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

Wij danken de leden van de lab Colas voor nuttige discussies en kritieken van het manuscript. Deze studie werd ondersteund door de NIH/NIEHS R44ES023521-02 en CIRM DISC2-10110 verleent aan Dr. Colas.

1. Id1 Virus voorbereiding en infectie
<ol><li>De overexpressing lentivirus Id1 door co transfecting pCMVDR8.74, pMD2.G en pCDH-EF1-Id1-PGK-PuroR genereren (Addgene: plasmide #107735) in HEK293T cellen. Virale deeltjes verzamelen, filtraat zuiveren van het supernatant en opslaan bij-80 ° C zoals Kitamura et al. (2003) 19. als alternatief, produceren Id1 lentivirus commercieel door pCDH-EF1-Id1-PGK-PuroR te sturen naar een lentivirus-producerende leverancier. Let op: Volg lentivirale...

<ol>
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Voor succesvolle differentiaties, zorg ervoor dat instructies boven op de voet volgen. Bovendien, markeren we hier sleutelparameters die differentiatie resultaten sterk te beïnvloeden. Voordat u begint een differentiatie, de volgende drie morfologische parameters moeten worden nageleefd: de morfologie van een stam van hPSCsId1, een hoge cellulaire verdichting en een hoge samenloop (> 90%) van de cultuur op dag 0. In dat opzicht optimale differentiatie voorwaarden zijn beste gemaakt door beplating los gezien h...

Grootschalige productie van menselijke pluripotente stamcellen (hPSCs)-afgeleide cardiale progenitoren en cardiomyocytes is een eerste vereiste voor stamcel gebaseerde therapieën1, modelleren van2,3 en de snelle karakterisering van ziekte nieuwe trajecten regulering van de cardiale differentiatie4,5,6 en fysiologie7,8. Hoewel een aantal studies9,10,11,12,13,14hebben,15 eerder beschreven hoogefficiënte de protocollen van de cardiale differentiatie van hPSCs, geen heeft gericht de hart veld oorsprong van resulterende cardiomyocytes, ondanks de identificatie van moleculaire verschillen tussen links (eerste hart veld) en rechts (tweede hart-veld) ventriculaire cardiomyocytes16 en het bestaan van hart veld-specifieke aangeboren hartziekten; dat wil zeggen, hypoplastic linker hart syndroom17 of arrhythmogenic recht ventriculaire dysplasie18. Dus, de generatie van cardiale voorlopercellen en cardiomyocytes van gedefinieerde hart veld oorsprong van hPSCs wordt steeds een noodzaak teneinde hun relevantie als therapeutische en ziekte modeling tools.
Dit protocol is gebaseerd op de constitutieve overexpressie van Id1, een onlangs geïdentificeerde5 eerste hart veld-opgeven cue thats in combinatie met Activin A, noodzakelijk en voldoende zijn om te starten van cardiogenesis in hPSCs. Met name Cunningham et al. (2017) 5 Toon dat Id1-geïnduceerde progenitoren specifiek express eerste hart-veld (HCN4, TBX5) maar niet het tweede hart veld markeringen (SIX2, ISL1) als ze cardiale differentiatie ondergaan. Bovendien, tonen de auteurs ook dat transgene muis embryo's ontbreekt de hele Id familie van genen (Id1-4), zonder dat zij eerste hart veld cardiale progenitorcellen, terwijl meer mediaal en posterieure cardiale progenitoren (vormen ontwikkelen tweede hart-veld) kan nog steeds vormen, waardoor suggereren dat Id eiwitten essentieel zijn voor het eerste hart veld cardiogenesis geactiveerd in vivo. Gunstig, Id1-geïnduceerde progenitoren kunnen worden cryopreserved en spontaan onderscheiden in cardiomyocytes ventriculaire-achtige kenmerken, met inbegrip van ventriculaire-specifieke markeringen (IRX4, MYL2) expressie weer te geven en ventriculaire-achtige actie potentieel.
Hier beschrijven we een eenvoudige en schaalbare methode voor het genereren van eerste hart veld-achtige (FHF-L) cardiale progenitoren en ventriculaire-achtige cardiomyocytes van Id1 overexpressing hPSCs. Een belangrijk kenmerk van dit protocol is de mogelijkheid om het afkoppelen van cardiale voorlopercellen generatie van latere cardiomyocyte productie met behulp van een handige cryopreservatie stap. Kortom, dit protocol gegevens de nodige maatregelen om (1) genereren Id1-overexpressing hPSCs, (2) genereren FHF-L cardiale progenitoren uit hPSCs (3) cryopreserve van de resulterende progenitoren en (4) hervatten FHF-L cardiale voorlopercellen differentiatie en genereren hoogverrijkt (> 70-90%) ventriculaire-achtige cardiomyocytes gewonnen.

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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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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

De generatie van grote hoeveelheden van functionele menselijke pluripotente stamcellen cardiale voorlopercellen en cardiomyocytes van gedefinieerde hart veld oorsprong is een vereiste voor cel-gebaseerde cardiale therapieën en modellering van de ziekte. We hebben onlangs aangetoond dat Id genen zowel noodzakelijk zijn en voldoende om aan te geven van de eerste veld voorlopercellen van het hart tijdens de ontwikkeling van de gewervelde. Deze differentiatie-protocol maakt gebruik van deze bevindingen en Id1 overexpressie gebruikt in combinatie met Activin A potent opgeven signalen te produceren eerste hart veld-achtige (FHF-L) progenitoren. Bovenal differentiëren resulterende progenitoren efficiënt (~ 70-90%) in ventriculaire-achtige cardiomyocytes. Hier beschrijven we een gedetailleerde methode 1) worden gegenereerd Id1-overexpressing hPSCs en 2) schaalbare hoeveelheden cryopreservable FHF-L progenitoren en ventriculaire-achtige cardiomyocytes onderscheiden.

Generatie van hPSCs Id1 lijnen hPSCs zijn besmet met een lentivirus bemiddelen Id1 overexpressie(Figuur 1). Zodra hPSCId1 worden gegenereerd, wordt transgenic expressie gekwantificeerd door qRT-PCR (Figuur 1B). Alleen hPSCId1 regels uitdrukken Id1 mRNA niveau groter is dan 0,005 vouw die van GAPDH<...

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

Hier beschrijven we een schaalbare methode, met behulp van een eenvoudige combinatie van Activin A en lentivirus-gemedieerde Id1-overexpressie, voor het genereren van eerste hart veld-achtige cardiale progenitoren en ventriculaire-achtige cardiomyocytes van menselijke pluripotente stamcellen.

Generatie van eerste hart veld-achtige cardiale progenitoren en ventriculaire-achtige Cardiomyocytes van menselijke pluripotente stamcellen

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.

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

Afleiding van zeer gezuiverde Cardiomyocyten van Human geïnduceerde pluripotente stamcellen Met behulp van kleine moleculen gemoduleerd Differentiatie en Latere Glucose Verhongering

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

Differentiatie van atriale cardiomyocyten uit pluripotente stamcellen Met behulp van de 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

Grootschalige productie van cardiomyocyten van menselijke pluripotente stamcellen met behulp van een zeer reproduceerbare Small-Molecule Based Differentiatie Protocol

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

Generatie van geïnduceerde pluripotente stamcellen uit menselijke Melanoom-Tumor-infiltrerende lymfocyten

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

Generatie van Integratie-free geïnduceerde pluripotente stamcellen uit menselijke perifere mononucleaire bloedcellen behulp Episomaal Vectors

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

Regie van differentiatie van primitieve en definitieve hematopoietische progenitorcellen van menselijke pluripotente stamcellen

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

Gedefinieerd en Scalable Generatie van hepatocyt-achtige cellen uit menselijke pluripotente stamcellen

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

Differentiatie van chondrocyten van perifere bloed afgeleide humane geïnduceerde pluripotente stamcellen

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

Generatie van gestandaardiseerde en reproduceerbaar reukkolf-type cerebrale Organoids van mens geïnduceerde pluripotente stamcellen

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

Generatie van 3D huid Organoid van koord bloed afkomstige geïnduceerde pluripotente stamcellen

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 ...