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.

large-scale-production-cardiomyocytes-from-human-pluripotent-stem

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 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 ...

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 different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based 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 cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

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

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 various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

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 a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

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

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

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 failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.