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

Vi takker medlemmer av laboratoriet Colas nyttig diskusjoner og kritiske vurderinger av manuskriptet. Denne studien ble støttet av NIH/NIEHS R44ES023521-02 og CIRM DISC2-10110 gir Dr. Colas.

1. Id1 Virus forberedelse og infeksjon
<ol><li>Generere Id1 overexpressing lentivirus med transfecting pCMVDR8.74, pMD2.G og pCDH-EF1-Id1-PGK-PuroR (Addgene: plasmider #107735) i HEK293T celler. Samle virus partikler, Filtrer, rense fra nedbryting og lagre-80 ° c som Kitamura et al. (2003) 19. også produsere Id1 lentivirus kommersielt ved å sende pCDH-EF1-Id1-PGK-PuroR til lentivirus-produserende leverandør. Forsiktig: Følg lentiviral sikkerhetsforskrifter og standardoperasjon p...

<ol>
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For vellykket atskillinger, må du nøye følge instruksjonene ovenfor. I tillegg markere her vi nøkkelparameterne som sterkt påvirker differensiering resultater. Før du starter en differensiering, følgende tre morfologiske parametere bør overholdes: en stilk morfologi av hPSCsId1, en høy mobilnettet komprimering og en høy samløpet (> 90%) av kultur på dag 0. I den forbindelse, optimal differensiering forhold er best laget av plating dissosiert hPSCsId1 som enkeltceller og tillate dem å da...

Storskala produksjon av menneskelig pluripotent stamceller (hPSCs)-avledede cardiac progenitors og cardiomyocytes er en forutsetning for stilk cellen-basert behandling1, sykdom modellering2,3 og rask karakterisering av romanen trasé regulerer hjerte differensiering4,5,6 og fysiologi7,8. Selv om en rekke studier9,10,11,12,13,14har,15 tidligere beskrevet høyeffektive CARDIAC differensiering protokoller fra hPSCs, ingen har adressert hjertet feltet opprinnelsen til resulterende cardiomyocytes, til tross for identifikasjon av betydelige molekylær forskjeller mellom venstre (første hjertet feltet) og høyre (andre hjertet felt) ventrikkel cardiomyocytes16 og eksistensen av hjertet Feltspesifikke medfødte hjertesykdommer; dvs hypoplastic venstre hjertet syndrom17 eller arrhythmogenic høyre ventrikkel dysplasia18. Dermed generering av cardiac progenitors og cardiomyocytes definert hjertet feltet opprinnelse fra hPSCs er blitt en nødvendighet for å øke relevansen som terapeutiske og sykdom modellverktøy.
Denne protokollen er avhengig av den grunnleggende overuttrykte Id1, en nylig identifisert5 første hjertet feltet-angir bunke som i kombinasjon med Activin A, er både nødvendig og tilstrekkelig å starte cardiogenesis i hPSCs. Cunningham et al. spesielt (2017) 5 viser at Id1-indusert progenitors spesielt uttrykke første hjertet-feltet (HCN4, TBX5), men ikke andre hjertet feltet indikatorer (SIX2, ISL1) som de gjennomgå cardiac differensiering. I tillegg viser forfatterne at transgene mouse embryoer mangler hele Id familien av gener (Id1-4), utvikle uten forming første hjertet feltet cardiac progenitors, mens mer mediale og posterior cardiac progenitors ( andre hjertet feltet) kan fortsatt danne, og dermed antyder at Id proteiner er avgjørende å starte første hjertet feltet cardiogenesis i vivo. Praktisk, Id1-indusert progenitors kan være cryopreserved og differensiere spontant i cardiomyocytes vise ventrikkel-lignende egenskaper, inkludert ventrikkel-spesifikke indikatorer (IRX4, MYL2) uttrykk og ventrikkel-lignende handling potensialer.
Her beskriver vi en enkel og skalerbar metode for å generere første hjertet felt-lignende (FHF-L) cardiac progenitors og ventrikkel som cardiomyocytes fra Id1 overexpressing hPSCs. Et viktig trekk ved denne protokollen er muligheten til å uncouple hjerte stamfar generasjon fra påfølgende cardiomyocyte produksjonen ved hjelp av en praktisk kryonisk bevaring trinn. I sammendraget, denne protokollen detaljer de nødvendige skritt for å (1) generere Id1-overexpressing hPSCs, (2) generere FHF-L cardiac progenitors fra hPSCs, (3) cryopreserve resulterende progenitors og (4) fortsette FHF-L cardiac stamfar differensiering og generere høyanriket (> 70-90%) slo ventrikkel-lignende cardiomyocytes.

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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>
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			<td height="20" style="height:20px;">Activin A</td>
			<td>Stem Cell Technologies</td>
			<td>Hu Recom Activin A</td>
			<td></td>
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			<td height="20" style="height:20px;">Antibiotic Antimycotic (Anti-Anti)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
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			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
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		<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>
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			<td height="20" style="height:20px;">B27 supplement w/o - vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587001</td>
			<td></td>
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			<td height="20" style="height:20px;">CDH5 antibody</td>
			<td style="width:144px;">R&#38;D Systems</td>
			<td>AF938</td>
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			<td height="20" style="height:20px;">CryoStor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>7930</td>
			<td style="width:444px;">Cryopreservation reagent</td>
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			<td height="20" style="height:20px;">DMEM high Glucose</td>
			<td>Mediatech</td>
			<td>10-013-CV&#160;</td>
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			<td height="20" style="height:20px;">DPBS w/ Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
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			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
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			<td height="20" style="height:20px;">FBS</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
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			<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>
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			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
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		<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>
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			<td height="20" style="height:20px;">mTeSR1 media kit</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
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			<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Puromycin&#160;</td>
			<td>Acros</td>
			<td>227422500</td>
			<td></td>
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		<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>
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			<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>
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generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

Generering av store mengder funksjonell menneskelige pluripotent stamceller-avledet cardiac progenitors og cardiomyocytes definert hjertet feltet Opprinnelsesland er en forutsetning for cellebasert cardiac terapier og sykdom modellering. Vi har nylig vist at Id gener er både nødvendig og tilstrekkelig til å angi første hjertet feltet progenitors under virveldyrenes utvikling. Denne differensieringen protokollen utnytter disse funnene og bruker Id1 overuttrykte i kombinasjon med Activin A som potent angir signaler for å produsere første hjertet felt-lignende (FHF-L) progenitors. Viktigere, skille resulterende progenitors effektivt (~ 70-90%) i ventrikkel-lignende cardiomyocytes. Her beskriver vi en detaljert metode 1) generere Id1-overexpressing hPSCs og 2) skille skalerbar mengder cryopreservable FHF-L progenitors og ventrikkel-lignende cardiomyocytes.

Generering av hPSCs Id1 linjer hPSCs er infisert med en lentivirus formidle Id1 overuttrykte (figur 1A). Når hPSCId1 genereres, er transgene uttrykk kvantifisert ved qRT PCR (figur 1B). Bare hPSCId1 linjer uttrykke Id1 mRNA på nivåer høyere enn 0.005 fold av som GAPDH skal brukes for diffe...

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

Her beskriver vi en skalerbar metode, bruke en enkel kombinasjon av Activin A og lentivirus-mediert Id1-overexpression, generere første hjertet felt-lignende cardiac progenitors og ventrikkel som cardiomyocytes fra menneskelige pluripotent stamceller.

Generasjon første hjertet felt-lignende Cardiac Progenitors og ventrikkel som Cardiomyocytes fra menneskelige Pluripotent stamceller

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

Avledning av Highly Purified kardiomyocytter fra menneskeskapte Pluripotent stamceller ved hjelp av små Molecule-modulert Differensiering og Følgende Glucose Sult

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

Differensiering av atrieflimmer cardiomyocytes fra Pluripotent stamceller ved hjelp av 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

Storskala produksjon av cardiomyocytes fra Menneskelig Pluripotent stamceller ved hjelp av en meget reproduserbar lite molekyl-Based Differensiering 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

Generering av Induced Pluripotent stamceller fra humane melanom Tumor-infiltrerende lymfocytter

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

Generasjon Integrerings--frie Utførte Pluripotent stamceller fra menneskelige perifere mononukleære blodceller Bruke episomale vektorer

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

Rettet differensiering av Primitive og Definitive blodkreft Progenitors fra menneskelige Pluripotent stamceller

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

Definert og Scalable Generation of hepatocytter lignende celler fra menneskelige Pluripotent stamceller

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

Differensiering av kondrocytter fra perifert blod-avledede humane inducerte pluripotente stamceller

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

Generasjon av standardiserte og reproduserbar Forebrain-type Cerebral Organoids fra menneske indusert Pluripotent stamceller

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

Generasjon 3D huden Organoid fra ledningen blod-avledet indusert Pluripotent stamceller

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 ...