This research was supported by NIH 4R00HL094708-03, MCW Research Affairs Committee New Faculty Award, and the Kern foundation (startup funds) at the Medical College of Wisconsin (RLG); Research Grants Council of Hong Kong Theme-based Research Scheme T13-706/11 (KRB); U01 HL099776, CIRM TR3-05556, CIRM DR2A-05394, and AHA Established Investigator Award (JCW); AHA Postdoctoral Fellowship 12POST12050254 (PWB). We thank Hope Campbell at the Flow Cytometry Core of the Blood Research Institute of Wisconsin for assistance with data collection and careful review of the manuscript.

1. Solution and Media Preparation
<ol>
	<li>hESC Qualified Matrix Coating Stock Solution
	<ol>
		<li>Slowly thaw hESC qualified matrix (5 ml) on ice at 4 &#186;C overnight. Dispense aliquots into pre-chilled, 1.5 ml sterile microcentrifuge tubes and immediately store at -20 &#186;C. 
		NOTE: The volume of the aliquot will vary based on lot and typically ranges 270-350 &#181;l. Manufacturer provides details regarding volume of aliquot required to achieve a 1x concentration upon dilution into 25 ml as described in step 2.1.</li>
	</ol>
	</li>
	<li>hPSC Media Stock Solutions
	<ol>
		<li>Use ultrapure water as a diluent unless otherwise indicated. Sterilize all components using a 0.22 &#181;m filter. Store the following as bulk solutions at 4 &#186;C: sodium bicarbonate (75 mg/ml); citric acid (10 mM, pH = 3).</li>
		<li>Sterilize all components using 0.2 &#181;m syringe filter and store each as aliquots at -20 &#186;C: Rho kinase (ROCK) inhibitor Y-27632 (10 mM in DPBS, 100 &#181;l aliquot); L-ascorbic acid 2-phosphate (64 mg/ml, 500 &#181;l aliquot); sodium selenite (70 &#181;g/ml, 100 &#181;l aliquot); transferrin (50 mg/ml, 107 &#181;l aliquot); fibroblast growth factor 2 (FGF2; 200 ng/&#181;l in DPBS, 250 &#181;l aliquot); transforming growth factor beta 1 (TGF&#946;1, 100 ng/&#181;l in cold 10 mM citric acid,10 &#181;l aliquot).</li>
	</ol>
	</li>
	<li>hPSC Media E8 Solution Composition
	<ol>
		<li>Prepare media using aliquots prepared in Step 1.2: DMEM/F12 (with L-Glutamine and HEPES; 500 ml), sodium bicarbonate (3.62 ml; final: 543 &#181;g/ml), L-ascorbic acid 2-phosphate (500 &#181;l; final: 64 &#181;g/ml), sodium selenite (100 &#181;l; final: 140 ng/ml), transferrin (107 &#181;l; final: 10.7 &#181;g/ml), insulin (1 ml; final: 20 &#181;g/ml), FGF2 (250 &#181;l; final: 100 ng/ml), TGF&#946;1 (10 &#181;l; final: 2 ng/ml).</li>
		<li>Combine all components, filter sterilize and store at 4 &#186;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>hPSC Media E8 with ROCK Inhibitor
	<ol>
		<li>Add 15 &#181;l ROCK inhibitor to 12 ml of E8 media prepared as above and mix well. Ensure that the final concentration is 10 &#181;M after addition of cells/media in step 3.7.</li>
	</ol>
	</li>
	<li>Stock Solutions of Wnt Modulators
	<ol>
		<li>Store the following aliquots at -20 &#186;C. CHIR 99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5&#8203;-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethy&#8203;l]amino]-3-pyridinecarbonitrile; 10 mM in DMSO, 15 &#181;l aliquot). IWR-1 (4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide; 10 mM in DMSO, 6 &#181;l aliquot).</li>
	</ol>
	</li>
	<li>Differentiation Media
	<ol>
		<li>Prepare differentiation media #1 with RPMI 1640 (with L-glutamine) with 2% B27 minus insulin supplement.</li>
		<li>Prepare differentiation media #2 with RPMI 1640 (with L-glutamine) with 2% B27 plus insulin supplement and 2% FBS.</li>
	</ol>
	</li>
	<li>Cell Wash Solution for Cell Culture
	<ol>
		<li>Use Dulbecco&#8217;s phosphate buffered saline (DPBS), without calcium or magnesium.</li>
	</ol>
	</li>
	<li>Cell Wash Solution for Flow Cytometry
	<ol>
		<li>Use phosphate buffered saline (PBS, without calcium or magnesium) with 1% FBS. Store at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Cell Maintenance Solution for Flow Cytometry
	<ol>
		<li>Use Hank&#8217;s Balanced Salt Solution (HBSS; without calcium or magnesium) with 5% FBS. Store at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Fixation Solutions for Flow Cytometry
	<ol>
		<li>Use Cytofix buffer as fixation solution for TNNI3 and IRX4 antibodies.</li>
		<li>Use 4% paraformaldehyde in 1x PBS (make fresh) as fixation solution for TNNT2 and MLC2a antibodies.</li>
		<li>Use 70% methanol/30% acetone as fixation solution for MLC2v antibody.</li>
		<li>Store all solutions at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Permeabilization Solutions for Flow Cytometry
	<ol>
		<li>Use Phosflow Perm Buffer III as permeabilization solution for TNNI3 and IRX4 antibodies. Store at room temperature (25 &#176;C).</li>
		<li>Use 0.2% Triton X-100 in 1x PBS as permeabilization solution for TNNT2, MLC2v, and MLC2a antibodies. Store at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Blocking Solution
	<ol>
		<li>Use 10% goat serum in 1x&#160;PBS. Make fresh and store at 4 &#186;C until use.</li>
	</ol>
	</li>
</ol>
2. Plate Coating
<ol>
	<li>Slowly thaw 1 aliquot of hESC qualified matrix at 4 &#186;C for 30 min, add to 25 ml of chilled DMEM/F12 and mix well in sterilized tissue culture hood immediately before coating 6-well plates.</li>
	<li>Add 1 ml per well of a 6-well plate under sterilized tissue culture hood (1 aliquot of the hESC qualified matrix is sufficient to coat four 6-well plates).</li>
	<li>Allow hESC qualified matrix to set for 30 min at room temperature under the hood. Aspirate the excess hESC qualified matrix and add 2 ml of E8 media with ROCK inhibitor to each well. Use plates immediately, or if desired, store at 4 &#176;C immediately after plating for up to 1 week and equilibrate to room temperature for 30 min prior to use.</li>
</ol>
3. Passaging and Maintenance of Undifferentiated hPSCs in Monolayer Culture
<ol>
	<li>Maintain hPSCs in monolayer culture in 6-well plates and perform all steps under a sterile tissue culture hood.</li>
	<li>Use hPSC lines that are well established (&#62;p20) and exhibit a homogeneous morphology without the presence of cells with a neural, epithelial- or fibroblast-like morphology. Ensure cells proliferate robustly, and on average, passage every three days at 75% confluence when seeded at 0.75 x&#160;106 per well.</li>
	<li>Passage hPSCs with dissociation to the single cell level at least 5x&#160;prior to start of differentiation to ensure maximum differentiation efficiency. To passage hPSCs, aspirate E8 media and wash cells twice with 4 ml of 1x DPBS (pre-warmed to room temperature). Add 1 ml of the cell detachment solution (pre-warmed to room temperature) to each well and leave the well undisturbed for 3-7 min, until cell boundaries begin to round-up.</li>
	<li>Use a cotton-plugged glass pipette with bulb to dislodge cells and transfer into a 15 ml conical tube containing 1 ml of DMEM/F12 media (to inactivate the cell detachment solution).</li>
	<li>Remove 10 &#181;l aliquot of cell solution and mix with 10 &#181;l of trypan blue in a microcentrifuge tube. Count cells (e.g. automated or manual hemocytometer) while remainder of cells are collected by centrifugation at 130 x g for 5 min at room temperature. Using this protocol, expect 70-80% viability using an automated hemocytometer and going forward, base all cell numbers on total cell counts.</li>
	<li>Aspirate media and resuspend the cell pellet in DMEM/F12 to a final concentration of 0.75 x 106 cells per 500 &#181;l.</li>
	<li>Add 500 &#181;l of the cell suspension to each well of a 6-well plate where each well contains 2 ml E8 media with ROCK inhibitor, prepared in step 2.3. Leaving plate on the worktop, gently move in a front-to-back and side-to-side motion to uniformly disperse cells across the well. Return cells to incubator at 5% CO2, 37 &#186;C.</li>
	<li>Beginning 24 hr after passaging, replace media daily using 2 ml/well E8 without ROCK inhibitor. Optimize seeding density to achieve 100% confluence prior to start of differentiation. Seed 0.75 x 106 cells/well four days prior to start of differentiation, but optimize for each cell line as needed.</li>
</ol>
4. Cardiomyocyte Induction of hPSCs by Selective Modulation of the Wnt Pathway
<ol>
	<li>Refresh media (2 ml/well) daily during days 0-7, and every other day after day 8.</li>
	<li>On day 0, begin differentiation process by replacing E8 media with differentiation media #1. Add 1.2 &#181;l CHIR (6 &#181;M final) to each well. Repeat on day 1.</li>
	<li>On days 2-3, replace with fresh differentiation media #1.</li>
	<li>On day 4, replace with fresh differentiation media #1. Add 1 &#181;l IWR-1 (5 &#181;M final) to each well. Repeat on day 5.</li>
	<li>On days 6-7, replace with fresh differentiation media #1.</li>
	<li>On day 8, replace with fresh differentiation media #2.</li>
	<li>Continue to replace with differentiation media #2 every other day for desired time of culture.</li>
	<li>If desired, passage cardiomyocytes using the cell detachment solution following steps outlined in 3.3-3.6, but substituting media with differentiation media #2. During passaging, dissociate cardiomyocytes into small clusters of ~3-10 cells. Seed at ~6 x 105 cells per well using hESC qualified matrix coated wells and differentiation media #2.</li>
</ol>
5. Collection of Cells for Flow Cytometry
<ol>
	<li>Perform all remaining aspects of steps 5-9 on the lab bench (i.e., not sterile).</li>
	<li>Aspirate growth media and wash cells twice with 1x PBS. Add 1 ml of the cell detachment solution (pre-warmed to room temp) to each well and leave undisturbed for 3-7 min, until cell boundaries begin to round-up. Use a cotton-plugged borosilicate glass disposable 9&#8221; pipette with bulb to dislodge cells and transfer into a 15 ml conical tube on ice.</li>
	<li>Throughout remainder of protocol, maintain cells on ice and perform centrifugations at 200 x g for 5 min at 4 &#186;C.</li>
	<li>Collect cells by centrifugation and aspirate solution. Resuspend cells in 10 ml cell wash solution using a 10 ml serological pipette with repeated trituration to ensure cell clumps are dispersed into single cells. Count cells as in step 3.5 while remainder of cells are collected by centrifugation.</li>
	<li>Resuspend cells in cell wash solution taking care to completely disaggregate cell pellet and aliquot 1 x 106 cells per round bottom tube. Collect cells by centrifugation and aspirate solution.</li>
</ol>
6. Fixation and Permeabilization of Cells for Intracellular Antigen Staining
<ol>
	<li>Add 100 &#181;l fixation solution to cell pellet. Add solution drop-wise with continuous gentle vortexing and then set on ice for 15 min.</li>
	<li>Add 3 ml cell wash solution. Collect cells by centrifugation and aspirate solution.</li>
	<li>Add 100 &#181;l permeabilization solution to cell pellet. Add solution drop-wise with continuous gentle vortexing then set on ice for 30 min. Use fixation and permeabilization conditions as outlined for each antibody in Table 1.</li>
	<li>Add 3 ml cell wash solution. Collect cells by centrifugation and aspirate solution. Repeat for a total of two washes after permeabilization.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="2">Primary Antibody (Clone)</td>
			<td rowspan="2">Immunogen/Epitope Recognized</td>
			<td rowspan="2">Istotype Control</td>
			<td colspan="2">Amount of primary antibody per 1 x 106 cells in 100 &#181;l</td>
			<td rowspan="2">Fixation Solution</td>
			<td rowspan="2">Permeabilization Solution</td>
			<td rowspan="2">Secondary Antibody</td>
			<td rowspan="2">Amount of secondary antibody/1 x 106 cells in 100 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>For percent positive measurements</td>
			<td>For Antigen Quantitation</td>
		</tr>
		<tr>
			<td>TNNI3 (284 (19C7)</td>
			<td>ISASRKLQL (human)</td>
			<td>Mouse IgG2b</td>
			<td>1.0&#160; &#181;g</td>
			<td>3.0 &#181;g</td>
			<td>BD Cytofix</td>
			<td>BD Phosflow perm III</td>
			<td>Goat anti-mouse IgG2b-Alexa488</td>
			<td>600 ng</td>
		</tr>
		<tr>
			<td>TNNT2 (1C11)</td>
			<td>Full length purified native human troponin T protein.</td>
			<td>Mouse IgG1</td>
			<td>1.0&#160; &#181;g</td>
			<td>2.0 &#181;g</td>
			<td>4% PFA in 1% PBS</td>
			<td>0.2% Triton X-100 in 1% PBS</td>
			<td>Goat anti-mouse IgG1 - Alexa 488</td>
			<td>600 ng</td>
		</tr>
		<tr>
			<td>MLC2v&#160; (330G5)</td>
			<td>FDPEGKG</td>
			<td>Mouse IgG2a</td>
			<td>2.0&#160; &#181;g</td>
			<td>3.0 &#181;g</td>
			<td>70% methanol/30% acetone</td>
			<td>0.2% Triton X-100 in 1% PBS</td>
			<td>Goat anti-mouse IgG2a - Alexa 647</td>
			<td>600 ng</td>
		</tr>
		<tr>
			<td>MLC2a&#160; (4E7)</td>
			<td>full length human recombinant protein of human MYL7 produced in E. coli</td>
			<td>Mouse IgG1</td>
			<td>0.5 &#181;g</td>
			<td>3.0 &#181;g</td>
			<td>4% PFA in 1% PBS</td>
			<td>0.2% Triton X-100 in 1% PBS</td>
			<td>Goat anti-mouse IgG1 - Alexa 488</td>
			<td>600 ng</td>
		</tr>
		<tr>
			<td>IRX4</td>
			<td>LQEHRKNP 
			YPTKGEKI 
			MLAIITKM 
			TLTQVST</td>
			<td>Rabbit IgG</td>
			<td>0.5 &#181;g</td>
			<td>0.5 &#181;g</td>
			<td>BD Cytofix</td>
			<td>BD Phosflow perm III</td>
			<td>Goat anti-rabbit IgG-PE</td>
			<td>600 ng</td>
		</tr>
	</tbody>
</table>
Table 1. Antibody Concentrations. Listed are the primary antibody, clone (if monoclonal), optimized concentrations and fixation and permeabilization conditions for each primary and secondary antibody used for flow cytometry analyses. As antibody stock concentrations can vary among vendors of the same clone, final concentrations of each antibody per 1 x 106 cells in a fixed assay volume, rather than dilutions, are provided. The immunogen used to generate the antibody, or epitope recognized by antibody, as provided by manufacturer, is listed but was not experimentally verified here.
7. Antibody Staining
<ol>
	<li>For every experiment, include one unstained control per fixation/permeabilization condition and the appropriate isotype control for each primary antibody used. Include non-cardiomyocyte cell types as negative controls (e.g., pluripotent stem cells or fibroblasts). Use isotype controls at the same concentration as corresponding primary antibody (see Table 1).</li>
	<li>Resuspend cells in 100 &#181;l blocking solution using a P200 pipette to disaggregate cells. Set samples on ice for 25 min with gentle rocking.</li>
	<li>Without removing blocking solution, add primary antibody or isotype control per guidelines in Table 1. Incubate 45 min on ice with gentle rocking.</li>
	<li>Add 3 ml cell wash solution. Collect cells by centrifugation and aspirate solution. Repeat for a total of two washes after primary antibody labeling.</li>
	<li>If a fluorophore-conjugated primary antibody is used, proceed to 8.1. When an unconjugated primary antibody is used (such as for all markers described here), perform labeling with secondary antibody that is conjugated to a fluorophore as follows.</li>
	<li>Resuspend cells in 100 &#181;l blocking solution using a P200 pipette to disaggregate cells.</li>
	<li>Add secondary antibody per guidelines in Table 1. Incubate 30 min on ice with gentle rocking.</li>
	<li>Add 3 ml cell wash solution. Collect cells by centrifugation and aspirate solution. Repeat for a total of two washes after secondary antibody labeling.</li>
</ol>
8. Preparation of Cells for Flow Cytometry
<ol>
	<li>Resuspend cells in 400 &#181;l cell maintenance solution using a P1000 pipette to disaggregate cells.</li>
	<li>Prepare a cell strainer cap on a round bottom tube by pre-wetting with 50 &#181;l cell maintenance solution. Set tube on ice. Use the cell strainer cap to prevent cell aggregates from clogging the flow cytometer.</li>
	<li>Transfer cell solution to cell strainer cap and allow cell suspension to drain by gravity. Tap bottom of tube gently on bench top as necessary so that cells are collected into tube and set back on ice as quickly as possible. Rinse strainer with 250 &#181;l cell maintenance solution to ensure maximal recovery of cells.</li>
	<li>Maintain cells on ice and protect from light until analyzed by flow cytometry.</li>
</ol>
9. Flow Cytometry Analysis
Detailed acquisition settings will vary among instruments. Fundamental parameters to consider for optimal data collection are described below.
<ol>
	<li>For optimal stream stability, ensure that the nozzle size exceeds 5-6x&#160;the diameter of the cell being analyzed. 
	NOTE: This may vary among instruments but is an important consideration both for analyzers and selecting appropriate nozzle size for cell sorting. The average diameter of day 10 cardiomyocytes is 11 &#181;m (ranging 8-14 &#181;m); thus, a standard 150 &#181;m sample injection tube on an analyzer works well.</li>
	<li>Immediately prior to data analysis, vortex each tube briefly to disperse cell aggregates.</li>
	<li>Optimize forward and side scatter voltage settings for each cell type based on unstained control for each fixation/permeabilization condition used in the experiment such that the populations of interest are on scale and centered (Figure 2A).
	<ol>
		<li>Maintain these settings throughout a single experiment and be consistent from experiment to experiment on the same instrument, as significant shifts may indicate potential problems with the flow cytometer or sample preparation.</li>
	</ol>
	</li>
	<li>For each fluorophore, analyze the isotype control and adjust appropriate laser voltage to determine the minimum intensity required to obtain a fluorescence histogram that displays both left and right edges of the peak. Maintain the laser settings for each isotype control when acquiring data on corresponding antibody-stained samples. 
	NOTE: Signal drift often occurs over time on the same instrument. It is critical to adjust laser settings at the beginning of each experiment based on the appropriate isotype control.</li>
	<li>Collect a minimum of 10,000 events. 50,000 events are preferred.</li>
	<li>For statistical analyses, gate the live cell population to exclude debris (Figure 2A). Determine percent positive cells within the gated population based on marker placement that allows &#8804;2% contribution from isotype control, as shown in Figure 2B,C.</li>
</ol>

The authors have nothing to disclose.

Critical to the success of the differentiation protocol is the use of high quality cultures of hPSCs that have been passaged at the single cell level for at least five passages prior to the start of differentiation. Similar differentiation efficiencies are routinely observed among various hPSC lines if they are 100% confluent at the start of differentiation, independent of cell line. Suboptimal efficiency is observed if the confluence of cells at the start of differentiation is &#8804;95% or &#62;100%. Therefore, seeding...

The generation of cardiomyocytes from hPSCs, including hESC and hiPSC, can function as an in vitro model of very early human cardiac developmental processes, providing insight into stages not otherwise accessible for mechanistic studies. This model system provides unique opportunities to study the molecular pathways that control cardiac lineage commitment and cell fate specification. In recent years, the ability to efficiently generate cardiomyogenic cells from hPSCs has greatly improved1-15. However, among protocols there is cell line variation with respect to the efficiency in generating cardiomyogenic cells and timing at which the cells express chamber-specific markers (e.g., ventricle and atria). Ideally, for future applications of this model system, more homogeneous populations of functionally defined cells are desired. In contrast to previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or Bone morphogenetic protein 4(BMP4) and robustly generates cultures highly positive for TNNI3, TNNT2, IRX4, MLC2v, and MLC2a by day 10 cells across all hESC and hiPSC lines tested to date. The strategy is technically simple to implement, especially compared to three-dimensional cultures, mass culture, or embryoid body based strategies4-9, and was recently defined in a study that describes a small molecule with selective toxicity to hPSCs (Boheler et al.)65. Features of this protocol include differentiation of hPSCs in monolayer culture using a single layer of a hESC qualified matrix (Matrigel), fully defined media using small molecules to modulate Wnt signaling (similar, yet distinct from1,2,7,13), and optimized flow cytometry staining methods for evaluation of differentiation efficiency and cell identity. In summary, advantages of this protocol compared to previous reports include its cost-effectiveness, reproducibility, and its high efficiency for generating cardiomyocytes among multiple hPSC lines, including hESC and hiPSC lines.
Flow cytometry is a powerful analytical tool for assessing the quality of cells in culture and determining subpopulation homogeneity, and with proper experimental design, can provide quantitative measurements. As with all antibody-based strategies, accurate interpretation of experimental results requires that elements of the assay design including antibody concentration and fixation and permeabilization conditions (when targeting intracellular antigens) are carefully tested for each antibody as sub-optimal conditions significantly affect efficiency of antibody binding, and therefore, interpretation of results. Importantly, if quantitation is required, monoclonal antibodies are essential, as polyclonal antibodies can recognize multiple epitopes and are prone to batch-to-batch variation. Currently, a variety of antibodies (polyclonal and monoclonal) and staining protocols have been described for the assessment of in vitro differentiation, making it difficult to compare efficiency of cardiomyogenesis among protocols1,2,9,11. For that reason, monoclonal antibodies are used when available for all flow cytometry analyses. Going forward, it is expected that standardization of these staining protocols, especially with regards to quantitation, should better permit comparison among differentiation strategies.
The choice of markers, and their corresponding antibodies, used to assess purity of in vitro cardiomyogenesis varies among reports. TNNT2 has been considered an indicator of cells committed to the cardiomyogenic fate and is routinely used to assess efficiency of cardiac differentiation protocols. However, TNNT2 is also expressed in skeletal muscle during early chick and rat development16,17 and it is present in human smooth muscle18. Thus, TNNT2 is not necessarily a specific marker of human cardiomyogenesis in vitro. MLC2v and MLC2a are routinely used as surrogate markers of ventricular and atrial subtypes, respectively. However, challenges with relying on MLC2v and MLC2a to determine cardiomyocyte subtype in the context of in vitro differentiation arise from the fact that these gene products may not be restricted to a specific chamber throughout cardiac development, from heart tube through adult. In the rodent looped heart, MLC2a mRNA is predominant in the atrial/inflow tract area and MLC2v mRNA is predominant in the ventricular/outflow tract regions. In the looped heart, co-expression of MLC2a and MLC2v mRNAs are observed in the inflow tract, atrioventricular canal, and the outflow tract19,20. By 3 days after birth, MLC2v mRNA is restricted to the ventricle and by 10 days after birth, MLC2a is restricted to the atria in the neonatal rat heart19. Therefore, interpretation of data regarding cardiomyogenesis efficiency and subtype identity must not only consider the presence and quantity of reference marker levels, but must consider the developmental stage(s) to which the timepoints of differentiation that are analyzed correspond. This is especially important considering that the maturation stage of cardiomyogenic cells generated by in vitro differentiation of hPSCs resembles most closely those of embryonic/fetal development21-25. Thus, relying on a marker&#8217;s spatial expression in the postnatal heart may not be appropriate for the assessment of hPSC-derived cells, at least in some cases.
In an effort to facilitate the development of more specific criteria for defining cardiomyocyte identity in vitro, TNNI3 is considered to be a valuable marker for evaluating cardiomyogenesis in vitro as it is restricted to cardiac muscle throughout embryogenesis in chick and zebrafish15,20 and is absent in human fetal skeletal muscle26. While TNNI1 is present in human fetal heart, TNNI3 is the only TNNI isoform present in normal adult heart27,28. Regarding cardiomyocyte subtype identity, IRX429-31 is an informative marker of cells with a ventricular fate. At the protein level, IRX4 has recently been shown to be restricted to the ventricle from linear heart tube through neonatal stages in the mouse32. Accordingly, optimized staining protocols for the analysis of TNNI3 and IRX4 by flow cytometry are described. To our knowledge, this is the first description of a method for efficient antibody-based staining and analysis of IRX4 levels in human cardiomyocytes by flow cytometry.

<table border="0" cellpadding="0" cellspacing="0" width="922">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Name of Reagent / Equipment</td>
			<td style="width:152px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:231px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Cell Culture</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Matrigel, hESC-qualified matrix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">354277</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Accutase cell detachment solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td style="width:111px;">7920</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Dubelcco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Dimethyl sulfoxide (DMSO, Hybri-Max, sterile-filtered)</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sodium bicarbonate</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">53817</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Citric acid</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">C2404-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Y-27632 dihydrochloride selective p160ROCK inhibitor</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">1254</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">A8960-5G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sodium selenite</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">S5261-10G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transferrin (Optiferrin &#8211; Defined, Animal-free, Recombinant Human)</td>
			<td style="width:152px;">Invitria</td>
			<td style="width:111px;">777TRF029</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Fibroblast growth factor 2 (FGF2)</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">4144-TC-01M</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transforming growth factor beta 1 (TGF&#946;1)</td>
			<td style="width:152px;">Peprotech</td>
			<td style="width:111px;">100-21</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">DMEM/F12 with L-Glutamine and 15 mM HEPES</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Insulin</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I9278-5ML</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">CHIR-99021 HCl</td>
			<td style="width:152px;">Sellekchem</td>
			<td style="width:111px;">S2924</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">IWR-1</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I0161</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">RPMI 1640 with L-Glutamine</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement Minus Insulin</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">A1895601</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement (with Insulin)</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Fetal bovine serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">10437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Flow cytometry reagents</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phosphate buffered saline (10X)</td>
			<td style="width:152px;">Quality Biological Inc.</td>
			<td style="width:111px;">119-069-151</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:431px;">Hank&#39;s balanced salt solution without Ca2+ and Mg2+</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14175-095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Cytofix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">554655</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Phosflow perm buffer III</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">558050</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">16% Paraformaldehyde</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:111px;">28906</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Methanol</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">179957-4L</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Acetone</td>
			<td style="width:152px;">Mallenckrodt Chemicals&#160;</td>
			<td style="width:111px;">2440-16</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Triton X-100</td>
			<td style="width:152px;">Amresco</td>
			<td style="width:111px;">M236-10ML</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Goat serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Trypan blue solution, 0.4%</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Materials</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (without cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352008</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (with 35 &#181;M cell strainer snap cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352235</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">2 ml rubber bulbs</td>
			<td style="width:152px;">Fischer Scientific</td>
			<td style="width:111px;">03-448-24</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">9&#34; borosilicate unplugged glass Pasteur pipets</td>
			<td style="width:152px;">BioExpress</td>
			<td style="width:111px;">P-2904-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">0.65 mL microcentrifuge tubes, graduated, green</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3259-G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">1.5 mL microcentrifuge tubes, graduated, red</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3260-R</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">TC20 automated cell counter</td>
			<td style="width:152px;">BioRad</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Counting slides for TC20</td>
			<td style="width:152px;">BioRad</td>
			<td style="width:111px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">6 well flat bottom tissue-culture treated plate</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">20 mL syringes</td>
			<td style="width:152px;">BD Plastipak</td>
			<td style="width:111px;">300613</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sterile syringe filters, 0.2 &#181;m polyethersulfone</td>
			<td style="width:152px;">VWR</td>
			<td style="width:111px;">28145-501</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">250 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431096</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">500 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431097</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Antibodies and Isotype Controls</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Anti-TNNI3 (clone: 284 (19C7))</td>
			<td style="width:152px;">Abcam</td>
			<td>ab19615</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-TNNT2 (clone: 1C11)</td>
			<td>Thermo Scientific</td>
			<td>MA1-16687</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL2 (MLC2v, clone: 330G5)</td>
			<td>Synaptic Systems</td>
			<td>310111</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL7 (MLC2a, clone: 4E7)</td>
			<td>Abcam</td>
			<td>AB131661</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-IRX4&#160;&#160;</td>
			<td>Bioss</td>
			<td>BS-9464R</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG1&#160;</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21121</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 647 Goat anti-mouse IgG2a</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21241</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG2b</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21141</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phycoerythrin(PE) Goat anti-rabbit IgG</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">F0110</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG1</td>
			<td>BD Biosciences</td>
			<td>557273</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG2a</td>
			<td>eBiosciences</td>
			<td>16-4724-81</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rabbit IgG-PE</td>
			<td>R&#38;D Systems</td>
			<td>IC105P</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">TaqMan Probesets for qRT-PCR</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ACTB</td>
			<td>Life Technologies</td>
			<td>Hs01060665</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Brachyury T</td>
			<td>Life Technologies</td>
			<td>Hs00610080</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CASQ2</td>
			<td>Life Technologies</td>
			<td>Hs00154286</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IRX4</td>
			<td>Life Technologies</td>
			<td>Hs01100809</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ISL1</td>
			<td>Life Technologies</td>
			<td>Hs00158126</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MESP1</td>
			<td>Life Technologies</td>
			<td>Hs01001283</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYH11 (SMMHC)</td>
			<td>Life Technologies</td>
			<td>Hs00224610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">MYH6</td>
			<td>Life Technologies</td>
			<td>Hs01101425</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL2 (MLC2v)</td>
			<td>Life Technologies</td>
			<td>Hs00166405</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL7 (MLC2a)</td>
			<td>Life Technologies</td>
			<td>Hs01085598</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYOG</td>
			<td>Life Technologies</td>
			<td>Hs01072232</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NKX2.5</td>
			<td>Life Technologies</td>
			<td>Hs00231763</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NPPA</td>
			<td>Life Technologies</td>
			<td>Hs01081097</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">POU5F1</td>
			<td>Life Technologies</td>
			<td>Hs04260367</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SOX17</td>
			<td>Life Technologies</td>
			<td>HS00751752</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI1</td>
			<td>Life Technologies</td>
			<td>Hs00913333</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI2</td>
			<td>Life Technologies</td>
			<td>Hs00268536</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI3</td>
			<td>Life Technologies</td>
			<td>HS00165957</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNT2</td>
			<td>Life Technologies</td>
			<td>Hs00165960</td>
			<td></td>
		</tr>
	</tbody>
</table>

high efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

On day 0, cells are 100% confluent with compact morphology and minimal cell debris. On days 1-2, it is common to observe significant cell death (40-50%), but attached cells will retain compact morphology (Figure 1A). During this time, media is orange and turbid. Pink media indicates excessive cell death, and in this case, confirm with trypan blue and discontinue if cell death exceeds 70%. Cells will recover during days 3-4 and density will increase. During days 5-6, minimal cell death occurs and dense pa...

Watch this Scientific Journal Video about High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry at JoVE.com

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

high-efficiency-differentiation-human-pluripotent-stem-cells-to

Research

JoVE Journal

Biology

30.0K Views.  Medical College of Wisconsin. The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population. 