We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

1. Ableitung von embryonalen Maus-CPCs aus Maus WSR <ol><li> Bereiten embryonalen Maus-Fibroblasten (MEFs) Feeder-Schicht. <ol><li> Warm MEF Medium (10% FBS in DMEM) auf 37 ° C. </li><li> Bereiten Gelatine beschichteten Platten. <ol><li> 1 ml der 0,1% Gelatine in Wasser in eine Vertiefung Platte mit 6 Vertiefungen oder 5 ml in eine 10 cm-Schale. </li><li> Lassen Platten oder Gerichte bei 37 ° C oder bei Raumtemperatur für mindestens 30 min. Streben die Gelatine vor der Verwendung. </li></ol></li><li> Thaw bestrahlt MEFs schnell in einem 37 ° C Wasserbad, unter leichtem Schütteln. </li><li> Übertragen die Zellen vorsichtig in ein 15 ml oder 50 ml konischen Röhrchen. 5 ml vorgewärmten MEF Medium. Schwenken Sie die Zellen langsam und Zentrifuge bei 200 xg für 5 min. </li><li> Zellen in MEF Medium und zählen lebensfähigen Zellen. Verwenden Sie die folgenden Mengen von MEF Medium: 2 ml für Zellen pro Vertiefung einer 6-Well-Platte und 10 ml für Zellen aus einer 10 cm Schüssel. </li><li> Platte Zellen in 6-Well-Platten oder 10 cm-Schalen bei 1-2x 10 6 pro Platte / Schale. </li><li> Die MEF Platten / Geschirr bei 37 ° C, 5% CO 2 mindestens 24 Stunden vor der Verwendung. HINWEIS: Entsorgen Sie MEF Platten / Geschirr älter als eine Woche. </li></ol></li><li> Bereiten Maus WSR <ol><li> Kultur der Isl1-cre; Rosa26YFP / Mef2c-cre; Rosa26YFP Maus WSR auf MEFs bis 90% konfluent. Verwendung ES-Zellmedium bestehend aus 410 ml DMEM, 75 ml FBS, 0,1 mM MEM NEAA, 2 mM L-Glutamin, 0,1 mM Pyruvat, 100 U Pen / Strep und 0,1 mM β-Mercaptoethanol. </li><li> Bereiten Gelatine beschichtete 6-Well-Platten, wie in 1.1.2 beschrieben. </li><li> Absaugen Medium von Platten und spülen Zellen mit DPBS. <ol><li> Saugen Sie DPBS und fügen Sie 0,4 ml 0,25% Trypsin in einem gut 6-Well-Platte. Zellen bei 37 ° C, 5% CO 2 für 5 min. 1 ml vorgewärmten ES-Medium zu Trypsin Reaktion zu beenden. </li></ol></li><li> Ernten Sie die Zellen und Zentrifuge Zellen bei 200 xg und saugen Medium. Zellen in 1 ml ES-Zellmedium und zähle die Zellen. &lt;/ Li&gt; <li> Platte WSR auf Gelatine-beschichteten Platten bei einer Dichte von 3 x 10 4 / cm 2. Bei 37 ° C, 5% CO 2 für ca. 2 Tage bei WSR erreichen ca. 90% konfluent. Ändern Sie das Medium jeden Tag. </li><li> Wenn WSR sind 90% konfluent, wiederholen Sie die Schritte 1.2.2-1.2.5 die MEF Feeder vollständig zu entfernen. </li></ol></li><li> Induzieren CPC Differenzierung von EB Bildung <ol><li> Bereiten Differenzierungsmedium wie folgt: 36 ml IMDM, 12 ml Ham-F12, 2 mM L-Glutamin, 0,34 ml BSA (7,5%), 0,25 ml N 2, 0,5 ml B27, 50 ug / ml AA und 0,45 mM 1-Thioglycerin. </li><li> Absaugen Medium von Zellen, und spülen Sie mit DPBS. Saugen Sie DPBS und fügen Sie 0,4 ml 0,25% Trypsin in einem gut 6-Well-Platte. Bei 37 ° C für 5 min. </li><li> 1 ml Differenzierungsmedium, um die Reaktion und die Pipette bis zu beenden und auf Zellclustern in einzelne Zellen zu verteilen. </li><li> Centrifuge WSR bei 200 xg für 5 min und entsorgen Sie das Medium. <ol><li> Resuspendieren 1 x 10 6 &lt;/ Sup&gt; WSR in 1 ml Differenzierungsmedium. Zählen Sie die Zellen, und die Kultur der Zellen in geringer Distanz Petrischale mit einer Konzentration von 1 x 10 5 Zellen / ml in Differenzierungsmedium bei 37 ° C für die ersten EB-Aggregation. </li></ol></li><li> Zwei Tage nach der EB Bildung, übertragen EVG vom Geschirr bis zu 50 ml Tuben. Spin bei 200 · g für 1 min. Entfernen Sie Medium und spülen EVG mit 10 ml DPBS ohne Ca 2+ und Mg 2+. </li><li> Saugen Sie DPBS und 1 ml 0,25% Trypsin. Bei 37 ° C für 5 min. Hinzufügen 4,5 ml Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. </li><li> Zentrifugiere die Zellen bei 200 g für 5 min. Absaugen Medium resuspendieren Zellen in 1 ml Differenzierungsmedium. </li><li> Zählen Sie die Zellen und verdünnte 2 x 10 6 Zellen in 1 x 10 5 Zellen / ml in Differenzierungsmedium. Hinzufügen VEGF, Bmp4 und Activin A zu dem Medium in einer Endkonzentration von 5 ng / ml, 0,8 ng / ml und 5 ng / ml. Culture Zellen in Petrischale für die zweite EB Bildung bei 37 ° C. </li><li> 40 Stunden nach der zweiten EB Bildung, übertragen EVG auf 50-ml-Röhrchen. Spülen mit DPBS einmal dissoziieren EBs mit 1 ml 0,25% Trypsin bei 37 ° C für 5 min. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. </li><li> Die Zellen in Stempro-34 Medium mit 5 ng / ml VEGF, 10 ng / ml bFGF und 12,5 ng / ml FGF10. Platte der Zellen bei 2 × 10 5 / cm 2 in Gelatine-beschichteten Platten. Kultur in 37 ° C Inkubator für etwa 32 Stunden vor dem CPC Isolation. </li></ol></li><li> Isolieren mCPCs <ol><li> Absaugen Medium und spülen Sie mit DPBS. 0,5 ml 0,25% Trypsin zu Kulturplatten bei 37 ° C für 5 min. Hinzufügen 4,5 ml Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. Sammeln Sie die Zellen durch Zentrifugieren und resuspendieren Zellen in 2% FBS / PBS für FACS-Reinigung. </li><li> Führen undifferenzierten WSR am Sortierer als negative Kontrolle. Ändern Spannung nach vorne Scat einstellenter (FSC) und Seitwärtsstreuung (SSC), um die gewünschte Population auszuwählen. Verwenden Vorwärtsstreuung gegenüber Seitenstreuung und Vorwärtsstreuhöhe gegen die Breite, um Schmutz und Wams aus. In den ausgewählten Subpopulation, nach der Verteilung der ESC steuert, ziehe eine positive Gate-YFP auf Histogramm, um die Zellautofluoreszenz zu beseitigen. Sammeln YFP + CPCs aus YFP + Gating. </li><li> Platte der gereinigten CPCs (YFP + Zellen) in 6-Well-Platten mit Differenzierungsmedium (1.3.1). Kultur weitere 3-5 Tage bei 37 ° C für die Differenzierung von CPC in Kardiomyozyten und SMCs. </li></ol></li></ol> 2. Gewinnung menschlicher embryonaler CPCs von Menschen WSR <ol><li> Bereiten Feeders <ol><li> Bereiten embryonalen Maus-Fibroblasten (MEF) Nährschicht wie oben bei einer Dichte von 2 x 10 4 / cm 2 beschrieben ist. </li></ol></li><li> Pflegen menschlichen WSR <ol><li> Pflegen menschlichen ISL1: cre; pCAG-flox-AUS-flox-GFP oder RFP WSR routinemäßig auf MEF mit regulären hES Medium (Knockout DMEM / F-12 385 ml, Knockout SR 100 ml, 2 mM L-Glutamin, 0,1 mM NEAA, 0,1 mM β-Mercaptoethanol, 10 ng / ml basischen FGF). Vor der Herzdifferenzierung, übertragen menschliche WSR in Feeder-freien Zustand für mindestens 2 Passagen. </li></ol></li><li> Bereiten menschlichen WSR in Einzug freien Zustand <ol><li> Bereiten beschichteten Platten für den menschlichen WSR Kultur. <ol><li> Auftauen Matrigel bei 4 ° C über Nacht. Setzen Sie einen 50-ml-Röhrchen auf Eis und 30 ml kaltem DMEM / F12-Medium in den Schlauch. </li><li> 1 ml Matrigel in das kalte Medium und gut mischen. 1 ml kaltem Matrigel-DMEM / 12-Medium in eine Vertiefung Platte mit 6 Vertiefungen oder 5 ml in eine 10 cm-Schale. </li><li> Lassen Platten / Geschirr bei 4 ° C über Nacht oder bei Raumtemperatur für 2 Std. Absaugen Medium vor der Verwendung. </li></ol></li><li> Split menschlichen WSR auf die Teller: absaugen Medien aus MEF Platte und spülen menschlichen WSR mit DPBS. Dann 1 ml Dispase (1 mg / ml) in eine Vertiefung einer Platte mit 6 Vertiefungen. Inkubieren Zellen in 37 ° CInkubator, bis Ränder Kolonien beginnen zu lösen. </li><li> Entfernen Dispase-Lösung. Spülen Sie mit DPBS zweimal, dann fügen Sie 2,5 ml / pro Vertiefung mTeSR Medium oder MEF-konditionierten Mediums auf die Platte. </li><li> Kratzen Sie Zellen mit einem Zellschaber und sorgfältig pipettieren nach oben und unten, um die menschliche ESC Kolonien in kleine Klumpen zu brechen. </li><li> Übertragen Sie die ESC-Klumpen und verdünnt 1: 3 in die beschichteten Platten. </li><li> 2 ml / Vertiefung mTeSR Medium oder MEF-konditionierten Mediums und ändern Medium täglich. </li><li> Kultur menschlichen WSR in mTeSR Medium oder MEF-konditionierten Mediums auf beschichteten Platten für mindestens 2 Passagen. </li></ol></li><li> Induzieren CPC Differenzierung von humanen WSR <ol><li> Wenn Zellen sind ~ 90% konfluent, saugen das Medium und spülen Sie mit DPBS. Inkubieren der HES in 0,5 mg / ml Dispase bei 37 ° C für 20 min. </li><li> Spülen Sie die hES mit DPBS zweimal Entfernen Zellen aus Platten mit einer Zellheber, dann resuspendieren Zellschellen in Differenzierungsmedium (DMEM / F12 mit 18% FBS, 0,1 mM NEAA, 2 mML-Glutamin, 0,1 mM β-Mercaptoethanol und 50 ug / ml Ascorbinsäure). </li><li> Übertragen Sie Zellen in 6-Well-Ultra-Low-Befestigungsplatten für EB-Bildung. </li><li> Ändern Sie am nächsten Tag Differenzierungsmedium. </li><li> Ersetzen Medium alle 3 Tage und pflegen EVG in Suspensionskultur. </li></ol></li><li> Isolieren HCPCS <ol><li> Sammeln EVG spätestens 9. Tag der Differenzierung der Menschen CPC Isolation. </li><li> Absaugen Medium und spülen EVG mit DPBS zweimal. 0,5 ml 0,25% Trypsin in jede Vertiefung einer 6-Well-Kulturplatten bei 37 ° C für 5 min. </li><li> Hinzufügen gleichen Volumen Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. Sammle die Zellen durch Zentrifugieren bei 200 xg für 5 Minuten und resuspendiere Zellen in 2% FBS / DPBS. Filterzellen mit 40 um Zellsieb und FACS-gereinigten RFP + CPC Zellen, wie in 1.4.3 beschrieben. </li><li> Platte nach HCPCS auf geltin beschichteten Platten. Kultur HCPCS in Differenzierungsmedium für 7-9 Tage in Kardiomyozyten und glatten Muskelzellen zu differenzieren. 7 Tage nach der Plattierung, die Kultur differenzierter Zellen mit 5% Knockout SR in DMEM / F12-Medium. </li></ol></li></ol>

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</ol>

The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Herzkrankheit bleibt die führende Ursache in der heutigen Welt und die Todesraten haben in den vergangenen zwei Jahrzehnten (American Heart Association) nahezu unverändert geblieben. Es gibt einen dringenden Bedarf für die Entwicklung neuer therapeutischer Strategien effektiv verhindern oder rückgängig Herzinsuffizienz. Ein vielversprechender Ansatz ist zellbasierte Therapie nach der raschen Entwicklung von Stammzellen biology 1. In dieser Hinsicht könnte multi CPCs eine ausgezeichnete Zellquelle für die Therapie aufgrund ihrer Fähigkeit, sich zu vermehren, aber nur, um die Herzlinie Differenzierung gebunden werden. Daher effiziente und robuste Methode zur Erzeugung und Isolierung CPCs ist von großer Bedeutung für die Herzzelltherapie-Studien. Dieses Protokoll konzentriert sich auf embryonale CPCs der frühen Embryogenese und wie ihre Generation von WSR identifiziert. Verschiedene CPCs wurden aus embryonalen und adulten Herzen isoliert worden, auch aus Knochenmark 2. Während der Embryonalentwicklung, Knochen morphogetische Proteine ​​(BMPs), flügellosen Typ MMTV Integrationsstelle Familienmitglieder (Wnts) und Nodal-Signale induzieren das Engagement der Mesp1 + multi Mesoderm 3. Mesp1 + Zellen in die embryonale CPCs 4 unterscheiden dann. Diese CPCs werden typischerweise durch HCN4 markiert, NK2 homeobox 5 (NKX2-5) Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5) und Myozyten-Enhancer Faktor 2C (Mef2c), bilden primären und zweiten Herzfelder, und während Kardiogenese 5-10 tragen zu der Hauptteile des Herzens. Sowohl NKX2-5 + und Isl1 + / + Mef2c CPCs Lage sind, in Kardiomyozyten differenzieren, glatte Muskelzellen (SMCs) und Endothelzellen 5-8. So sind diese CPCs werden die zu Herzgefäßsystem sowie Herzgewebe zu geben und sind eine ideale Zellquelle für zellbasierte Herztherapie. Folglich erzeugen CPCs in vitro war ein wichtiger Forschungsschwerpunkt in der Herz-Kreislauf-Studien. Seit WSR haben unbegrenzte Erweiterungsmöglichkeiten einnd stellen die ICM-Zellen im Blastozystenstadium, ist die Differenzierung von WSR in embryonale CPCs nach der Naturembryogenese als eine logische und effektiven Ansatz zur CPCs zu erhalten. Eine weit angelegte Herangehensweise an CPCs von WSR erhalten, ist die Wirtschafts- und Sozialräte in EBs 11 aggregieren. Die Differenzierung Effizienz zu verbessern, sind definierte chemische und Wachstumsfaktoren auf Basis der Kenntnis der Herzentwicklung verwendet worden, 12-14. Es gibt jedoch keine definitive CPC Marker, insbesondere keine Zelloberflächenmarkern, die allgemein auf dem Gebiet akzeptiert. Um dieses Problem anzugehen, WSR sind so konstruiert, Isl1 + oder Mef2c + CPCs und ihre Derivate mit fluoreszierenden Reporter mit Cre / loxP-System markieren. Die cre-Rekombinase in unter der Kontrolle Isl1 / Mef2c Promotor / Enhancer geklopft. Das modifizierte fluoreszierende Protein RFP oder YFP-Gen von einem konstitutiven Promotor angetrieben wird durch Exzision flox Stoppcodon mit cre-Rekombinase aktiviert werden(ISL1: cre; pCAG-flox-AUS-flox-GFP oder RFP / Isl1-cre; Rosa26YFP / Mef2c-cre; Rosa26YFP) 5,6. Sobald die Wirtschafts- und Sozialräte in zweiten Herzfeldes CPCs unterschieden werden Isl1 / Mef2c Promotor / Enhancer angetrieben cre die fluoreszierenden Reportern zu aktivieren und CPCs können durch FACS-Aufreinigung angereichert werden. Kurz gesagt, wird EB Aggregationsmethode verwendet werden, um ESC Differenzierung einzuleiten. Differenzierung Effizienz zu verbessern, werden die differenzierten Zellen mit Ascorbinsäure (AA) und Wachstumsfaktoren wie Bmp4, Activin A und VEGF 13,15 behandelt. Dieses Protokoll ermöglicht robuste und effiziente CPC Differenzierung sowohl mit Maus und Mensch WSR.

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derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

Das Protokoll zeigt Ableitung CPCs aus mehreren ES-Zelllinien. WSR werden aggregiert, um EBs bilden in CPCs zu unterscheiden. WSR werden routinemäßig auf MEF Feeder (1A, F) gehalten und Zuführungen vor Differenzierung entfernt. Nach Aggregation der WSR in EBs in Differenzierungsmedium (1B, G), werden die zweiten geformten EVGs Bmp4 und Activin A behandelt, um das Mesoderm Differenzierung in Zelllinien der Maus (1C) zu verbessern. WSR in Herzlinie differenziert wie fl...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Ableitung von Herzvorläuferzellen aus embryonalen Stammzellen

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.1K Aufrufe.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Serie: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Ableitung von hämatopoetischen Stammzellen aus embryonalen Stammzellen der Maus

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Forschung

Biologie

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.

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.

Die Differenzierung der Atrial Kardiomyozyten aus pluripotenten Stammzellen Mit dem BMP-Antagonisten Grem2

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

Entwicklungsbiologie

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Beurteilung Stem Cell DNA-Integrität für kardiale Zelltherapie

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetik

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In-vitro- Differenzierung humaner mesenchymaler Stammzellen in funktionellen Cardiomyocyte-ähnlichen Zellen

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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 a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Generation der ersten Herz Bereich-wie kardiale Stammväter und ventrikuläre wie Herzzellen aus menschlicher pluripotenter Stammzellen

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

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Induktion der endothelialen Differenzierung in kardialen Vorläuferzellen unter niedrigen Serum-Bedingungen

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In Vitro-Generierung von Mausherzfeldspezifischen Herzvorläuferzellen

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Generation of Human Cardiomyocytes: Eine Differenzierung Protokoll von Feeder-freien menschlichen induzierten pluripotenten Stammzellen

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

High Efficiency Differenzierung von humanen pluripotenten Stammzellen zu Kardiomyozyten und Charakterisierung mittels Durchflusszytometrie

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...