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
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 ...

<ol>
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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. 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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 cardiogen...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#946;-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&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</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 (CaCl2)</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&#160;</td>
			<td>Innovative Cell Technologies Inc.</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SL2&#160;</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&#160;</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 </sup...

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 ...

The overall goal of this experiment is to differentiate human pluripotent stem cells into cardiomyocytes with high efficiency and reproducibility. This protocol can be scaled up in a bioreactor or modified for differentiating cell lines, which are sensitive to single cell culture. This method can help answer key questions in cardiac disease modeling using human induced pluripotent stem cells.The main advantages of this technique are that it is cost-effective and has high efficiency, reproducibility, and applicability for both human embryonic and induced pluripotent stem cells. Our demonstrator today is Hananeh Fonoudi, currently a PhD student at the Victor Chang Cardiac Research Institute, coming to us from the Royan Institute. Before coating, use a cleaning brush and distilled water to thoroughly remove any dust or culture residue from a 100 milliliter spinner flask with a glass pendulum.Next, fill the flask with 70%ethanol. After 30 minutes, rinse the flask with distilled water, and fill it with five molar sodium hydroxide. The next morning, rinse the flash under running water for five minutes, and fill it with one molar hydrochloric acid.After 15 minutes, rinse the flask under running water for another five minutes, followed by two rinses with double-distilled water. Place the flask in a biosafety cabinet to dry. Then, add 1.5 milliliters of siliconizing solution to the flask, and rotate the flask horizontally to cover all of the surfaces with silicon.Heat the coated flask in a dry oven for one hour at 100 degrees Celsius. When the flask has cooled, rinse it three times for 15 minutes with deionized water. Then, autoclave sterilize the container at 121 degrees Celsius for 20 minutes.After passaging undifferentiated spheroids, using the single cell passaging method at least three times, use a five milliliter pipette to transfer all of the undifferentiated spheroids into a 15 milliliter conical tube. Wash the wells with one milliliter of human embryonic stem cell medium to collect any remaining spheroids, and pool the wash with the harvested spheroids. Allow the spheroids to sediment for five minutes to form a loose pellet.Then, aspirate the supernatant, taking care not remove any spheroids. Next, rinse the spheroids with one milliliter of PBS for five minutes, and incubate them in 5 milliliters of Trypsin EDTA at 37 degrees Celsius. After four to five minutes, stop the reaction with one milliliter of human embryonic stem cell medium, and use a P1000 pipette to dissociate the spheroids into a single cell suspension.After counting by trypan blue exclusion, resuspend the cells at a 200, 000 cell per milliliter concentration, in conditioned medium supplemented with basic fibroblast growth factor and ROCK inhibitor, and transfer 100 milliliters of the cell suspension into the siliconized bioreactor flask. Place the flask on the agitator at 35 RPM. After 24 hours, increase the agitation to 40 RPM.After 48 hours, stop the agitation for five to ten minutes until all of the reformed spheroids have settled to the bottom of the flask, and replace half of the medium with conditioned medium supplemented with basic fibroblast growth factor. Then, restart the agitator at 40 RPM for another 24 hours, replacing half of the medium by the same process every day for the next three days. On day five, stop the agitator for five to ten minutes.Then, transfer all of the spheroids to a 50 milliliter tube in 50 milliliters of medium or less. Discard any of the remaining medium in the flask, taking care that no spheroids remain in the vessel. Then, carefully remove the supernatant from the settled spheroids, and wash cell clusters in 25 milliliters of PBS with calcium and magnesium.After five to ten minutes, remove the wash, and resuspend the spheroids in 40 milliliters of RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, ROCK inhibitor, and polyvinyl alcohol. Transfer all of the spheroids back to the bioreactor flask. Then, add fresh RPMI-B27 medium to a total volume of 100 milliliters, and restart the agitation at 40 RPM.After 24 hours, follow the same process to change media from RPMI-B27 medium. After an additional 24 hours, follow the same process and change media with RPMI-B27 plus IWP2, purmorphamine, and SB43. Leave for 48 hours.First beating is usually observed after seven days. For the fast differentiation of human pluripotent stem cell lines that have not been adapted to single cell culture, first treat to 70 to 80%confluent human pluripotent stem cell culture, grown on mouse embryonic fibroblast cells, with 5 milliliters of dissociation solution for 30 to 60 seconds. Dissociation solution should only be left until the MEFs round up and hPSC colony edges become clear.If the cells are left too long, they will not differentiate correctly. At the appropriate time, quickly replace the dissociation solution with 75 milliliters of collagenase type IV, and incubate the cells for five to 15 minutes at 37 degrees Celsius, checking the culture under the microscope every few minutes. When whole colonies begin to detach, replace the collagenase with 1.5 milliliters of human embryonic stem cell medium, and use a P1000 pipette to gently detach whole cell colonies from the bottom of the plate.Cells should be carefully detached so that the colonies do not break up into small pieces. Then, use a five milliliter pipette to transfer the cells into a 15 milliliter conical tube, and wash the wells with one milliliter of human embryonic stem cell medium, pooling any remaining cells in the 15 milliliter tube. After five minutes of sedimentation, carefully replace the supernatant with two milliliters of fresh human embryonic stem cell medium supplemented with basic fibroblast growth factor.Using a new five milliliter pipette, transfer the cells into an ultra-low attachment plate, and incubate the colonies for six to 12 hours. At the end of the incubation, use a five milliliter pipette to carefully transfer the colony aggregates into a new 15 milliliter tube, and collect any remaining aggregates with an RPMI-B27 medium wash. When the colonies have settled, replace the supernatant with fresh RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, and carefully transfer the colonies to laminin-coated plates for attachment at 37 degrees Celsius.After 24 hours, change the medium with RPMI-B27 medium. After another 24 hours, change the medium with RPMI-B27 containing IWP2, purmorphamine, and SB43. First beating is usually observed after seven days.To date, the static suspension differentiation protocol has been tested on five human embryonic stem cell and four human induced pluripotent stem cells lines resulting in more than 90%beating spheroids from each line, demonstrating the high reproducibility of this protocol among different human pluripotent stem cell lines. The differentiating spheroids exhibit a similar behavior in a stirred suspension bioreactor environment as in the static system, with approximately 100%of spheroids observed to beating at day 10. Indeed, beating spheroid sections collected at day 30 reveal the cytoplasmic and nuclear expression of cardiac troponin T and the cardiac transcription factor NKX2.5 respectively.Further, when undifferentiated spheroids are replaced with undifferentiated cell aggregates, beating clusters appear seven days after differentiation. With 42 tested human induced pluripotent stem cell lines generating on average more than 60%cardiac troponin T positive cell populations by day 15. The beating clusters also demonstrate a similar cardiac troponin T cytoplasmic and NKX2.5 nuclear expression as observed for the stirred suspension bioreactor-generated spheroids.While attempting this procedure, it is important to remember to use hPSCs which has been adapted to single cell culture for at least three passages prior to the differentiation in the bioreactor. This procedure can be combined with other methods for enriching cardiomyocyte to acquire a pure population of cells. This technique has paved the way for commercialization of hPSC-derived cardiomyocyte.It has the potential for use in clinical, pharmaceutical, tissue engineering, and organ or organoid development applications. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells into cardiomyocyte and to scale up the culture in bioreactor.

Research

JoVE Journal

Developmental Biology

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 ...

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 ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...