Name: large-scale production of cardiomyocytes from human pluripotent stem cells using a highly reproducible small molecule-based differentiation protocol
Uploaded: 2016-07-25T14:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol at JoVE.c

This study was funded by grants provided from Royan Institute, Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), the National Health and Medical Research Council of Australia (NHMRC; 354400), the National Heart Foundation of Australia/Heart Kids Australia (G11S5629), and the New South Wales Cardiovascular Research Network. HF was supported by a University International Postgraduate Scholarship from the University of New South Wales, Australia. RPH was supported by a NHMRC Australia Fellowship. The authors express their gratitude to the human subjects who participated in this research.

1. Preparation of Culture Media, Coating of Cell Culture Plates and Maintenance of Undifferentiated hPSCs
<ol>
	<li>Media Preparation 
	Note: Sterilize media using a 0.22 &#181;m filtration device and store at 4 &#176;C protected from light for up to 4 weeks. Reagent names, suppliers and catalog numbers are listed in Materials Table.
	<ol>
		<li>For Mouse Embryonic Fibroblasts (MEF) Medium, combine 445 ml DMEM, 50 ml Fetal Bovine Serum (FBS) and 5 ml cell culture media (e.g., Glutamax).</li>
		<li>For hESC Medium, combine 390 ml Knockout-DMEM (KO-DMEM), 100 ml Knockout Serum Replacement (KO-SR), 5 ml cell culture media, 5 ml MEM minimum essential amino acids solution and 0.5 ml 55 mM &#946;-mercaptoethanol. 
		CAUTION: &#946;-Mercaptoethanol is toxic. Avoid inhalation, ingestion and skin contact.</li>
		<li>For RPMI-B27 (RB) Medium, combine 475 ml RPMI 1640, 10 ml B27 minus insulin, 5 ml cell culture media, 5 ml MEM minimum essential amino acids solution, 5 ml penicillin/streptomycin and 0.5 ml 55 mM &#946;-mercaptoethanol.</li>
		<li>For the Dissociation Solution (DS), combine 10 ml 0.05% trypsin, 4 ml KO-SR, 1 ml collagenase type IV (1 mg/ml), 5 ml KO-DMEM and 20 &#181;l CaCl2(1 M).</li>
		<li>For Feeder-cell Conditioned Medium, add 15 ml hESC medium (without bFGF) to a T75 flask with con&#64258;uent feeder cells (MEF or human foreskin fibroblasts) which have been previously inactivated by treatment with either mitomycin C or by irradiation. Harvest conditioned medium after 24 hr and replace with fresh hESC medium. Cells can be used for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Coating of Cell Culture Plates
	<ol>
		<li>ECM Gel Coating:
		<ol>
			<li>Upon purchase, thaw the ECM gel extract at 4 &#176;C until it is in an evenly consistent liquid state, as per the manufacturer&#39;s instructions. Dilute at a ratio of 1:2 in cold KO-DMEM medium, aliquot and store at -20 &#176;C. 
			Note: During the preparation of the ECM gel, keep all materials required for use in the aliquotting and coating procedure cold. The concentration of ECM gel varies with batch number. Ensure the concentration is noted on aliquots. Aliquots can be kept at -20 &#176;C for up to 6 months.</li>
			<li>Thaw an ECM gel aliquot at 4 &#176;C. When thawed, add cold KO-DMEM medium for a final concentration of 0.34 mg/ml. Pipette well and add 0.75 ml per one well of a 6-well plate. Incubate for 1 hr at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Mitotically Inactivated Mouse Embryonic Fibroblast Feeder Cell (MEF) Coating 
		Note: Preparation of MEF feeder cells has been described previously. 14
		<ol>
			<li>Coat a 6-well cell culture plate with Attachment Factor (AF) or 0.1% gelatin (see step 1.2.3) at RT&#160;for 5 min (0.75 ml per one well of a 6-well plate). Remove and leave plate in the biosafety cabinet (BSC) to dry.</li>
			<li>Thaw MEF by transferring a frozen vial to a 37 &#176;C water bath for approximately 2 - 3 min.</li>
			<li>Add 7 ml MEF medium to a 15 ml tube. When the vial of cells is thawed, slowly transfer the contents to the 15 ml tube. Centrifuge at 300 x g for 4 min. Aspirate the supernatant and resuspend the cells in MEF medium.</li>
			<li>Count the cells and plate 1 &#215; 106cells per 6 well plate (~ 1.7 x 105 cells/well). Transfer to a 37 &#176;C / 5% CO2 incubator and allow cells to attach O/N before use.</li>
		</ol>
		</li>
		<li>Gelatin Coating:
		<ol>
			<li>Dissolve 1 g of gelatin powder in ultrapure water to make a 0.1% (w/v) solution. Incubate for 15 min at 37 &#176;C then autoclave the solution at 121 &#176;C for 15 min. When cooled, add to the required number of wells (0.75 ml per one well of a 6-well plate) and incubate for 15 min at 37 &#176;C. 
			Note: Any remaining 0.1% gelatin solution can be kept at 4 &#176;C for up to 1 month.</li>
		</ol>
		</li>
		<li>Laminin Coating:
		<ol>
			<li>Thaw laminin (1 mg/ml) at 4 &#176;C until thawed. To 5 &#181;l of laminin solution, add 1 ml of cold PBS. Pipette well then add to the required number of wells (0.75 ml per one well of a 6-well plate) and incubate for 1 hr at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Silicon Coating of Spinner Flask Internal Surface:
		<ol>
			<li>Thoroughly wash a 100 ml spinner flask (with 1 glass pendulum) with distilled water, using a cleaning brush to remove any dust and/or culture residue. Fill with 70% ethanol and after 30 min rinse with distilled water. Add 5 M NaOH and leave O/N.</li>
			<li>Remove NaOH solution and rinse the flask under running water for 5 min. Fill the flask with 1 M HCl and leave for 15 min. Wash the flask with running water for 5 min, then twice with double distilled water to completely remove any trace of HCl. Leave the flask to dry completely in the BSC.</li>
			<li>Add 1.5 ml of siliconizing solution to the flask and rotate horizontally to cover all surfaces. Repeat the same procedure for the glass pendulum.</li>
			<li>Heat the coated flask in a dry oven at 100 &#176;C for 1 hr or allow to dry O/N at RT. If heated in an oven, leave to cool to RT for 30 - 60 min.</li>
			<li>Rinse the flask 3 times with deionized water for 15 min each, then sterilize by autoclaving at 121 &#730;C for 20 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Maintenance of Undifferentiated hPSCs 
	Note: For each of the protocols described, unless specifically stated, cells are grown and cultured in wells of 6-well culture plates and volumes will be given appropriate for this format.
	<ol>
		<li>hPSCs Cultured as Undifferentiated Spheroids in a Static Suspension System 
		Note: Cells used in these steps should already be adapted to single-cell culture. 15
		<ol>
			<li>Start experiment with hPSCs cultured on ECM gel in a 6-well culture plate at approximately 70 - 80% confluency. Remove any differentiated colonies using the stereomicroscope in the BSC. Add 10 &#181;M ROCK inhibitor Y-27632 and incubate at 37 &#176;C for 1 hr. 
			Note: For removing the differentiated colonies, use a pipette tip to detach and gently remove all the differentiated parts manually under the stereomicroscope. Be careful not to remove undifferentiated colonies.</li>
			<li>Remove cells from incubator and aspirate medium. Wash the cells with 1 ml PBS, remove and add 0.5 ml cell dissociation enzyme. Incubate the cells for 4 - 5 min at 37 &#176;C.</li>
			<li>Add 1 ml hESC medium and harvest the cells using a cell scraper. Dissociate the cells into single cells using a p1,000 pipette. Count the viable cells using Trypan blue and a hemocytometer.</li>
			<li>Resuspend cells to 2 &#215; 105 viable cells/ml in feeder-cell conditioned medium supplemented with 100 ng/ml bFGF and 10 &#181;M ROCK inhibitor Y-27632. Transfer cells to ultra-low attachment plates using a 5 ml pipette (3 ml per well of 6-well plate).</li>
			<li>After 2 days, carefully remove cells from incubator and aspirate half of the medium (approximately 1.5 ml). Replace with fresh conditioned medium supplemented with 100 ng/ml bFGF .</li>
			<li>Change half of the medium every day with conditioned medium supplemented with 100 ng/ml bFGF until day 5. Now, either further culture cells, passage for continued undifferentiated culture (step 1.3.1.2 - 5), or use for cardiac differentiation (Section 2). If continuing culture, passage cells every 4 - 8 days.</li>
		</ol>
		</li>
		<li>hPSCs Cultured on Feeder Cells and Passaged Using Collagenase Type IV
		<ol>
			<li>Before passaging hPSCs, remove any differentiated colonies using the stereomicroscope in the BSC. Aspirate the medium and wash cells with 1 ml PBS per well. Remove then add 0.75 ml collagenase type IV (1 mg/ml) and incubate at 37 &#176;C for 5 - 15 min, as required to have the edges of the colonies starting to peel off.</li>
			<li>Aspirate collagenase and add 1 ml of hESC medium per well. Detach colonies to small clusters using a cell scraper, then transfer the cells using a p1,000 pipette into a 15 ml tube. Be careful not to break up the clusters into small pieces.</li>
			<li>Wash the well with 1 ml hESC medium to collect any remaining cells and add to 15 ml tube. Centrifuge at 100 x g for 2 min. Aspirate the supernatant and resuspend cells in hESC medium supplemented with 100 ng/ml bFGF.</li>
			<li>Remove a MEF coated plate from the incubator and aspirate the MEF medium. Transfer the cells at a ratio between 1:3 and 1:10, as appropriate. Change medium daily with fresh hESC medium supplemented with 100 ng/ml bFGF. Passage cells every 4 - 8 days.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Differentiation of hPSCs as Spheroids in a Static Suspension System
<ol>
	<li>Start experiment using day 5 undifferentiated spheroids as prepared in Section 1.3.1. 
	Note: At this stage, the average size of spheroids should be 175 &#177; 25 &#181;m. A minimum of 50 spheroids should be used when starting a differentiation experiment.</li>
	<li>Transfer spheroids into a 15 ml tube using a 5 ml pipette. Wash the well with 1 ml RB medium to collect any remaining spheroids and add to the same 15 ml tube. Leave the cells for 5 min until spheroids have sedimented to form a loose pellet (DO NOT centrifuge). Aspirate the supernatant, being careful not to take any spheroids.</li>
	<li>Add 3 ml RB medium supplemented with 12 &#181;M CHIR99021. Transfer the cells back into one well of a 6-well ultra-low attachment plate. After 24 hr, repeat step 2.2. 
	Note: RB medium is light sensitive. When working with RB medium, keep the BSC light switched off.</li>
	<li>Add 3 ml RB medium to the cell pellet. Transfer the cells back into one well of a 6-well ultra-low attachment plate. After 24 hr, repeat step 2.2.</li>
	<li>Add 3 ml RB medium supplemented with IWP2, purmorphamine and SB431542 (5 &#181;M each). Transfer the cells back into one well of a 6-well ultra-low attachment plate. After 2 days repeat step 2.2.</li>
	<li>Resuspend the cells in 3 ml of RB medium and transfer to a gelatin-coated plate to allow the cells to attach. 
	Note: At this stage, cells can also be kept in suspension. In order to continue with static suspension culture, transfer the cells back to an ultra-low attachment plate.</li>
	<li>Change the medium with 3 ml RB medium after 2 days. The cultures will start to beat next day. Change the medium every 3 - 4 days with RB medium.</li>
</ol>
3. Differentiation of hPSCs as Spheroids in a Stirred Suspension Bioreactor
<ol>
	<li>Start experiment with undifferentiated spheroids cultured in static suspension for at least 3 passages (See Section 1.3.1). Add 10 &#181;M ROCK inhibitor Y-27632 and incubate at 37 &#176;C for 1 hr.</li>
	<li>Remove cells from incubator and transfer all spheroids into a 15 ml tube using a 5 ml pipette. Wash the well with 1 ml hESC medium to collect any remaining spheroids and add to the same 15 ml tube. Leave the cells for 5 min until spheroids have sedimented to form a loose pellet (DO NOT centrifuge). Aspirate the supernatant, being careful not to take any spheroids.</li>
	<li>Wash the cells by adding 1 ml PBS. Leave for 5 min until spheroids have sedimented to form a loose pellet (DO NOT centrifuge). Remove supernatant and add 0.5 ml 0.05% trypsin plus 0.53 mM EDTA. Incubate the cells for 4 - 5 min at 37 &#176;C.</li>
	<li>Remove cells from the incubator and add 1 ml hESC medium. Using a p1,000 pipette, dissociate the cells into single cells, count the cells using Trypan blue and a hemocytometer. Resuspend to 2 &#215; 105 viable cells/ml in conditioned medium supplemented with 100 ng/ml bFGF and 10 &#181;M ROCK inhibitor Y-27632.</li>
	<li>Transfer 100 ml of cell suspension into the siliconized bioreactor flask with 1 pendulum (see Section 1.2.5). Start with 35 rpm agitation and after 24 hr increase to 40 rpm.</li>
	<li>After 48 hr, stop agitation for 5 - 10 min until all the spheroids have settled to the bottom of the flask. Replace half of the medium with conditioned medium supplemented with 100 ng/ml bFGF. Repeat the same process every 24 hr until day 5. 
	Note: At this stage, undifferentiated spheroids with an average size of 175 &#177; 25 &#181;m should be formed.</li>
	<li>Stop agitation for 5 - 10 min until all the spheroids have settled to the bottom of the flask. Transfer all spheroids to a 50 ml tube in a minimum amount of medium (less than 50 ml). Discard any remaining medium in the bioreactor flask, carefully ensuring no spheroids are remaining in the vessel.</li>
	<li>Leave for 5 - 10 min until all the spheroids have settled at the bottom of the 50 ml tube then carefully remove the supernatant without disrupting the loose cell pellet. Wash with 25 ml PBS with calcium and magnesium or RB medium, then leave again for 5 - 10 min until all the spheroids have settled at the bottom of the 50 ml tube to again form a loose pellet.</li>
	<li>Carefully remove the supernatant and add 40 ml RB medium supplemented with 12 &#181;M CHIR99021, 10 &#181;M Rock inhibitor and 0.1% poly vinyl alcohol (PVA). Resuspend the spheroids and transfer all cells back to the bioreactor flask. Add medium to make the total volume 100 ml. Restart agitation at 40 rpm for 24 hr. 
	Note: RB medium is light sensitive. When working with RB medium keep the BSC in light switched off mode.</li>
	<li>Repeat Steps 3.7 - 3.8.</li>
	<li>Carefully remove the supernatant and add 40 ml RB medium supplemented with 0.1% PVA. Resuspend the spheroids and transfer all cells back to the bioreactor flask. Add medium to make the total volume 100 ml. Restart agitation at 40 rpm for 24 hr.</li>
	<li>Repeat Steps 3.7 - 3.8.</li>
	<li>Carefully remove the supernatant and add 40 ml RB medium supplemented with IWP2, purmorphamine and SB431542 (5 &#181;M each). Resuspend the spheroids and transfer all cells back to the bioreactor flask. Add medium to make the total volume 100 ml. Restart agitation at 40 rpm for 2 days.</li>
	<li>Repeat Steps 3.7-3.8.</li>
	<li>Carefully remove the supernatant and add 40 ml RB medium only. Resuspend the spheroids and transfer all cells back to the bioreactor flask. Add medium to make the total volume 100 ml. Restart agitation at 40 rpm. Beating spheroids should be observed 48 - 72 hr later.</li>
	<li>Change half of the medium every 3 - 4 days with RB medium only by stopping the agitation for 5 - 10 min until all the spheroids have settled to the bottom of the vessel. Carefully remove 50 ml of medium without disturbing the spheroids and carefully replace with 50 ml of fresh RB medium.</li>
</ol>
4. Differentiation of hPSCs Using Cultures not Adapted to Single Cell Passaging
Note: This approach is specifically useful for the fast differentiation of a high number of hPSC lines without having to adapt to single-cell culturing techniques, an enormously labor-intensive and time consuming process. This technique is applicable to cell lines which are highly sensitive to enzymatic cell dissociation, such as newly established hPSC lines.&#160;
<ol>
	<li>Start experiment with hPSCs cultured on MEF (as in Section 1.3.2) which are approximately 70 - 80% confluent. Remove any differentiated colonies using the stereomicroscope in the BSC.</li>
	<li>Remove medium and add 0.5 ml Dissociation Solution (DS). Incubate for 0.5 - 1 min until MEF cells have rounded up and the edges of hPSCs colonies become clear. Quickly remove DS and add 0.75 ml collagenase type IV (1 mg/ml).</li>
	<li>Incubate the cells for 5 - 15 min at 37 &#176;C. Check the cells under the microscope during the incubation period, and when whole colonies are starting to lift off and detach, remove collagenase and add 1.5 ml hESC medium. 
	Note: If after 15 min most of the colonies are still attached, put the cells back to incubator. Check the cells under the microscope every 5 min. Be careful not to leave the cells in collagenase for more than 30 min. If there are many floating colonies in the collagenase solution, do not remove the collagenase; just add 1 ml hESC medium.</li>
	<li>Using a p1,000 pipette, gently pipette colonies to detach as whole colonies. Do not break the colonies into small pieces. Transfer the cells into a 15 ml tube using a 5 ml pipette. Wash the well with 1 ml hESC medium to collect any remaining cells to add to the same tube. Leave for 5 min until all colonies have sedimented (DO NOT centrifuge).</li>
	<li>Carefully remove the supernatant and add 2 ml hESC medium supplemented with 100 ng/mL bFGF. Transfer the cells into an ultra-low attachment plate using 5 ml pipette (1 ml into each well of a 24-well plate and 3 ml into each well of a 6-well plate). Incubate at 37 &#176;C/5% CO2 for at least 6 hr (maximum of 12 hr).</li>
	<li>During this time, prepare the required number of laminin-coated wells/plates for step 4.7. Recommended cell transfer ratios are 1 confluent well of a 6-well plate to 2 wells of a 24-well plate (or equivalent amount per surface area).</li>
	<li>After 6 hr, carefully transfer the colony aggregates into a 15 ml tube using 5 ml pipette. Wash the plate with RB medium to collect any remaining aggregates and add to the same 15 ml tube (0.5 ml per well for 24-well and 1 ml per well for 6-well plates). Wait 5 min until the aggregates sediment, then carefully aspirate the supernatant. Be careful not to disturb the loose pellet.</li>
	<li>Add 2 ml RB medium supplemented with 12 &#181;M CHIR99021 and carefully transfer with a 5 ml pipette to your pre-prepared laminin coated wells (as in Section 1.2.4) to allow the cells to attach.</li>
	<li>After 24 hr, carefully remove the medium in each well and add 1 ml RB medium only. 
	Note: Some of the aggregates will have only attached very weakly to the culture plate; therefore it is important to not remove any large aggregates which may have come loose during the medium change procedure. It is important, however, to remove as much of the small cell debris as possible.</li>
	<li>After 24 hr, carefully remove the medium and add RB medium supplemented with IWP2, purmorphamine and SB431542 (5 &#181;M each).</li>
	<li>Change the medium after 2 days with RB medium only. Cells will start to beat next day.</li>
	<li>Change the medium every 3 - 4 days with RB medium only.</li>
</ol>

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R., Samadian, A., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocess+development+for+mass+production+of+size-controlled+human+pluripotent+stem+cell+aggregates+in+stirred+suspension+bioreactor.">Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor.</a> Tissue Eng Part C Methods. 18, 831-851 (2012).</li><li>Larijani, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+undifferentiated+human+embryonic+and+induced+pluripotent+stem+cells+in+suspension.">Long-term maintenance of undifferentiated human embryonic and induced pluripotent stem cells in suspension.</a> Stem Cells Devt. 20, 1911-1923 (2011).</li><li>Baharvand, H., Larijani, M. R., Yousefi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+expansion+of+undifferentiated+human+embryonic+and+pluripotent+stem+cells+in+suspension.">Protocol for expansion of undifferentiated human embryonic and pluripotent stem cells in suspension.</a> Methods Mol Biol. 873, 217-226 (2012).</li><li>Burridge, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nat Methods. 11, 855-860 (2014).</li><li>Minami, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+molecule+that+promotes+cardiac+differentiation+of+human+pluripotent+stem+cells+under+defined,+cytokine-+and+xeno-free+conditions.">A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions.</a> Cell reports. 2, 1448-1460 (2012).</li><li>Buskirk, A. R., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+small-molecule-dependent+switches+to+modulate+biological+functions.">Creating small-molecule-dependent switches to modulate biological functions.</a> Chem Bio. 12, 151-161 (2005).</li><li>McKinsey, T. A., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+therapies+for+cardiac+hypertrophy:+moving+beneath+the+cell+surface.">Small-molecule therapies for cardiac hypertrophy: moving beneath the cell surface.</a> Nat Rev Drug Discov. 6, 617-635 (2007).</li><li>Niebruegge, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+embryonic+stem+cell-derived+mesoderm+and+cardiac+cells+using+size-specified+aggregates+in+an+oxygen-controlled+bioreactor.">Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor.</a> Biotechnol Bioeng. 102, 493-507 (2009).</li><li>Bauwens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cardiomyogenic+induction+in+human+pluripotent+stem+cells.">Geometric control of cardiomyogenic induction in human pluripotent stem cells.</a> Tissue Eng Part A. 17, 1901-1909 (2011).</li><li>Hwang, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwell-mediated+control+of+embryoid+body+size+regulates+embryonic+stem+cell+fate+via+differential+expression+of+WNT5a+and+WNT11.">Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11.</a> Proc Natl Acad Sci U S A. 106, 16978-16983 (2009).</li><li>Nguyen, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+generation+of+cardiospheres+promotes+robust+enrichment+of+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">Microscale generation of cardiospheres promotes robust enrichment of cardiomyocytes derived from human pluripotent stem cells.</a> Stem Cell Reports. 3, 260-268 (2014).</li><li>Kempf, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+expansion+and+cardiomyogenic+differentiation+of+human+pluripotent+stem+cells+in+scalable+suspension+culture.">Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture.</a> Stem Cell Reports. 3, 1132-1146 (2014).</li><li>Hemmi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+massive+suspension+culture+system+with+metabolic+purification+for+human+pluripotent+stem+cell-derived+cardiomyocytes.">A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes.</a> Stem Cells Transl Med. 3, 1473-1483 (2014).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell stem cell. 12, 127-137 (2013).</li><li>Nunes, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biowire:+a+platform+for+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Nat meth. 10, 781-787 (2013).</li><li>Devalla, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial-like+cardiomyocytes+from+human+pluripotent+stem+cells+are+a+robust+preclinical+model+for+assessing+atrial-selective+pharmacology.">Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology.</a> EMBO Mol Med. 7, 394-410 (2015).</li></ol>

The Victor Chang Cardiac Research Institute does not engage in, nor does it condone, the destruction of human embryos for research. Its contribution to this study was limited to work on human induced pluripotent stem cells.
The authors declare they have no competing financial interests.

Cardiomyocytes derived from hPSCs are an extremely attractive source for use in human disease modeling, drug screening/toxicity testing and, perhaps in the future, regenerative therapies. One of the major hurdles to using these cells however, is the ability to provide enough high quality material for their effective use. Using our described protocol, we offer a method that overcomes this limitation.
Recently, synthetic small molecules targeting specific signaling pathways involved in cardiogenesis have been described to enhance cardiac differentiation 16,17,22,23. These are now being used as an alternative to recombinant cytokines and serum, containing many undefined factors and showing batch variation. The main advantage of a small molecule protocol, other than its specificity, is that it is inexpensive and component factors generally have an extended shelf-life in comparison to media containing cytokines or growth factors. As the small molecules used in our reported protocol are low molecular weight agents, they are structurally and functionally defined and can diffuse easily through the cell membrane 24,25.
So far, different methods have been applied to hPSCs in order to establish robust, scalable differentiation techniques; however, most of these methods have been established as 2D and small-scale static cultures, which may offer poor scalability and homogeneity. Techniques such as forced aggregation, micro-printing technologies and micro-carrier cultures 26-29 are now coming into use, but in spite of some advances in this area, these methods are still far from providing the cell numbers required for their use in high-demand systems. Limited or unproven reproducibility and scalability, and the high costs due to the necessity for expensive media (mTeSR1 or StemPro-34) or micro-carriers for both expansion of hPSCs and their directed differentiation to cardiomyocytes 30,31, are the drawbacks of using these methods in high throughput hPSCs technologies.
In this study, we have reported a simple, robust and scalable protocol for the production of hPSC-derived cardiomyocytes. The reproducibility of this protocol has been validated with more than 40 different hPSC lines, the most extensive validation reported to date. Compared to previously reported suspension protocols, this method shows great advantages in terms of scalability, reproducibility, affordability, efficacy and functionality of the cardiomyocytes generated. The success of our differentiation protocol is thoroughly dependent on the high quality of the hPSCs used for study. Therefore it is crucial to check each line&#39;s karyotype and the high and sustained expression of pluripotency markers by immunostaining and PCR before starting the experiment. Particularly, when culturing cells in the bioreactor, it is crucial to use hPSCs that have been passaged at the single-cell level for at least three passages prior to the start of differentiation. In our protocols we have used spheroids with an average size of 175 &#177; 25 &#181;m, which were spheroids generated after 5 days. The day which the spheroids meet this size restriction may vary depending on the growth rate of individual hPSC lines; therefore it is important that the size, more than the day of growth, is taken into consideration.
This protocol also can be manipulated to become suitable for sensitive cell lines and large cohorts of hPSC lines. Although this protocol results in highly enriched cardiomyocytes, purity may be improved by combining the differentiation protocol with a metabolic selection method such as lactate-enriched medium 32. Additionally, improvements in the maturation and functionality of the derived cardiomyocytes described in this protocol may be applied by the generation of three-dimensional, aligned cardiac tissues 33. One of the limitations of this protocol is that particular subtypes of cardiomyocytes are not specifically generated, simply a mixture of atrial, ventricular and nodal cells. Further investigation in this field is needed to develop differentiation protocols which can produce highly enriched subtype-specific cardiomyocytes in large-scale. Although some small-scale protocols have been developed to date, the methods would need to be rigorously tested to ensure scale-up is possible 34.
Development of such integrated platforms can be considered as an important step towards the commercialization of hPSC-derived cardiomyocytes technologies for clinical, pharmaceutical, tissue engineering, and in vitro organ/organoid development applications.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability of self-renewal and the capacity to differentiate into cells of the three embryonic germ layers 1,2. Due to these characteristics, hPSCs provide a valuable and unlimited source for the generation and scalable production of disease-relevant cell types for modeling human disease 3-5, for high-throughput drug screening and toxicity assays 6,7 and potentially for clinical applications 8. Generation of cardiomyocytes from hPSCs provides the opportunity to specifically investigate the mechanisms of complex human cardiovascular diseases and their possible treatments, previously beyond the scope of our capabilities due to the lack of relevant animal models and/or the availability of affected primary tissues.
All of the aforementioned applications of hPSCs necessitate the production of massive numbers of highly enriched and functional cardiomyocytes. Thus, the availability of an efficient, reproducible and scalable in vitro cardiac differentiation protocol suitable for multiple hPSC lines is crucial. Conventional cardiomyocyte differentiation protocols have employed different strategies such as embryoid body formation 9, co-culture techniques 10, induction with cocktails of cytokines 11 and protein transduction methods 12. In spite of advances in these techniques, most still suffer from poor efficiency, require expensive growth factors, or offer limited universality when attempting to use multiple hPSC lines. To date, these challenges have set limits to the production of hPSC-derived cardiomyocytes for cell therapy studies in animal models, as well as in the pharmaceutical industry for drug discovery 13. Therefore, the development of robust and affordable techniques for large-scale production of functional hPSC-derived cardiomyocytes in scalable culture systems would largely facilitate their commercial and clinical applications.
In this manuscript, we report the development of a cost-effective and integrated cardiac differentiation system with high efficacy, reproducibility and applicability to hESCs and hiPSCs generated from a variety of sources and culture methods, including a method for the large-scale production of highly enriched populations of hPSC-derived cardiomyocytes using a bioreactor. Additionally, we have optimized this protocol for hPSC lines not adapted to feeder free and/or single cell culture, such as newly established hiPSCs or large cohorts of hPSC lines relevant to analysis of disease mechanism.

<table><tbody><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829018</td><td></td></tr><tr><td>Knockout Serum Replacement (KO-SR)</td><td>Life Technologies</td><td>10828028</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>MEM Non-essential Amino Acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Basic Fibroblast Growth Factor (bFGF)</td><td>Miltenyi Biotec</td><td>130-093-843</td><td></td></tr><tr><td>RPMI1640</td><td>Life Technologies</td><td>11875093</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Life Technologies</td><td>14190144</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14287072</td><td></td></tr><tr><td>Attachment Factor (AF)</td><td>Life Technologies</td><td>S006100</td><td></td></tr><tr><td>ECM Gel</td><td>Sigma-Aldrich</td><td>E1270</td><td></td></tr><tr><td>Laminin</td><td>Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11965-092&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Fatal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>B27 minus insulin</td><td>Gibco</td><td>A18956-01</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>0.05% Trypsin/EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Life Technologies</td><td>17140-019</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Mitomycin C</td><td>Bioaustralis</td><td>BIA-M1183</td><td></td></tr><tr><td>CHIR99021</td><td>Miltenyi Biotec</td><td>130-104-172</td><td></td></tr><tr><td>IWP2</td><td>Miltenyi Biotec</td><td>130-105-335</td><td></td></tr><tr><td>SB431542</td><td>Miltenyi Biotec</td><td>130-095-561</td><td></td></tr><tr><td>Purmorphamine</td><td>Miltenyi Biotec</td><td>130-104-465</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Miltenyi Biotec</td><td>130-104-169</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Poly Vinyl Alcohol (PVA)</td><td>Sigma-Aldrich</td><td>363073</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Trypan Blue</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Accumax&amp;nbsp;</td><td>Innovative Cell Technologies Inc.</td><td>AM105</td><td></td></tr><tr><td>Sigmacote&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SL2&amp;nbsp;</td><td></td></tr><tr><td>CELLSPIN</td><td>Integra Biosciences</td><td>183 001</td><td></td></tr><tr><td>Spinner flask with 1 pendulum, 100 ml&amp;nbsp;</td><td>Integra Biosciences</td><td>182 023</td><td></td></tr><tr><td>Mouse Embryonic Fibroblasts (MEF)</td><td>Prepared in-house (or commercially available)</td><td></td><td></td></tr><tr><td>Human pluripotent stem cell (hPSC) lines</td><td>Prepared in-house (or commercially available)</td><td></td><td></td></tr></tbody></table>

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.

In order to establish a simple method for the large-scale differentiation of cardiomyocytes from hPSCs, we created a protocol in which cells were treated initially with a WNT/&#946;-catenin activator (CHIR99021)16 and subsequently with inhibitors of the WNT/&#946;-catenin and transforming growth factor-&#946; (TGF-&#946;) pathways (IWP216 and SB43154217, respectively) and finally an activator of the sonic hedgehog (SHH) pathway (purmorphamine)17 (Figure 1A). In our differentiation strategy, approximately 50% of the spheroids (175 &#177; 25 &#181;m spheroid diameter size) started beating 7 days after initiation of differentiation, which then increased to 100% by day 10. Interestingly, using this protocol, differentiated spheroids have been observed to continue beating up to 60 days after differentiation initiation18. Population studies on dissociated spheroids examined by flow cytometry revealed that at day 15, more than 90% of the population contained cardiac troponin T positive (cTnT+) cells, while less than 12% of the cells were positive for smooth muscle and endothelial markers (3.1% von Willibrand Factor (vWF+), 8.4% alpha smooth muscle actin-positive (aSMA+))18. To date, the static suspension differentiation protocol has been tested on 5 hESC and 4 hiPSC lines, with outputs resulting in approximately 90% of beating spheroids from each line, showing the high reproducibility of this protocol among different hPSC lines (Figure 1B).
In order to develop an integrated platform for large-scale production of human cardiomyocytes, we applied our static suspension differentiation strategy to a stirred suspension bioreactor (Figure 2A). The differentiating spheroids showed similar behavior in the bioreactor environment as in the static system (Figure 2B) and approximately 100% of spheroids were observed to be beating at day 10 18. Immunostaining in sections of beating spheroids collected at day 30 using antibodies against cTnT and the cardiac transcription factor NKX2-5, showed cytoplasmic and nuclear expression of these proteins, respectively (Figure 2C). The total yield of cells in the dynamic suspension culture was approximately 90 - 100 million cells in 100 ml working volume after 15 days of differentiation culture. With this being the case, the cardiomyocyte yield can reach up to approximately 54-90 million cells (with the observed 60 - 90% differentiation efficacy) from 20 million starting hPSCs, inoculated into the hPSCs expansion phase as single cells. We additionally examined the electrophysiological properties of differentiated cardiomyocytes using the single-cell patch clamp method. Beating spheroids from the bioreactor cultures were dissociated into single cells at day 30 and action potentials recorded on representative cells using the whole cell patch clamp technique. Data showed the presence of the three main cardiac cell types (atrial-, nodal- and ventricular-like cells) in the population of single cells18.
For the two differentiation techniques mentioned above, hPSCs need to be adapted to feeder-free and/or single-cell suspension culture19-21. Some hPSC lines however, are highly sensitive to enzymatic cell dissociation and show significant cell viability loss after dissociation, such as can be observed in newly established hiPSC lines. Additionally, when working with large cohorts of lines, adapting all to feeder-free and single-cell culture would be enormously labor intensive and time consuming. To address these issues, undifferentiated spheroids were replaced with undifferentiated cell aggregates (Figure 3A). Our data show that using this modified technique, beating clusters appeared 7 days after differentiation initiation. The reproducibility of this modified version was confirmed using 42 hiPSC lines, where all tested lines generated on average more than 60% cTnT+ cells by day 15 for each experiment performed (Figure 3B). Immunostaining using antibodies against cTnT and NKX2-5 in dissociated beating clusters showed cytoplasmic and nuclear expression, respectively (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/54276/54276fig1.jpg" /> 
Figure 1. Schematic of Cardiomyocyte Differentiation from hPSCs in Static Suspension and Representative Data. (A) Experimental design of hPSCs differentiation to cardiomyocytes in static suspension system. (B) Evaluation of the efficiency of cardiac differentiation is measured by counting the number of beating spheroids (%). Shown here are the averages of at least 3 differentiation experiments from each of 5 hESC and 4 hiPSC lines18. Overall average of beating spheroids from all lines is approximately 90%. Error bars represent SD (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54276/54276fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54276/54276fig2.jpg" /> 
Figure 2. Overall Schematic of Cardiomyocytes Differentiation from hPSCs in Dynamic Suspension and Representative Data. (A) Experimental design of hPSCs differentiation to cardiomyocytes in dynamic suspension system. RI: Rock inhibitor (B) Phase contrast image of day 5 spheroids (Representative result from Royan H5 hESC line). Scale bar 200 &#181;m (C) Immunostaining of hESC derived-beating spheroids sectioned at day 30 for NKX2.5 and cTnT. Scale bar 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54276/54276fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54276/54276fig3.jpg" /> 
Figure 3. Overall Schematic of Cardiomyocytes Differentiation from hPSCs using Cultures not Adapted to Single Cell Passaging. (A) Experimental design of hPSCs differentiation to cardiomyocytes using cultures not adapted to single cell passaging. (B) Flow cytometry analysis of dissociated aggregates show on average more than 60% cTnT+ cells in 42 tested hiPSC lines. (C) Immunostaining of dissociated cardiomyocytes derived from hiPSCs for NKX2.5 and cTnT (Representative result from 646-4 hiPSC line). Scale bar 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54276/54276fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol at JoVE.c

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

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 ...

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Nanyang Technological University

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Developmental Biology

10.5K Views.  Royan Institute for Stem Cell Biology and Technology, ACECR. 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.

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

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Bioengineering

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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

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