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 (Video) | JoVE

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.

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 ...

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

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

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

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 (Video) | JoVE

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

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System (Video) | JoVE

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells (Video) | JoVE

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 (Video) | JoVE

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

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells (Video) | JoVE

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells (Video) | JoVE

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

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells (Video) | JoVE

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

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

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells (Video) | JoVE

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