Name: generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells
Uploaded: 2018-06-19T14:00:00
Description: Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

Nous remercions les membres du laboratoire Colas pour discussions utiles et critiques du manuscrit. Cette étude a été financée par le NIH/NIEHS R44ES023521-02 et CIRM DISC2-10110 accorde aux Dr Colas.

1. infection et préparation de Virus Id1
<ol><li>Générer des lentivirus sur-exprimant Id1 par transfection co pCMVDR8.74, pMD2.G et pCDH-EF1-Id1-PGK-PuroR (Addgene : plasmide #107735) dans les cellules HEK293T. Recueillir des particules virales, filtrer, purifier du surnageant et stocker à-80 ° C comme Kitamura et al. (2003) 19. vous pouvez également produire Id1 lentivirus commercialement en envoyant pCDH-EF1-Id1-PGK-PuroR à un fournisseur produisant des lentivirus. ATTENTIO...

<ol>
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Pour les différenciations réussies, assurez-vous de suivre attentivement les instructions indiquées ci-dessus. En outre, ici nous mettons en évidence les paramètres clés qui influencent les résultats de la différenciation. Avant de commencer une différenciation, les trois paramètres morphologiques suivantes doivent être respectées : une morphologie de la tige de hPSCsId1, un compactage cellulaire élevé et une confluence élevée (> 90 %) de la culture au jour 0. À cet égard, des conditions opt...

Production à grande échelle des cellules souches pluripotentes humaines (hPSCs)-dérivées de cellules souches cardiaques et les cardiomyocytes est une condition sine qua non pour les thérapies basées sur les cellules souches1, modélisation2,3 et la caractérisation rapide de la maladie nouvelles voies réglementant la différenciation cardiaque4,5,6 et physiologie7,8. Bien qu’un certain nombre d’études9,10,11,12,13,14,,,15 ont décrits précédemment très efficace protocoles de différenciation cardiaques de hPSCs, aucune a abordé l’origine domaine de cœur des cardiomyocytes qui en résulte, en dépit de l’identification des importantes différences moléculaires entre gauche (premier champ du coeur) et droite (second champ de coeur) cardiomyocytes ventriculaires16 et l’existence de maladies cardiaques congénitales par coeur domaine spécifique ; c'est-à-dire, hypoplasie du coeur gauche syndrome17 ou arythmogène dysplasie ventriculaire droite18. Ainsi, la génération des progéniteurs cardiaques et les cardiomyocytes d’origine champ défini de coeur de hPSCs devient une nécessité afin d’augmenter leur pertinence en tant que thérapeutique et outils de modélisation de la maladie.
Ce protocole repose sur la surexpression constitutive ID1, récemment identifié5 premier cœur en spécifiant le champ repère qui, en combinaison avec l’activine A, est nécessaire et suffisante pour initier cardiogenesis dans hPSCs. Notamment, Cunningham et al. (2017) 5 démontrent que les progéniteurs induite par l’Id1 spécifiquement premier champ de cœur (HCN4, TBX5) mais pas second marqueurs de champ de cœur (SIX2, ISL1) car ils subissent une différenciation cardiaque. En outre, les auteurs montrent également que des embryons de souris transgéniques dépourvus de toute la famille de gènes (Id1-4), de Id développe sans former premier cœur champ cardiaque progéniteurs, en plus des progéniteurs cardiaques médial et postérieur ( deuxième champ de cœur) peut encore former, ce qui laisse croire que les protéines Id sont essentiels d’initier une première heart champ cardiogenesis in vivo. Idéalement, les progéniteurs induite par l’Id1 peut être cryoconservés et spontanément se différencient en cardiomyocytes ventriculaires-comme caractéristiques, notamment de l’expression des marqueurs spécifiques ventriculaire (IRX4, MYL2) et potentiels d’action ventriculaire-like.
Nous décrivons ici une méthode simple et évolutive pour générer des premier cœur comme champ (FHF-L) cardiaques progéniteurs et cardiomyocytes ventriculaires ressemblant de Id1 sur-exprimant hPSCs. Une caractéristique importante de ce protocole est la possibilité de découpler la génération de progéniteurs cardiaques du cardiomyocyte ultérieures de production utilisant une étape pratique de cryoconservation. En résumé, ce protocole détaille les étapes nécessaires pour (1) générer Id1-surexprimant hPSCs, (2) générer des cellules souches cardiaques FHF-L de hPSCs, (3) cryoconservé progéniteurs qui en résulte et (4) reprendre la différenciation des progéniteurs cardiaques FHF-L et générer hautement enrichi (> 70 – 90 %) battant cardiomyocytes ventriculaires-like.

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			<td height="20" style="height:20px;width:204px;">ACTC1 antibody</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:119px;">A7811</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>Stem Cell Technologies</td>
			<td>Hu Recom Activin A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic Antimycotic (Anti-Anti)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CDH5 antibody</td>
			<td style="width:144px;">R&#38;D Systems</td>
			<td>AF938</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoStor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>7930</td>
			<td style="width:444px;">Cryopreservation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM high Glucose</td>
			<td>Mediatech</td>
			<td>10-013-CV&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">DPBS w/ Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
			<td></td>
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			<td height="20" style="height:20px;">FBS</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FluoVolt membrane potential kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10488</td>
			<td>For optical action potential acquisition, please refer to McKeithan et al. 2017</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel, Growth Factor Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td style="width:444px;">Coating reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 media kit</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS w/o Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puromycin&#160;</td>
			<td>Acros</td>
			<td>227422500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td style="width:444px;">Enzyme-free dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAGLN antibody</td>
			<td style="width:144px;">Abcam</td>
			<td>ab14106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thiazovivin</td>
			<td>Stem Cell Technologies</td>
			<td>72254</td>
			<td style="width:444px;">RHO/ROCK pathway inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605 -010</td>
			<td style="width:444px;">1X enzyme-containing dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrodes solution mix packets&#160;&#160;</td>
			<td>Sigma</td>
			<td>T2145-10X1L</td>
			<td>(For optical action potential acquisition, please refer to McKeithan et al. 2017)</td>
		</tr>
	</tbody>
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generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

La génération de grandes quantités de pluripotentes humaines fonctionnels dérivés de cellules souches cardiaques progéniteurs et les cardiomyocytes d’origine champ défini de coeur est une condition sine qua non pour la modélisation de la maladie et les thérapies cellulaires cardiaques. Nous avons récemment démontré que les gènes Id sont à la fois nécessaire et suffisante pour spécifier la premières progéniteurs de champ de cœur au cours du développement des vertébrés. Ce protocole de différenciation s’appuie sur ces constatations et utilise Id1 surexpression en combinaison avec l’activine A comme puissants repères indiquant pour produire des progéniteurs de (FHF-L) premiers cœur comme champ. Ce qui est important, résultant des progéniteurs différencient efficacement (~ 70 – 90 %) dans les cardiomyocytes ventriculaires ressemblant. Nous décrivons ici une méthode détaillée pour 1) générer Id1-surexprimant hPSCs et 2) se différencient des quantités évolutives des progéniteurs de FHF-L cryopreservable et des cardiomyocytes ventriculaires ressemblant.

Génération de hPSCs ID1 lignes hPSCs sont infectés par un lentivirus médiation surexpression Id1 (Figure 1A). Une fois que hPSCId1 sont générés, expression du transgène est quantifiée par qRT-PCR (Figure 1B). HPSC uniquement les lignes deId1 exprimant Id1 ARNm à des concentrations supérieure...

Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

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

Nous décrivons ici une méthode évolutive, en utilisant une combinaison simple d’activine A et induite par le lentivirus Id1-surexpression, pour générer des premiers progéniteurs cardiaques de coeur comme champ et cardiomyocytes ventriculaires-comme des cellules souches pluripotentes humaines.

Génération du premier cœur comme champ progéniteurs cardiaques et Cardiomyocytes ventriculaires-comme des cellules souches pluripotentes humaines

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

The main advantage of this simple technique is to generate cardiac progenitors and cardiomyocytes of defined heart field origin and subtypes. In turn, resulting cells can be used for a wide range of research application, including basic developmental biology, proof-of-concept cell-based therapy, and cardiac disease modeling studies. Demonstrating the procedure will be Sean Spiering and Michael Yu from my laboratory.To begin, coat enough wells of a 96-well plate with 50 microliters of coating reagent to accommodate subsequent cell seeding and viral infection steps, then add one milliliter of dissociation reagent to a well in the six-well plate to detach the pluripotent stem cells seeded. Then, use a sterile pipette to transfer the cell suspension in a 15-milliliter plastic centrifuge tube. In the tube, add five milliliters of PBS containing calcium and magnesium to neutralize the dissociation reagent.Then centrifuge the cells at 200 times g for three minutes. Post-centrifugation, discard the supernatant and then gently flick the tube for the pellet to be dislodged. Then dissolve the cell pellet in one milliliter of stem cell medium.Next, use an automated cell counter to count the number of cells. Once the counting is done, seed 20, 000 viable cells in each well of a 96-well plate containing 50 microliters of stem cell medium supplemented with two-micromolar RHO/ROCK pathway inhibitor. After 24 hours, monitor the cell attachment, then replace the medium with 100 microliters of stem cell medium one hour prior to the lentiviral infection.Next, thaw the Id1 lentivirus on ice, then add three microliters of purified lentivirus per well. After adding the lentivirus, transfer the plate back to cell culture incubator at 37 degrees Celsius with 5%carbon dioxide. 24 hours post-viral infection, add 100 microliters of fresh stem cell medium to the infected cells.After 48 hours, replace the virus-containing medium with 200 microliters of fresh stem cell medium supplemented with one microgram per milliliter of puromycin. Every day, keep changing the medium with fresh 200 microliters of stem cell medium supplemented with one microgram per milliliter of puromycin. Start the process of differentiation on Day 0 by replacing the stem cell medium with 1.5 milliliters of induction medium with Activin A in a 12-well plate.24 hours after differentiation has started, replace the medium with two milliliters of induction medium without Activin A.On the third day, replace with two milliliters of induction medium without Activin A.On the fifth day, aspirate the entire medium from the well containing the FHF-L CPs, then immediately add one milliliter of warm enzyme-containing dissociation reagent in the well, then incubate the culture plate at 37 degrees Celsius. After a minute, shake the plate inside the incubator and remove it from the incubator after two minutes, then add one milliliter of 10%fetal bovine serum-containing medium in the well. Detach the cells from the plate by gently pipetting the cells up and down.Next, transfer the cell suspension to a centrifuge tube and subject to centrifugation. After the centrifugation, dissolve the cell pellet in two milliliters of cryopreservation reagent, then count the cell number. Then transfer the cells in the cryopreservation vials and leave the vials in the cooling device.Then transfer the device at minus 80 degrees Celsius overnight. After 24 hours, transfer the vials in liquid nitrogen for long-term storage. Just before thawing the FHF-L CPs, coat the wells of the 12-well plate with two milliliters of coating reagent.Next transfer the five-days old FHF-L CPs from the liquid nitrogen in a dry ice container. From the dry ice, transfer the vial in a water bath at 37 degrees Celsius for two minutes to ensure complete thawing. After the cells are completely thawed, transfer the cells to a centrifuge tube, then pipette 10 drops of pre-warmed cardiogenic medium every 30 seconds to the cell solution.Keep adding the cardiogenic medium til the cryopreservation and cardiogenic medium ratio reaches one to three. Next subject the cells to centrifugation. Post-centrifugation remove the supernatant and carefully flick the tube, then dissolve the cell pellet in cardiogenic medium supplemented with two-micromolar RHO/ROCk pathway inhibitor.Further dilute the cell suspension with cardiogenic medium to achieve the desired cell concentration. Then seed the cells on the culture plate containing two milliliters of cardiogenic medium. Leave the plate in the tissue culture hood for 20 minutes before transferring to the incubator.Monitor the cell attachment on the sixth day of differentiation. On the seventh day, aspirate half of the cardiogenic medium and replace it with fresh medium. From here on out, replace 50%of the cardiogenic medium every other day, then, on the 15th day, quantify the cardiac differentiation.Here the expression level of Id1 mRNA is quantified in the hESC and hPSC cell lines. Only hPSC lines expressing Id1 mRNA at levels greater than 0.005-fold GAPDH are used for differentiation. Shown here are brightfield images post-differentiation of optimal, sub-confluent, and over-confluent hPSCs at Day 0.Upon further differentiation, the cell confluency reaches 75%after 24 hours. At Day 3, the differentiating cells form a cluster, and, by Day 5, the cells remain in tightly-connected clusters with homogenous morphology. At Day 6, the FHF-L CPs cover more than 90%of the surface area of the wells.In fact, by Day 12, the cardiomyocytes start to beat and display cardiac contractions. Further into the differentiation process, there are increased mRNA expression levels of MYL2 and IRX4 after Day 15. Both proteins represent ventricular-specific markers within the cardiomyocytes.Here the action potential trace represents the rapid upstroke and downstroke velocities as well as the ventricular plateau. These rates were characteristic of ventricular-like cardiomyocytes at Day 25 of differentiation. In summary, it takes about two weeks to generate Id1 over-expressing human pluripotent stem cells, then it takes about five days to generate first heart field-like progenitors and about 10 days to generate beating ventricular-like cardiomyocytes.

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JoVE Journal

Developmental Biology

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

Dérivation des cardiomyocytes hautement purifiée de anthropiques cellules souches pluripotentes en utilisant de petits différenciation de Molecule-modulé et ultérieur glucose famine

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

Recherche

Biologie du développement

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

Différenciation des atrial cardiomyocytes à partir de cellules souches pluripotentes Utilisation de la BMP Antagonist Grem2

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

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

Production à grande échelle de cardiomyocytes à partir pluripotentes humaines cellules souches en utilisant un petit protocole Différenciation Molecule-Based hautement reproductible

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Génération de Induced cellules souches pluripotentes à partir de mélanome humain Lymphocytes tumorales infiltrant

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Génération d&#39;Intégration-libres induits des cellules souches pluripotentes à partir de cellules mononucléaires sang périphérique humain utilisant épisomique Vecteurs

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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

Réalisé la différenciation des progéniteurs hématopoïétiques primitives et définitif des cellules souches pluripotentes humaines

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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Définition et génération évolutive des cellules hépatocytes-like de pluripotentes humaines Cellules souches

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Différenciation des chondrocytes provenant des cellules souches pluripotentes induites par le sang périphérique dérivé du sang

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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

Génération de type prosencéphale standardisées et reproductibles organoïdes cérébrale de l’homme induites par les cellules souches pluripotentes

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

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Génération de peau 3D Organoïde de dérivés du sang de cordon induite par les cellules souches pluripotentes

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...