ml). Place the medium in a 37 &#176;C water bath for at least 15 min.</li>
	<li>Remove the microwell plate from the incubator. Inspect the aggregates under the microscope. Compared to immediately post-centrifuge aggregation, they should appear intact with smooth edges (more round and less square than the previous day).</li>
	<li>Remove the supernatant without disturbing the aggregates inside the microwells:
	<ol>
		<li>Hold the microwell plate horizontally level (i.e., do not tilt it). For each well on the microwell plate, place the tip of a P1000 micropipettor at the surface of the culture medium and against the edge of the well.</li>
		<li>Slowly remove the medium, being careful not to disturb the aggregates at the bottom of the microwells. After reducing the medium level to about 1 to 2 mm from the textured microwell surface, slowly tilt the plate to collect medium at one side of the well (once the volume is sufficiently low, fluid motion is greatly reduced and it is easier to avoid aggregates getting lifted out of their individual microwells). Slowly pipette out the remaining medium in the well.</li>
	</ol>
	</li>
	<li>To add fresh Stage 1 Induction Medium while ensuring the aggregates remain in their individual microwells: Draw 1 ml of Stage 1 induction medium with a P1000 micropipettor. Hold the pipette tip against the inside edge of the well and very slowly dispense the medium against the inside wall of the well. Repeat for the remaining wells.</li>
	<li>Return the plate to the incubator under hypoxic conditions for 3 days.</li>
</ol>
5. Cardiac Induction Stage 2
<ol>
	<li>On day 4, place wash medium (volume = number of wells x 2 ml) in a 37 &#176;C water bath for a minimum of 15 min.</li>
	<li>Prepare the necessary volume of Stage 2 Induction Medium (volume = number of wells x 1 ml): Per ml of Complete Cardiac Induction Medium, add 2 &#181;l of VEGF stock solution (final concentration = 10 ng/ml) and 0.5 &#181;l IWP-2 stock solution (final concentration = 5 &#181;M). Place the prepared Stage 2 Induction medium in a 37 &#176;C water bath for a minimum of 15 min.</li>
	<li>Using a 5 ml serological pipette, harvest the aggregates from each well of the microwell plate and collect the aggregate suspension in a 15 ml conical tube (collect up to 10 wells per 15 ml tube).</li>
	<li>Allow the aggregates to settle for 15 min in a hypoxic incubator. 
	NOTE: This step is important to separate single cells and cellular debris from the intact aggregates.</li>
	<li>Aspirate the supernatant carefully and resuspend the aggregates in 10 ml of the pre-warmed Wash Medium to remove residual inductive cytokines (e.g., Activin A is a potent signaling molecule even at very low concentrations).</li>
	<li>Centrifuge the aggregates at 50 x g for 2 min. Aspirate the supernatant. Resuspend the pelleted aggregates in the pre-warmed Induction 2 Medium.</li>
	<li>Transfer the aggregate suspension to a 24-well ultra-low attachment (ULA) plate at 1 ml per well. Observe the aggregates under the microscope as uniform, tight cell clusters. Incubate under hypoxic conditions until day 6.</li>
</ol>
6. Cardiac Induction Stage 3
<ol>
	<li>On day 6, prepare the necessary volume of Stage 3 Induction Medium (volume = number of wells x 1 ml). For 1 ml of Complete Cardiac Induction Medium, add 2 &#181;l VEGF stock solution (final concentration = 10 ng/ml), and 0.5 &#181;l of bFGF stock solution (final concentration = 5 ng/ml). Place prepared Stage 3 induction medium in a 37 &#176;C water bath for a minimum of 15 min.</li>
	<li>Use a 5 ml serological pipette to transfer the aggregates to 15 ml conical tubes, pooling up to 10 ml of aggregates per tube.</li>
	<li>Allow 10 min for the aggregates to settle. Aspirate the supernatant and resuspend the aggregates in the pre-warmed Stage 3 Induction Medium. Using a 5 ml serological pipette, redistribute the aggregates into a 24-well ULA plate at 1 ml per well.</li>
	<li>On day 10, prepare the necessary volume of Stage 3 Induction Medium (volume = number of wells x 1 ml) as per Step 6.1.</li>
	<li>Use a 5 ml serological pipette to transfer the aggregates to 15 ml conical tubes, pooling up to 10 ml of aggregates per tube.</li>
	<li>Allow 10 min for the aggregates to settle. Aspirate the supernatant and resuspend the aggregates in Stage 3 Induction Medium. Using a 5 ml serological pipette, redistribute the aggregates into a 24-well ULA plate at 1 ml per well.</li>
	<li>Incubate under hypoxic conditions for two days. On day 12, begin incubating the cells at normoxic oxygen levels for the remainder of the culture period (37 &#176;C, 20% O2, 5% CO2). 
	NOTE: After this time point, cells are no longer cultured under hypoxic conditions.</li>
	<li>Repeat this complete medium exchange (Steps 6.2 and 6.3) every 4 days, beginning day 14 until cell harvest (typically, peak cardiomyocyte concentrations are observed after day 14 of differentiation).</li>
</ol>
7. Flow Cytometry Analysis of Cardiac Troponin T (cTnT) Expression Frequency of Microwell Culture Output
<ol>
	<li>Dissociate the aggregates as follows:
	<ol>
		<li>Use a 5 ml serological pipette to transfer 1 well of aggregates to a 15 ml conical tube. Centrifuge the aggregates at 50 x g for 2 min and carefully aspirate the supernatant.</li>
		<li>Add 1 ml of freshly dissolved 1 mg/ml Collagenase Type II solution to the aggregates. Transfer the aggregate suspension to a 1.5 ml microcentrifuge tube. Incubate the aggregates in Collagenase Type II overnight at room temperature.</li>
		<li>The following day, use a P1000 micropipettor to gently dissociate the aggregates into a homogenous single cell suspension. If the aggregates do not readily dissociate, settle the aggregates, aspirate the supernatant, and incubate the aggregates in 700 &#956;l of Dissociation enzyme for 1 to 2 min at RT. Gently pipette the aggregates 1 to 2 times with a P1000 micropipettor to dissociate.</li>
		<li>Dilute the dissociation enzyme: Add 700 &#956;l of Wash Medium containing 14 &#956;l of 1 mg/ml DNAse stock solution. Take a 10 &#956;l sample to perform a cell count, and centrifuge the remaining suspension using a bench-top microcentrifuge at 300 x g for 2 min.</li>
	</ol>
	</li>
	<li>Perform a cell count: Stain the 10 &#956;l counting sample with an equal volume of Trypan Blue and count using a hemocytometer.</li>
	<li>Remove the 1.5 ml microcentrifuge tube containing the remaining cells from the microcentrifuge. Aspirate the supernatant and resuspend the cells, at a concentration of 200,000 to 500,000 cells per 100 &#956;l, in Hanks Balanced Salt Solution containing 2% Fetal Bovine Serum (HF).</li>
	<li>For each condition, transfer 100 &#956;l of cell suspension per well to 2 wells of a 96-well plate (one well will be stained with the cTnT antibody and the other will be the secondary antibody control).</li>
	<li>Centrifuge the plate at 300 x g for 2 min. Remove the supernatant with a multichannel micropipettor.</li>
	<li>Fix the cells: Add 200 &#956;l of Fixation Solution (kit component) per well and incubate the plate for 15 min at RT.</li>
	<li>Centrifuge the plate at 300 x g for 2 min. Use a multichannel micropipettor to carefully withdraw the supernatant from the wells. Dispose the supernatant in a paraformaldehyde waste container.</li>
	<li>Wash the fixed cells twice: Add 200 &#956;l of HF to each well. Centrifuge the plate at 300 x g for 2 min. Aspirate the supernatant, and repeat the wash step once more. Fixed cells can be stored for up to one week in HF at 4 &#176;C.</li>
	<li>Permeabilize the cells:
	<ol>
		<li>Centrifuge the plate at 300 x g for 2 min.</li>
		<li>Add 100 &#956;l of Permeabilization Solution (kit component) to each well and incubate the plate at room temperature for 5 min.</li>
		<li>Centrifuge the plate at 300 x g for 2 min and aspirate the supernatant.</li>
	</ol>
	</li>
	<li>Prepare a master mix of anti-cTnT in HF at the optimal concentration for the given lot number (The optimal dilution must be determined by titration and typically ranges from 1:500 to 1:2,000).</li>
	<li>For each condition (2 wells per condition), add 100 &#956;l of master mix to one well (stained sample) and 100 &#956;l of plain HF to the other well (secondary antibody control). Incubate the cells at 4 &#176;C for 30 min.</li>
	<li>Centrifuge the cells at 300 x g for 2 min. Aspirate the supernatant and add 200 &#956;l of HF per well. Repeat this wash step one more time.</li>
	<li>Prepare a master mix of the secondary antibody. Transfer a volume of HF that corresponds to 100 &#956;l for every well (secondary antibody control and cTnT-stained) being treated. Add 1 &#956;l of goat anti-mouse-APC secondary antibody per 200 &#956;l of HF (1:200 dilution).</li>
	<li>Stain the samples. Add 100 &#956;l of staining solution to each well (both the secondary antibody control and anti-cTnT-stained wells). Incubate cells in the dark at 4 &#176;C for 30 min (keep the plate covered or in the dark after adding fluorescent secondary antibody to avoid photobleaching).</li>
	<li>Centrifuge the cells at 300 x g for 2 min. Aspirate the supernatant and add 200 &#956;l of HF per well. Repeat this wash step one more time.</li>
	<li>Transfer the samples to 5 ml round bottom flow cytometer analysis tubes and perform flow cytometry for signal in the APC channel using the standard protocols for the instrument19.</li>
</ol>

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Bioeng. 102, 493-507 (2009).</li></ol>

M.U. has a financial interest in the underlying microwell technology.

It has been observed that efficient cardiac differentiation of pluripotent stem cells is a highly variable process. While it is not surprising that different cell lines exhibit varying propensities for differentiation capacity to specific cell types, it has been observed that cardiac differentiation efficiency fluctuates dramatically between replicate runs using the same cell line6. The protocol described here addresses one major source of this variability by directly controlling the input cell number per aggregate. To further reduce variability between runs, it is recommended that hPSC lines adapted for single cell passaging are used, as this form of hPSC expansion and maintenance results in more consistent pluripotent populations with respect to expression frequencies of pluripotency markers (e.g., Oct4, Nanog, Tra-1-60, etc.).
The protocol as written here specifies an aggregate size of 1,000 cells for optimal cardiac induction from the HES-2 embryonic stem cell line. To apply this protocol to different cell lines, it is critical that an initial aggregate size screen be performed to determine the cell line-specific optimal aggregate size. While it does not directly impact the procedures to be followed here, we remind the reader that changes in aggregate size and overall cell density are expected to affect oxygen delivery. This may become a relevant consideration in downstream applications. Additionally, apoptotic cell death is a concern during dissociation of hPSCs to single cells. Therefore, it is critical to ensure that ROCK inhibitor is present during forced cell aggregation in the microwells. Finally, it is critical that on day 4 of differentiation the aggregates are well washed to remove trace Activin A, present in the Induction 1 Medium, prior to resuspension in Induction 2 Medium. After day 4 of differentiation, Activin A promotes endoderm differentiation at the expense of mesoderm induction20.
The main application of this technique is to screen aggregate sizes that promote efficient cardiac differentiation. However, one of the limitations of the current technique is that it is challenging to scale cardiac production to clinically relevant levels using microwell plates. Scale up of cardiac differentiation is typically carried out in bulk culture conditions in stirred suspension bioreactors21. Therefore, once the microwell system has been used to determine acceptable ranges of aggregate size for efficient cardiac induction, the next step to scale up is to determine bioreactor impeller speeds that can generate the desired cell aggregate size.
One of the significant differences of this technique with respect to other methods for aggregate-based cardiac differentiation is that it enables direct investigations into modulating the effects of endogenous signaling in aggregates as well as the co-culture of inductive/inhibitory tissue types with the hPSCs in the aggregate13. These types of investigations can inform process development of large scale cardiac production.

In vitro cell culture can be performed under a number of modalities, but is typically carried out either in two-dimensional adherent conditions or in three-dimensional suspension conditions that more fully recapitulate in vivo systems. Consequently there is a growing trend in many fields of research to develop robust methods for generating three-dimensional tissue constructs. In scenarios where cell types and processes require a supportive extracellular matrix (ECM) surface and adhesion signals, three-dimensional culture may be enabled via scaffolded constructs, where cells are cultured on or in an exogenous supporting matrix1. Cells and processes that do not require adhesion to a supportive matrix can be carried out in suspension as unscaffolded systems composed primarily or exclusively of cells (which may then proceed to generate their own endogenous matrices)2,3. Here, we present a protocol for cardiac differentiation of human pluripotent stem cells (hPSCs &#8212; stem cells capable of becoming any cell type in the body, whether from embryonic or other sources) in size-controlled, uniform, unscaffolded aggregates.
Differentiation of hPSCs as suspension aggregates is plagued by large variations in aggregate size both within a run and between runs. This variability is a consequence of the method typically employed to generate these aggregates, which involves mechanical dissociation of cell colonies. To reduce this variability, a number of approaches have been used to control the number of cells per aggregate as well as aggregate diameter and uniformity. Examples include formation of aggregates in microcentrifuge tubes4 or as hanging drops5, micropatterning defined two-dimensional hPSC colonies6 which can then be transferred to suspension, or centrifugation of cells into U- or V-bottom multi-well plates7,8,9. However, all these approaches are limited by their low throughput of aggregate generation. Well-based systems employ a similar approach to V-bottom plate systems, however the smaller size of the microwells (in this protocol each having a width of 400 &#181;m) enables the generation of larger numbers of uniform aggregates from a single culture-plate well (standard diameter of ~ 15.5 mm containing ~ 1,200 microwells) than would be generated from a whole V-bottom plate10. Well-based aggregate formation has been used in a number of settings including differentiation of hPSCs to ectodermal11, endodermal12, mesodermal13 and extraembryonic14 fates; chondrogenesis from mesenchymal stem cells15; generation of uniform substrates for toxicological screening16; and investigations of mechanobiology17.
One major challenge in developing robust manufacturing protocols for the production of hPSC-derived cardiomyocytes has been the lack of reproducibility in cardiac differentiation efficiency between runs. We previously demonstrated that this variability can be attributed to heterogeneity in the starting hPSC population, which comprises both self-renewing hPSCs and differentiating cells that express genes associated with endoderm and neural differentiation6,18. Signals secreted by these differentiating cells impact cardiac induction. Specifically, extraembryonic endoderm promotes cardiac induction, while neural progenitors inhibit cardiac induction. Upon hPSC aggregation, cells within the aggregate differentiate and organize so that undifferentiated hPSCs are surrounded by a layer of extraembryonic endoderm cells that develop on the aggregate surface13. By controlling aggregate size, we can modulate the ratio of cardiac-inducing endoderm cells to undifferentiated hPSCs (surface area to volume ratio) and optimize this ratio for maximum cardiac induction13.

<table><tbody><tr><td>Biological safety cabinet</td><td></td><td></td><td></td></tr><tr><td>Pipette aid</td><td></td><td></td><td></td></tr><tr><td>Serological pipettes (5 to 25 ml)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Aspirator</td><td></td><td></td><td></td></tr><tr><td>Aspirator or Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>15 and 50 ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>Fume hood</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;micro;m syringe filter</td><td></td><td></td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 5% O&lt;sub&gt;2&lt;/sub&gt;&lt;sub&gt;,&lt;/sub&gt; and humidity controlled cell culture incubator&amp;nbsp;</td><td></td><td></td><td>Hypoxic (low oxygen) incubator</td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 20% O&lt;sub&gt;2&lt;/sub&gt;&lt;sub&gt;,&lt;/sub&gt; and humidity controlled cell culture incubator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Low speed centrifuge with a swinging bucket rotor fitted with a plate holder&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>P2, P20, P200, and P1000 micropipettors and associated tips&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope with 4X, 10X and 20X phase objectives&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Ultra-Low Attachment (ULA) 24 well plates&amp;nbsp;</td><td>Corning/Costar</td><td>3473</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>Bench-top microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma-Aldrich</td><td>A4403</td><td></td></tr><tr><td>Sterile Ultrapure distilled water</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>-20 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>Monothioglycerol</td><td>Sigma-Aldrich</td><td>M6145</td><td>Toxic; Aliquoting of MTG is strongly recommended to minimize oxidation due to repeated opening. Aliquots can be stored at 4 &amp;deg;C for up to 3 months, -20 &amp;deg;C is recommended for long-term storage.</td></tr><tr><td>StemPro-34 Medium</td><td>Thermo Fisher Scientific</td><td>10639-011</td><td>Basal Cardiac Induction Medium; The supplement is stored at -20 &amp;deg;C and the basal medium at 4 &amp;deg;C.</td></tr><tr><td>Transferrin</td><td>Roche</td><td>10652202001</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D Technologies</td><td>314-BP</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Technologies</td><td>233-FB</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D Technologies</td><td>293-VE</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D Technologies</td><td>338-AC</td><td></td></tr><tr><td>IWP-2</td><td>Reagents Direct</td><td>57-G89</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>14190</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Thermo Fisher Scientific</td><td>15561</td><td></td></tr><tr><td>Hydrochloric acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>258148</td><td>Corrosive</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>12660</td><td></td></tr><tr><td>100x Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140</td><td></td></tr><tr><td>100x L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828010</td><td></td></tr><tr><td>TrypLE Select</td><td>Thermo Fisher Scientific</td><td>12563</td><td>Dissociation enzyme</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Trypan Blue</td><td></td><td></td><td></td></tr><tr><td>Aggrewell 400 plates</td><td>StemCell Technologies</td><td>27845</td><td>Microwell Plates</td></tr><tr><td>Aggrewell Rinsing Solution</td><td>StemCell Technologies</td><td>7010</td><td>Microwell Rinsing Solution</td></tr><tr><td>Y-27632 ROCK Inhibitor</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>Collagenase Type II</td><td>Sigma-Aldrich</td><td>C6885&amp;nbsp;</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermo Fisher Scientific</td><td>14025092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>12483</td><td></td></tr><tr><td>96 well plate (for FACS staining)</td><td></td><td></td><td></td></tr><tr><td>Intraprep Permeabilization Reagent</td><td>Beckman Coulter</td><td>IM2389</td><td>Kit with 2 parts: Fixation Solution and Permeabilization Solution; Toxic</td></tr><tr><td>cTnT antibody</td><td>Neomarkers&amp;nbsp;</td><td>MS-295</td><td></td></tr><tr><td>goat anti-mouse-IgG APC antibodyThermoFisher</td><td>Molecular Probes</td><td>A865</td><td></td></tr><tr><td>5 ml round bottom flow cytometry tubes</td><td>FACS machine dependent</td><td></td><td></td></tr></tbody></table>

aggregate size optimization in microwells for suspension-based cardiac differentiation of human pluripotent stem cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Size-controlled aggregates of hPSCs can effectively be formed using the microwell system, dependent only on the concentration of cells and the microwell surface area. Following a short centrifugation, the appropriate numbers of cells (1,000 in this protocol) are brought together in each microwell (Figure 1A). Importantly, these cells reestablish intracellular connections within 24 hr, and should no longer fill the well, but appear as compact aggregates with smooth edges (Figure 1B). These aggregates provide the starting materials for further differentiation towards a cardiac fate. If the cells fail to form tight clusters, this suggests possible cell death following dissociation and reaggregation, and the suitability of single cell passaging and ROCK inhibitor concentrations for a particular cell line should be examined. The following three days in microwells show little change in aggregate morphology, although some growth is evident. When removed from the microwells, aggregates should maintain their round, tightly packed morphology and be of a similar size to one another (Figure 1C). Culture in ULA 24-well plates will permit further cell expansion and growth.
By day 8, after exposure first to Activin signaling, and Wnt inhibition, the aggregates will begin to appear as larger and brighter aggregates (Figure 1D). During this period, considerable cellular debris will be evident at the bottom of each well and must be removed by allowing aggregates to settle before removing media. Occasionally, many aggregates will fuse together. This will not inhibit the differentiation of other aggregates in the well, although these &#34;super aggregates&#34; tend not to exhibit the morphological changes seen with smaller aggregates and are less likely to undergo complete differentiation.
Continued differentiation results in notable morphological changes to the aggregates with an increased size and the appearance of organized fibrous regions. By day 12, contracting aggregates can be observed. These will always be made up of large, transparent cells and often include extensive extracellular matrix outside of the aggregate (Figure 2). While aggregate-wide contractions indicate a successful differentiation, cardiac marker expression may also be observed in aggregates that do not appear to contract. Following dissociation of aggregates and immunolabeling, a majority of cells will be positive for the cardiomyocyte marker cTnT by flow cytometry (Figure 3). The expression of this marker is stable in the cells and may be observed in aggregates as late as day 19 of differentiation.
<img alt="Figure 1" src="/files/ftp_upload/54308/54308fig1.jpg" /> 
Figure 1: Timeline of hPSC differentiated towards a cardiac fate. Immediately after aggregation, cells nearly fill each microwell (A). One day later, the aggregates appear condensed and smooth (B). This morphology persists even when the aggregates are removed from the microwells and plated into well plates (C). By day 8, aggregates begin to expand and appear lighter in color (D). Scale bar: 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54308/54308fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54308/54308fig2.jpg" /> 
Figure 2: Aggregates Begin Contracting by Day 12 of Differentiation. After six days in Cardiac Induction Stage 3 Medium, forceful aggregate-wide contractions were observed (top panel: relaxation, middle panel: contraction). The lower panel is derived from subtracting the upper and middle panels, with most significant differences appearing as black or white (arrowheads). Scale bar: 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54308/54308fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54308/54308fig3.jpg" /> 
Figure 3: Immunolabeling for Cardiac Troponin T in Differentiated hPSCs. At day 17, most of the cells are positive for cardiac troponin T by flow cytometry (filled histogram). Also shown are cells stained with secondary antibody alone (unfilled histogram). <a href="https://www.jove.com/files/ftp_upload/54308/54308fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells at JoVE.com

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

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Research

JoVE Journal

Developmental Biology

6.7K Views.  University of Toronto. Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Video: Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

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

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

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

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

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Bioengineering

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

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

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

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

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

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

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 ...

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...