Name: differentiation of atrial cardiomyocytes from pluripotent stem cells using the bmp antagonist grem2
Uploaded: 2016-03-10T15:00:00
Description: Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

This work was supported by NIH grants HL083958 and HL100398 (A.K.H.) and 2T32HL007411-33 &#34;Program in Cardiovascular Mechanisms: Training in Investigation&#34; (J.B.).

1. Preparation of Cell Culture Media, Solutions, and Reagents.
<ol>
	<li>Prepare 500 ml of Mouse embryonic stem cell (mESC) media by mixing and sterile filtering (0.2 &#956;m pore size) 445 ml Glasgow Minimum Essential Medium (GMEM), 50 ml heat inactivated fetal bovine serum (FBS), 5 ml 100X concentrated L-Glutamine replacement, 5 &#181;l recombinant mouse Leukemia Inhibitory Factor (LIF) at 1 x 107 units per ml, and 1.43 &#181;l &#223;-Mercaptoethanol (final concentration is 50 &#181;M). Store at 4 &#730;C ...

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B., 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=Gremlin+2+promotes+differentiation+of+embryonic+stem+cells+to+atrial+fate+by+activation+of+the+JNK+signaling+pathway.">Gremlin 2 promotes differentiation of embryonic stem cells to atrial fate by activation of the JNK signaling pathway.</a> Stem Cells. 32 (7), 1774-1788 (2014).</li><li>Kattman, S. J., Witty, A. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell Stem Cell. 8 (2), 228-240 (2011).</li><li>M&#252;ller, I. I., Melville, D. 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J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+Real-Time+quantitative+PCR+and+the+2&#8722;&#916;&#916;CT+method.">Analysis of relative gene expression data using Real-Time quantitative PCR and the 2&#8722;&#916;&#916;CT method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Beck, H., Semisch, M., Culmsee, C., Plesnila, N., Hatzopoulos, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egr-1+regulates+expression+of+the+glial+scar+component+phosphacan+in+astrocytes+after+experimental+stroke.">Egr-1 regulates expression of the glial scar component phosphacan in astrocytes after experimental stroke.</a> American Journal of Pathology. 173 (1), 77-92 (2008).</li><li>Graham, E. L., Balla, C., Franchino, H., Melman, Y., del Monte, F., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation, culture, and functional characterization of adult mouse cardiomyoctyes.</a> Journal of Visualized Experiments. (79), (2013).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recording+of+ion+channels+expressed+in+Xenopus+oocytes.">Patch clamp recording of ion channels expressed in Xenopus oocytes.</a> Journal of Visualized Experiments. (20), (2008).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. 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(50), (2011).</li><li>Antonchuk, J., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+embryoid+bodies+from+human+pluripotent+stem+cells+using+AggreWell&#8482;+plates.">Formation of embryoid bodies from human pluripotent stem cells using AggreWell&#8482; plates.</a> Basic Cell Culture Protocols. 946, 523-533 (2013).</li><li>Horii, T., Nagao, Y., Tokunaga, T., Imai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells.">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li></ol>

This protocol routinely produces cultures with a high percentage of CMs that are characteristic of the atrial lineage. As with any differentiation protocol, the quality of the mESCs prior to differentiation should be given particular attention. mESCs should be routinely monitored for proper morphology (Figure 1A). Any spontaneous differentiation that occurs prior to formation of EBs will severely limit the efficiency of cardiogenesis and should be removed before passaging (Figure 1B). EB...

Pluripotent stem cells are a powerful tool for generating and studying cells from a host of difficult to access tissues for basic research and pre-clinical studies, especially in humans1-5. Proper modulation of developmental signaling pathways can direct the differentiation of pluripotent stem cells to the desired phenotypic fate. Many protocols have been developed to generate cardiomyocytes (CMs) from pluripotent stem cells6-14. These protocols generally involve modulation of TFG&#946; superfamily (Activin, BMP, and TGF&#946;) and Wnt pathways through timed addition of exogenous growth factors and/or small molecules10,12-15. These protocols are generally effective at increasing the percentage of cells that become CMs, but lack specificity, generating a mixed population of cells representing atrial, ventricular, and nodal/conduction system lineages. In order to study specific cardiac subtypes a more directed differentiation approach is required.
Gremlin2 (Grem2, also called Protein Related to Dan and Cerberus or PRDC for short) is a secreted BMP antagonist that is necessary&#160;for proper cardiac differentiation and atrial chamber formation during cardiac development in zebrafish16. Treating differentiating embryonic stem cells with Grem2 at differentiation day 4, just after peak expression of mesodermal markers T-Brachyury and Cerberus like 1, increases the yield of CMs and generates a pool of cells predominantly of the atrial lineage14.
Recombinant Grem2 is used to treat the differentiating cells and can be made using standard protein production techniques17 or can be purchased commercially. It is highly soluble in aqueous solutions and may be added exogenously to cultures at the desired time point.
Differentiation can be tracked using RT-qPCR to quantify expression of markers representative of cardiovascular progenitors, cardiac progenitors, and committed CMs. Immunofluorescence can also be used to identify and visualize spatial distribution of cardiac cell types.
Applications that require pure populations are more readily carried out when using a reporter system to identify and isolate CMs. For this purpose, we have introduced the &#945;MHC-DsRedNuc construct into the mouse CGR8 ES cell line14. CGR8 cells grow and remain pluripotent without feeder cells, facilitating expansion and differentiation assays18. The ES cell line contains a DSRed fluorescent protein coding sequence with a nuclear localization signal under the cardiac-specific alpha Myosin Heavy Chain (&#945;Mhc or Myh6) gene promoter. Using these cells, CMs can be easily identified and isolated for quantification,&#160;cell sorting,&#160;electrophysiology, drug screens, and studying mechanisms of atrial differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="479">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:217px;">GMEM</td>
			<td style="width:116px;">Life Technologies</td>
			<td style="width:83px;">11710</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Life Technologies</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamax</td>
			<td>Life Technologies</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIF</td>
			<td>EMD Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#223;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Sigma</td>
			<td>I3390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-Essential Amino Acids</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Tissue Culture Plates</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 cm Bacterial Petri Dishes</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 cm Bacterial Petri Dishes</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well tissue culture plates</td>
			<td>Sarstedt</td>
			<td>83.3920</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gremlin 2 recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>2069-PR-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile filter units</td>
			<td>Thermo Fisher</td>
			<td>09-741-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin (from porcine skin)</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X PBS, Sterile</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA&#160;</td>
			<td>Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% Trypsin-EDTA solution</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no Calcium, no Magnesium</td>
			<td>Life Technologies</td>
			<td>14200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Prior to differentiation, pluripotent stem cells should be compact and free of spontaneous differentiation. The cells shown in Figure 1A are ready to be singularized and used for hanging drops as described in 5.1 of the protocol section. The cells shown in Figure 1B are spontaneously differentiating and should not be used for preparation of hanging drops.
The panels included in Figure 2<...

Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

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

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 overall goal of this differentiation protocol is to generate homogeneous populations of atrial cardiomyocytes in culture. So this method can help answer key questions in the study of cardiac development and cardiac diseases such as atrial arrhythmia. The main advantages of this technique are that it produces homogeneous cultures of atrial cardiomyocytes in vitro and that it can be done with minimal training.Up to four days before setting up the mouse embryonic stem cell, or mESC cultures, code 10-centimeter tissue dishes with 5 milliliters of 0.2%gelatin solution each, in six well plates with 1 milliliter of gelatin per well in a sterilized tissue culture hood. On the day of the culture, remove the extra gelatin solution, and add 10 milliliters of medium to one of the gelatin-coated 10-centimeter dishes. Then, use cryotongs to gently swirl a vial of 1 times 10 to the 6 mESC's in a 37-degree Celcius water bath until only a small ice crystal remains in the center of the vial.Dry the vial with a disposable, lint-free wipe and sterilize it with 70%ethanol. Then place the vial in the tissue culture hood and use a P1000 pipette to add the ESC's to the dish. Place the cells in a 37-degree Celcius humidified cell culture incubator with 5%carbon dioxide, and gently move the plate front to back and side to side to evenly distribute the cells.At the end of the incubation, use a brightfield microscope to confirm the cell attachment. Some debris and dead cells will be observed. Replace the medium with 10 milliliters of fresh mESC medium, and return the cells to the incubator.When the culture is 60 to 70%confluent, rinse the plate with 5 milliliters of sterile DPBS without magnesium and calcium, and incubate the cells in 2 milliliters of trypsin EDTA at 37 degrees Celcius for five minutes. While the cells are incubating, aspirate the excess gelatin solution from one new 10-centimeter dish per passaged culture, and add 10 milliliters of medium to each plate. Then collect the detached cells by centrifugation in a 15 milliliter tube, and re-suspend the pellet in 10 milliliters of mESC medium.Depending on the desired split ratio, add 0.5 to 1 milliliter of cell suspension to each 10 centimeter dish and return the cells to the incubator, gently distributing the cells in each dish as just demonstrated. When the passaged cells are 70%confluent, detach them with trypsin EDTA as just demonstrated, then resuspend the single-cell suspension in 5 milliliters of embryoid body or EB medium. Count the mESC's and then dilute them in the appropriate volume of EB medium to a final concentration of 2.5 times 10 to the 3rd cells per milliliter.Next, add 2 milliliters of PBS to the bottom of one sterile 10-centimeter bacterial petri dish per hanging drop culture, and transfer the EB suspension into a sterile solution basin. Now, remove the lid from one of the prepared dishes and use a multichannel pipette, equipped with six tips, to carefully deposit 60 20-microliter drops of EB suspension onto the inner surface. Gently place the lid back onto the dish, taking care not to dislodge the hanging drops and place the dishes into the cell culture incubator for two days.To wash down the EB'ss, carefully remove the lid from the hanging drop culture, and tilt it at a 45-degree angle. Then, using a 5-milliliter serological pipette containing room-temperature EB medium, gently wash the EB's into a pool at the bottom of the lid. Carefully inspect the lid for EB's that are stuck to the surface, then transfer the EB's into an uncoated, 6-centimeter petri dish.Return the EB's to the tissue culture incubator for another two days, checking the plates daily for EB aggregation and attachment. Before plating the EB's, replace the excess gelatin coating solution in each well of the prepared six-well plate, with 2 milliliters of freshly prepared, room temperature Grem2 treatment medium. Then use a P1000 pipette set to 100 microliters to transfer 30 EB's to each well, then return the plate to the incubator overnight, evenly dispersing the cells with gentle movement as demonstrated.After the EB's have attached to the bottom of the wells, replace the Grem2 medium every two days until day 10. Then, feed the cells with fresh EB medium every two days to maintain a healthy cell culture. Prior to their hanging drop culture, the pluripotent stem cells should be compact and free of spontaneous differentiation.If spontaneous differentiation is observed, the cells should not be used in the hanging drop cultures. Quality EB is generally formed best within evenly spaced, well rounded hanging drops. By differentiation day two, the EB's should be visible to the naked eye, as spherical arrangements of differentiating stem cells at the tips of the hanging drops.At day four, well-formed EB's should be free floating and homogeneous in size and shape. Once plated, the EB's will flatten as the cells migrate out from the EB onto the surface of the tissue culture plate, as observed here on differentiation days eight and 10. In non-treated control wells, small, slowly contracting patches are observed as early as day six and as late as day nine.While in Grem2-treated wells, large patches with a relatively fast contracting rate are common. RT-QPCR analysis of gene expression in the differentiated cells typically reveals high levels of cardiac structural and regulatory genes in Grem2-treated cultures. If the alpha-MHC DsRed nuke cell line is used, the increased yield of cardiomyocytes is observed as a higher number of DsRed-labeled nuclei in the Grem2-treated wells.Grem2 treatment also biases the differentiation toward an atrial-like phenotype, as confirmed by RT-QPCR and patch clamp measurements of cellular action potential duration. Once mastered, the differentiating stem cells can be treated with Grem2 in as little as 60 minutes and beading cells are usually observed as early as day seven. While attempting this procedure, it's important to remember to consistently add Grem2 at day four after the peak expression of gastrulation markers such as brachyury.Following this procedure, other methods like electrophysiology and QPCR can be performed to understand the functional and molecular characteristics of the cells that have been generated. After watching this video, you should have a good understanding of how to generate atrial cardiomyocytes from mouse ESC's, using Grem2.

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 ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

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 ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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 First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

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 ...