Name: pluripotent stem cell derived cardiac cells for myocardial repair
Uploaded: 2017-02-03T15:00:00
Description: Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

This work was supported by US Public Health Service grants NIH RO1s HL67828, HL95077, HL114120, and UO1 HL100407-project 4 (to JZ), an American Heart Association Scientist Development Grant (16SDG30410018) and a Research Voucher Award from University of Alabama at Birmingham Center for Clinical and Translational Science (to WZ).

Alle eksperimentelle prosedyrer er utført i samsvar med Animal retningslinjer ved University of Alabama i Birmingham. 1. Skille hiPSCs inn hiPSC-CMS <ol><li> Belegge brønnene i en 6-brønns plate med på forhånd avkjølt vekstfaktor-redusert gelatinøse proteinblanding ved 4 ° C til over natten. Aspirer geléaktige proteinblandingen før bruk. Seed de hiPSCs på de på forhånd belagte plater, og kulturen cellene (1 x 10 5 celler per brønn) i 5% CO2 og 37 ° C i ...

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Forbedret Yield / Purity av hiPSC-CMS Konvensjonelle protokoller for differensiering menneskelige stamceller til CMS er ofte begrenset av lav rente og renhet; for eksempel, fikk bare 35-66% av hESC-CMS via Percoll separasjon og hjerte kroppen dannelsen uttrykt treg myosin tung kjede eller cTnT 6. Renheten av differensierte hiPSC-CM populasjoner kan økes vesentlig ved å velge for ekspresjon av et reportergen som har vært knyttet til promoteren til en CM-spesifikt prote...

Menneskeskapte pluripotente stamceller (hiPSCs) er blant de mest lovende agenter for regenerativ celleterapi fordi de kan bli differensiert i et potensielt ubegrenset utvalg og mengde av celler som ikke er avvist av pasientens immunsystem. Imidlertid kan deres evne til selv-replikasjon og differensiering også føre til tumordannelse og følgelig hiPSCs må være fullt differensiert i spesifikke celletyper, slik som kardiomyocytter (CMS), endotelceller (ECS) og glatte muskelceller (SMC ), før administrasjon. En av de enkleste og mest vanlige fremgangsmåter for administrering celle er direkte intramyocardial injeksjon, men antallet av transplanterte celler som er innpodet med den native myokardialt vev er usedvanlig lav. Mye av denne slitasje kan tilskrives den cytotoksiske miljøet i ischemisk vev; imidlertid, når muse-embryonale stamceller (ESCS) ble injisert direkte inn i hjertemuskelen av uskadde hjerter, oare ~ 40% av de 5 millioner celler som leveres ble opprettholdt i 3-5 timer 1, noe som antyder at en betydelig del av den administrerte cellene kommet ut av administrasjonsstedet, kanskje fordi de ble presset ut gjennom nålen sporet ved de høye trykk som produseres i løpet av hjerteinfarkt sammentrekning. Her presenterer vi nye og vesentlig mer effektive metoder for å generere hiPSC-avledet cardiomyocytes (hiPSC-CMS) 2, endotelceller (hiPSC-ECS) 3, og glatte muskelceller (SMC) 4. Spesielt er dette hiPSC-SMC protokollen den første til å etterligne det brede spekter av morfologiske og funksjonelle egenskaper observert i somatisk SMC 5 ved å rette cellene mot en overveiende syntetisk eller kontraktile SMC fenotype. Vi gir også en fremgangsmåte for celle-levering som forbedrer innpoding frekvensen av injiserte celler ved å skape en cytokin-inneholdende fibrin patch over injeksjonsstedet. Plasteret ser ut til å forbedre både celleansamlinger, ved å tette kanylen spor for å hindre at celler fra å gå ut av hjertemuskelen, og celleoverlevelse, ved å frigjøre insulin-lignende vekstfaktor (IGF) over en periode på minst tre dager.

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Protocol 1</td>
			<td style="width:180px;"></td>
			<td style="width:155px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td>Stem cell technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth-factor-reduced matrigel</td>
			<td>Corning lifescience</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Stem cell technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement, serum free</td>
			<td>Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI1640</td>
			<td>Fisher Scientific</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BMP-4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">bFGF</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase IV</td>
			<td>Fisher Scientific</td>
			<td>NC0217889</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:163px;">Hanks Balanced Salt Solution (Dextrose, KCl, KH2PO4, NaHCO3, NaCl, Na2HPO4 anhydrous)</td>
			<td>Fisher Scientific</td>
			<td>14175079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10438018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well plate</td>
			<td>Corning Lifescience</td>
			<td>356721</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm dish</td>
			<td>Corning Lifescience</td>
			<td>354732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene</td>
			<td>Fisher Scientific</td>
			<td>15040066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>F8630-5g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T7009-1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMB2 medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VEGF</td>
			<td>ProSpec-Tany</td>
			<td>CYT-241</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EPO</td>
			<td>Life Technologies</td>
			<td>PHC9431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TGF-&#223;</td>
			<td>Peprotech</td>
			<td>100-21C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGM2-MV medium</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB-431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31</td>
			<td>BD Bioscience</td>
			<td>BDB555445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD144</td>
			<td>BD Bioscience</td>
			<td>560411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>12565269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorff Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR99021</td>
			<td>Stem cell technologies</td>
			<td>720542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDGF-&#223;</td>
			<td>Prospec</td>
			<td>CYT-501-10ug</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olive oil</td>
			<td>Sigma-Aldrich</td>
			<td>O1514</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGF</td>
			<td>R&#38;D</td>
			<td>291-G1-01M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>15561020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heating plate</td>
			<td>Fisher Scientific</td>
			<td>SP88850200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"><img alt="ezh" src="/files/ftp_upload/55142/ezh.jpg" />-aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0420000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM medium</td>
			<td>Fisher Scientific</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td>Fisher Scientific</td>
			<td>1482748</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Datex-Ohmeda</td>
			<td>47810</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia ventilator</td>
			<td>Ohio Medical</td>
			<td>V5A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defibrillator</td>
			<td>Physiol Control</td>
			<td>LIFEPAK 15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5T MRI</td>
			<td>General Electric</td>
			<td>Signa Horizon LX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7T MRI</td>
			<td>Siemens</td>
			<td>10018532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gadolinium Contrast Medium (Magnevist)</td>
			<td>Berlex</td>
			<td>50419-188-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-0 silk suture</td>
			<td>Ethilon</td>
			<td>685H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 silk suture</td>
			<td>Ethilon</td>
			<td>622H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-0 monofilament suture</td>
			<td>Ethilon</td>
			<td>627H</td>
			<td></td>
		</tr>
	</tbody>
</table>

pluripotent stem cell derived cardiac cells for myocardial repair

Menneskeskapte pluripotent stamceller (hiPSCs) må være fullt differensiert i spesifikke celletyper før administrering, men konvensjonelle protokoller for å differensiere hiPSCs inn cardiomyocytes (hiPSC-CMS), endotelceller (hiPSC-ECS), og glatte muskelceller (SMC) er ofte begrenset av lavt utbytte, renhet og / eller dårlig fenotypisk stabilitet. Her presenterer vi nye protokoller for generering hiPSC-CMs, -ECs og -SMCs som er vesentlig mer effektiv enn konvensjonelle metoder, så vel som en fremgangsmåte for å kombinere celleinjeksjon med et cytokin holdig lapp skapt over administreringsstedet. Plasteret forbedrer både retensjonen av de injiserte cellene, ved å tette kanylen spor for å hindre at cellene fra å bli presset ut av myokard, og celleoverlevelse, ved å frigjøre insulin-lignende vekstfaktor (IGF) over en lengre periode. I et svin modell av myokardiskemi-reperfusjonsskade, frekvensen av engraftment var mer enn to ganger større nårceller ble administrert med cytokin-holdige patch sammenligne til cellene uten lappen, og behandling med både celler og lappen, men ikke med cellene alene, var forbundet med betydelige forbedringer i hjertefunksjon og infarktstørrelsen.

Karakterisering av Differensiert hiPSC-CMS, -ECs, og -SMCs Differensial kapasitet på hiPSCs ble evaluert to, tre, fire. Flowcytometri analyser av hjerte troponin T (cTnT) ekspresjon tyder på at renheten av slutt hiPSC-CM befolkning kan overstige 90% (figur 1A, 1B, panel B1). Nesten alle cellene uttrykt...

Watch this Scientific Journal Video about Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair at JoVE.com

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Vi presenterer tre nye og mer effektive protokoller for å differensiere menneskeskapte pluripotent stamceller til kardiomyocytter, endotelceller og glatte muskelceller og en leveringsmåte som bedrer innpoding av transplanterte celler ved å kombinere celleinjeksjon med patch-mediert cytokin levering.

Pluripotente stamcelle Avledet hjerteceller for Hjerteinfarkt Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Research

JoVE Journal

Developmental Biology

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Generasjon av murine pacemaker Cell Aggregater Basert på ES-Cell-programmering i kombinasjon med Myh6-arrangøren-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generasjon Integrerings- fritt menneskeskapte Pluripotent stamceller Bruke Hair-avledet Keratinocytter

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Feeder-fri Utledning av Melanocytter fra Menneskelig Pluripotent stamceller

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

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 ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Aggregate Størrelse Optimalisering i mikro for Suspension basert Cardiac Differensiering av menneskelige Pluripotent stamceller

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

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 ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Kondrogen pelletsdannelse fra Cord Blood-avledede Induced Pluripotent Stamceller

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt sebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

Bruker Human indusert Pluripotent Stamcelle-avledet Hepatocyte-lignende celler for medisiner

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...