1. Empfehlungen Vor dem Start <ol><li> Verwenden Sie keine PSCs mit Mykoplasmen kontaminiert, weil sie nicht richtig in Sinusknotenzellen differenzieren. Test auf Mykoplasmen-Kontaminationen vor dem Start des Protokolls. Tun Sie dies mit Hilfe eines PCR-Kits für die schnelle, hochempfindlichen Nachweis von Mykoplasmen und folgen Sie dem Hersteller `Protokoll. </li><li> Für jede Petrischale (Schritt 2.3.4), Wappen eine 10 cm 2 Zellkulturschale mit sterilem 7 ml 0,1% Gelatine aus Kaltwasserfischen Haut für 1 Stunde bei 37 ° C. Entfernen Sie die Gelatine und lassen Sie die Schale trocken unter sterilen Bedingungen in einer Sterilbank. </li><li> Bevor Sie die Differenzierung Protokoll zu starten benötigen Sie einen Doppel transfiziert stabile Maus-ES-Zelllinie, Klon, der die folgenden Merkmale: i) Die konstitutive Überexpression von TBX3 mit einem Säugetier-Expressionsvektor. Ii) Der G418-Resistenz-Gen unter der Kontrolle des Myh6-Promotor 5. </li><li> Die undifferenzierten Zellen PSCs sollten zusammen pflegen werdend mit bestrahlten Feeder-Zellen unter Standardbedingungen wie beschrieben ein. </li></ol> 2. Differenzierung Vorgehens <ol><li> Zwei Tage vor der Differenzierung - entfernen PSCs von Feeder-Zellen. <ol><li> Saugen Sie das Medium, waschen Sie die Zellen mit 10 ml phosphatgepufferter Salzlösung PBS, 7 ml Collagenase IV-Lösung und Inkubation der Zellen für 10 min bei 37 ° C. Legen Sie eine sterile 40 um Filter in ein 50 ml Röhrchen. </li><li> Die PSC Kolonien vorsichtig spülen (zu vermeiden, um die Feeder-Zellen zu entfernen) durch Pipettieren der Collagenase-Lösung auf und ab 5 mal. </li><li> Übertragen der Zellsuspension auf eine 40 &amp; mgr; m-Filter, spült das Filter dreimal mit 8 ml PBS. Drehen Sie sich um den Filter und stellen Sie es auf den Kopf in einer Petrischale. Entfernen Sie die PSC Zellkolonien durch Pipettieren von 10 ml PBS, um den Boden des Filters. </li><li> Übertragen der Zellsuspension in ein 15 ml Zentrifugenrohr und für 5 min bei 300 x g. </li><li> Entfernen Sie die PBS, die Zellen zu suspendieren in 1 ml Accutase und incubate bei 37 ° C für 5 min. </li><li> 10 ml PBS, mischen Sie die Zell-Lösung pipettiert es nach oben und unten 5-mal und Zentrifuge für 5 Minuten bei 300 x g. </li><li> Entfernen Sie die PBS, die Zellen in 10 ml Kultivierungsmedium zu suspendieren und bestimmen die Zellzahl. </li><li> Seed 15.000 Zellen / cm2 auf einem 75 cm 2 Filterkolben und pflegen sie für 2 Tage bei 37 ° C, 5% CO2. Nach 2 Tagen sollte der Kolben 50 bis 70% konfluent sein. </li></ol></li><li> Tag 0 - Beginn der Differenzierung <ol><li> Entfernen Sie das Medium und waschen Sie die Zellen mit 10 ml PBS. </li><li> Saugen Sie das PBS, 2 ml Accutase und inkubieren Sie die Zellen bei 37 ° C für 5 min. Legen Sie eine sterile 40 um Filter in ein 50 ml Röhrchen. </li><li> 10 ml PBS, übertragen Sie die Zellsuspension auf die 40-um-Filter, spülen Sie den Filter durch die Zugabe von 10 ml PBS und zentrifugieren die Durchfluss für 5 Minuten bei 300 x g. </li><li> Die Zellen in 10 ml Differenzierungsmedium und Bestimmung der Zellzahl. </li><li> Verdünnen ter Zellsuspension mit Differenzierungsmedium bis zu einer Endkonzentration von 20.000 Zellen / ml. </li><li> Je 20 ml Wasser und 5 ml hängenden Tropfen (HD) Lösung in einer quadratischen Petrischale zur Austrocknung des HDs vermeiden. HINWEIS: Bei allen 24 Well-Platte mit iSABs (siehe Abschnitt 2.8.6.4/2.8.7) beginnen mit 16 Petrischalen. </li><li> Pipettieren von bis zu 50 ml Zellsuspension in ein Fach ein. </li><li> Drehen um den Deckel der Petrischale. Zeigen 180 HDs, jede mit 20 ul (400 Zellen / HD) Zellsuspension auf dem Deckel mit einem 12-Kanal-Pipette. </li><li> Vorsichtig um den Deckel ab und legen Sie sie auf die Petrischale. HINWEIS: Die Geschwindigkeit der Drehung um die Deckel von großer Bedeutung. Wenn der Deckel in der Umgebung zu langsam oder zu schnell gedreht wird, ist die Oberflächenspannung der Zellsuspension nicht hoch genug ist, um zu halten den hängenden Tropfen auf dem Deckel. Bevor Sie versuchen, HDs erstmals Praxis Drehen um den Deckel mit 20 ul Tropfen Medium zu erzeugen. </li><li> Kultivieren der Zellen für 2 Tage bei 37 ° C, 5% CO 2 lassen sich bilden EBs. HINWEIS: Legen Sie eine Petrischale mit Wasser auf der Oberseite eines Stapels von 5 Petrischalen gefüllt. Andernfalls ist die HD im oberen Petrischale wird trocknen. </li></ol></li><li> Tag 2 <ol><li> Vorsichtig um den Deckel des quadratischen Petrischale ab und übertragen Sie die EBs aus zwei Petrischalen (360 EVG) abgeleitet, um ein 50 ml Röhrchen. </li><li> Warten Sie 10 min die EVG am Boden des Röhrchens absetzen zu lassen (Sie Zentrifuge nicht die EVG). </li><li> Saugen Sie so viel Medium wie möglich auszusetzen, die EVG in 10 ml Differenzierungsmedium und übertragen Sie die Suspension auf eine 10 cm Petrischale. </li></ol></li><li> Tag 2-6 Suspensionskultur <ol><li> Pflegen Sie die EVG in Suspension für 4 Tage bei 37 ° C, 5% CO 2, zu ändern Medium nach 2 Tagen. HINWEIS: Auch wenn die Petrischale ist nicht beschichtet (im Gegensatz zu einer Zellkulturschale) Das EBS legen oft an die Oberfläche. Kontrollieren Sie die Petrischalen für angebracht EVG jeden Tag und bei Bedarf nehmen Sie den EVGvon der Oberfläche durch vorsichtiges Pipettieren. Optional: Schütteln Sie die Petrischalen kontinuierlich während der Suspensionskulturperiode. Seien Sie vorsichtig mit der Geschwindigkeit der Shaker. Die EVG sollte weder zu akkumulieren in der Mitte noch schweben an der Grenze der Petrischalen. </li></ol></li><li> Tag 6 <ol><li> Übertragen Sie die EVG aus einer Petrischale mit einem Gelatine beschichtete 10 cm 2 Zellkulturschale. </li></ol></li><li> Tag 7-12 <ol><li> Die Zellen sollten beginnen, nach 8-12 Tagen zu schlagen. Überprüfen Sie für den Sieg gegen Schwerpunkte der Zellen täglich unter dem Mikroskop. Die Zellen beginnen, um eine Schicht zu bilden. </li><li> Ändern Medium täglich. HINWEIS: Nach dem Ändern Medium, die Zellen benötigen 3-4 Stunden zu erholen und wieder anfangen zu schlagen. </li></ol></li><li> Tag 12-15 Auswahl der Sinusknotenzellen. <ol><li> Drei Tage, nachdem die Zellen fingen (um Tag 12-15), saugen die mittel- und Pipette 10 ml frischem Differenzierungsmedium, das 400 ug / ml G418 zu den Zellen. </li></ol></li><li&gt; Vier Tage nach Beginn der Selektion dissoziieren die Zellschicht. HINWEIS: Es ist möglich, dass die Zellschicht bereits von der Oberfläche abgelöst. <ol><li> Entfernen Sie das Medium vorsichtig ohne Absaugen der Zellschicht. </li><li> 10 ml PBS und entfernen Sie die PBS vorsichtig ohne Absaugen der Zellschicht. </li><li> 10 ml Kollagenase IV Lösung, übertragen die Zellen in ein 50 ml-Röhrchen und Inkubation der Zellen bei 37 ° C für 5 min. </li><li> Gründlich mischen die Suspension durch Auf- und Abpipettieren 10 Mal und inkubieren Sie die Zellen bei 37 ° C für 5 min. Eine Suspension von kleinen Clustern auftreten sollte. Wenn es noch große Teile der Schicht nach links, dann 2.8.4 noch zwei weitere Male wiederholen Schritt. </li><li> Mit 20 ml PBS und zentrifugieren für 10 min bei 300 x g. </li><li> Saugen Sie die PBS sorgfältig. HINWEIS: Wenn iSABs sind erzeugt mit Schritt 2.8.7 werden. Wenn ISAB abgeleitete Sinusknoteneinzelzellen erzeugt werden sollen weiter mit Schritt 2.9. </li><li> Suspend die Zellenin 12 ml Differenzierungsmedium, das 400 ug / ml G418 und entkernen auf 6 Vertiefungen von Gelatine 24 Vertiefungen (2 ml / Vertiefung). </li></ol></li><li> Generierung von Einzelknotenzelle. <ol><li> Aussetzung der Zellen aus Schritt 2.8.6 in 5 ml Accutase und bei 37 ° C für 5 min. </li><li> Gründlich mischen die Suspension durch Auf- und Abpipettieren 10 Mal und inkubieren Sie die Zellen bei 37 ° C für 5 min. </li><li> Mit 20 ml PBS und Zentrifuge 5 min bei 300 x g. </li><li> Die Zellen in 12 ml Differenzierungsmedium, das 400 ug / ml G418 und entkernen auf 6 Vertiefungen einer Gelatine 24 Vertiefungen (2 ml / Vertiefung). </li></ol></li><li> Tag 2 nach der Dissoziation, saugen Sie den mittleren und waschen Sie die Zellen dreimal mit PBS. 1 ml Differenzierungsmedium / well. </li><li> Tag 4-8 nach der Dissoziation, jeden 2. Tag ändern Medium. Am Tag 10 nach der Dissoziation Verwendung iSABs oder ISAB abgeleitete Sinusknoteneinzelzellen sind zur weiteren Analyse zur Verfügung. </li></ol>

<ol>
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Die Autoren haben nichts zu offenbaren.

Die Fähigkeit zur Produktion von Stammzell eine Herzschrittmacherzellen kann Rekonstitution der richtigen Herzrhythmus in dem Sinne von &quot;biologischen Schrittmacher&quot; ermöglichen. Ebenso wird Drogentests in vitro von deren Verfügbarkeit profitieren. PSCs können zu jedem Zelltyp des Säugetierkörpers einschließlich Herzmuskelzellen mit Herzschrittmacher Zelleigenschaften 8,9,10,11,12,13 geben. Allerdings, in der Regel die Zellpopulationen im &quot;Embryoid Bodies&quot; sind sehr heterogen, was zw...

Der Begriff &quot;Sick-Sinus-Syndrom&quot;, fasst mehrere Erkrankungen, die zu einer Verschlechterung der Herzschrittmacher-System. Es besteht aus pathologischer, symptomatische Sinusbradykardie, Sinusknoten-Block, Sinusstillstand sowie die Tachykardie Bradykardie-Syndrom. Dadurch wird ein &quot;krankes Sinus-Syndrom&quot; oft durch allgemeinen Herzerkrankungen begleitet, wie eine koronare Herzkrankheit, Herzmuskelerkrankungen oder Herzmuskelentzündung. Derzeit werden therapeutische Ansätze über die Implantation elektrischen Schrittmacher basiert. Allerdings geht dies zusammen mit einer Reihe von Risiken wie Infektionen und Batterieausfall. Insgesamt ist die Häufigkeit von Komplikationen wie vor sehr hoch bei Patienten mit einem Herzschrittmacher implantiert. Weiterhin, im Gegensatz zu dem endogenen Schrittmacher, diese Geräte nicht auf Hormonstimulation reagieren. Eine zukünftige Alternative kann von der Verfügbarkeit von &quot;biologischen Schrittmacher&quot; für die PSCs dienen könnte verlassenAls geeignetes Zellquelle und dem auch für In-vitro-Drogen-Test von hohem Wert sein. Dennoch liegt ein großes Problem in der sehr seltenen Auftreten von Sinusknotenzellen in embryonale Körper (EVG) - dies in der Regel nicht ~ 0,5% 1 überschreitet. Zuvor wurde gezeigt, dass &quot;zukunfts Programmierung&quot; auf bestimmte Subtypen Kardiomyozyten möglich ist über die Überexpression von verschiedenen frühen Herz-Kreislauf Transkriptionsfaktoren wie Mesoderm spezifische-posterior 1 (MesP1) und NK2 Transkriptionsfaktor bezogen, locus 5 (Nkx2.5) 2, 3. Für normale Größe und Funktion der Sinusknoten (SAN) ist der T-Box-Transkriptionsfaktor TBX3 entscheidend, was gezeigt worden ist, um den Schrittmacher-Gen-Programm zu initiieren und Differenzierung des SAN 4 steuern. Obwohl diese verbesserte das Aussehen des Funktionsschrittmacherzellen, deren Inhalt noch nicht ~ 40% in der gesamten Zellpopulation cardiomyocytic überschreiten. Daher wurde eine zusätzliche Myh6-Promotor basiert antibiotische Selektion Schritt 5 von uns vorstellen. Dies führt letztlich zu noch unbeobachtet Kardiomyozyten Aggregate (&quot;induzierte sinuatrialer Stellen;&quot; iSABs &quot;), die stark erhöhte schlagen Frequenzen (&gt; 400 min) in vitro zeigen, zum ersten Mal die denen eines murinen Herzen und vergleichbar mit in vitro kultivierten Sinusknotenzellen aus einer Maus-Herz 6 isoliert. Unter Isoprenaline Verwaltung selbst schlagen Frequenzen von 550 Schlägen pro Minute erreicht. Bemerkenswert ist, iSABs bestehen aus mehr als 80% Funktionsknotenzellen, wie aus physiologischen umfangreiche Analysen 7. Kürzlich wurden mehrere Ansätze für die Sinusknotenzellen erzeugen mit Gleich Umprogrammierung 19, Oberflächenmarkierungen 14 oder pharmakologische Behandlung mit kleinen Molekülen 16,17 wurden beschrieben. Doch keines dieser Verfahren führte zu einer so hohen Reinheit von Schrittmacherzellen und Schlagen Frequenzen nahe dem murinen erKunst als in iSABs beobachtet. Darüber hinaus wird in einem ex vivo Modell der kultivierten adulten Maus ventrikuläre Scheiben, die ihren spontanen Schlagaktivität verloren haben, sind die iSABs Lage die Integration in die Scheibe Gewebe und damit spontan aktiv bleiben und kräftig Tempo der Herzscheiben zu Kontraktionen 7. Ein detailliertes Protokoll für die Erzeugung dieser iSABs ist in diesem Papier beschrieben.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Name of the Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s liquid medium with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0465</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM liquid medium without Na-pyruvate, with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0435</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLS type IV, CLS IV</td>
			<td>Biochrom AG</td>
			<td>C4-22</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Biochrom AG</td>
			<td>K 0293</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS Superior</td>
			<td>Biochrom AG</td>
			<td>S 0615</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium pyruvate (100 mM)</td>
			<td>Biochrom AG</td>
			<td>L 0473</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G 418-BC liquid (ready-to-use solution), sterile</td>
			<td>Biochrom AG</td>
			<td>A 2912</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoir PP f.multichannel pip. 60ml,sterile</td>
			<td>Brand</td>
			<td>703409</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>eBioscience,Inc.</td>
			<td>00-4555-56</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Xplorer/Xplorer plus, electronic pipette</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Cell Strainer 40&#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>GE Healthcare</td>
			<td>P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Greiner BioOne</td>
			<td>663102</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS without Ca and Mg</td>
			<td>PAN-Biotech</td>
			<td>P04-36500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>PAN-Biotech</td>
			<td>P30-3302</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
			<td>LIF-250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quadratic petri dishes</td>
			<td>Roth</td>
			<td>PX67.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from cold water fish skin</td>
			<td>SigmaAldrich</td>
			<td>G7765</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Mercaptoethanol</td>
			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture dishes</td>
			<td>TPP</td>
			<td>93100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture flask</td>
			<td>TPP</td>
			<td>90076</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture test plates (24 well)</td>
			<td>TPP</td>
			<td>92424</td>
		</tr>
	</tbody>
</table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

Die Behandlung des &quot;Sick-Sinus-Syndrom&quot; wird auf Kunstherzschrittmachern basiert. Diese mit Gefahren, wie zB Batterieausfall und Infektionen. Außerdem fehlt ihnen Hormonempfindlichkeit und das gesamte Verfahren ist kostenintensiv. &quot;Biologische Herzschrittmacher&quot; von PSCs erzeugt wird, kann eine Alternative sein, aber der typische Inhalt der Schrittmacherzellen in embryonale Körper (EVG) ist extrem niedrig. Die beschriebenen Protokoll verbindet &quot;forward-Programmierung&quot; von murinen PSCs über den Sinusknoten Induktor TBX3 mit Myh6-Promotors auf der Basis Antibiotika-Selektion. Dies führt zu Kardiomyozyten Aggregate konsequent von&gt; 80% physiologisch funktionellen Schrittmacherzellen. Diese &quot;induzierten Sinusknoten-Stellen&quot; (&quot;iSABs&quot;) werden spontan kontra bei noch unerreichten Frequenzen (400-500 bpm), die Knoten Zellen aus Mäuseherzen isoliert und können murine Myokard ex vivo zu stimulieren. Mit dem beschriebenen Protokoll hochreines SinusKnoteneinzelzellen erzeugt werden, die beispielsweise für in vitro Arzneimitteltests verwendet werden kann. Weiterhin können die nach diesem Protokoll erzeugt iSABs ein entscheidender Schritt in Richtung Herzgewebetechnik geworden.

Das beschriebene Protokoll ermöglicht Generation iSABs mit einer Schlagfrequenz von etwa 450 bpm von PSCs (im Film dargestellt), die in der Nähe ist es, die Maus Herzschlagfrequenz. Nach Dissoziation iSABs (Schritt 2.8.8) die beobachteten Einzelzellen zeigen eine typische Form der Zellen der Sinusknoten (Spindel und Spinnenzellen), wie in Abbildung 1 dargestellt. Diese Zellen hoch Proteine ​​exprimieren, die bekannt sind für die Funktion zu sein, des Sinusknotens wie hyperpolarisations-aktivierte...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

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

Dieses Protokoll beschreibt die Funktionssinusknotengewebe von Maus pluripotente Stammzellen (PSC) zu produzieren. T-Box3 (TBX3) Überexpression sowie Herz-Myosin-Schwerketten (Myh6) Promoter Antibiotika-Selektion führt zu hochreinem Schrittmacher Zellaggregate. Diese &quot;Verursachen-Sinusknoten-Stellen&quot; (&quot;iSABs&quot;) enthalten mehr als 80% Schrittmacherzellen, zeigen stark erhöhte schlagRaten und können Myokard ex vivo zu stimulieren.

Generierung von murinen Herzschrittmacher Zellaggregate Basierend auf ES-Zell-Programmierung in Kombination mit Myh6-Promoter-Auswahl

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

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K Aufrufe.  University of Rostock.  Dieses Protokoll beschreibt die Funktionssinusknotengewebe von Maus pluripotente Stammzellen (PSC) zu produzieren. T-Box3 (TBX3) Überexpression sowie Herz-Myosin-Schwerketten (Myh6) Promoter Antibiotika-Selektion führt zu hochreinem Schrittmacher Zellaggregate. Diese "Verursachen-Sinusknoten-Stellen" ("iSABs") enthalten mehr als 80% Schrittmacherzellen, zeigen stark erhöhte schlagRaten und können Myokard ex vivo zu stimulieren. 

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

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Menschliche pluripotente Stammzelle Basierend Entwicklungstoxizität Assays für chemische Sicherheit Screening und Systembiologie Datengenerierung

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Forschung

Entwicklungsbiologie

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

Generation von Aggregaten von embryonalen Stammzellen der Maus, die Symmetriebrechung, Polarisation und Emergent Collective Verhalten anzeigen<em&gt; In-vitro-</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

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

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Aggregate Größenoptimierung in Mikrotiterplattenvertiefungen für Abhänge-basierte Herz Differenzierung von humanen pluripotenten Stammzellen

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

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Eine neuartige Mamma-Fat-Pad-Transplantationstechnik zur Visualisierung der Gefäßerzeugung von vaskulären endothelialen Stammzellen

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Quantitative ganze-Mount Immunfluoreszenz Analyse der kardialen Vorläuferzellen Populationen in Mausembryonen

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Generation von Gerüst-frei, dreidimensionale Insulin mit dem Ausdruck Pancreatoids vom Maus Pankreas Stammväter In-vitro-

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Analyse der Entwicklung der kardialen Kammer in der Maus Embryogenese mit ganzen Mount Epifluoreszenz

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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 ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generierung von Mosaik-Mammary-Organoiden durch Differential-Trypsinisierung

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generierung eines humanen iPSC-basierten Blut-Hirn-Barriere-Chips

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...