Dieses Manuskript ist dem Andenken von Dr. Wenlan Wang, der am 26. Mai 2011 verabschiedet gewidmet. Wir danken Dr. Douglas A. Kerr für die großzügige Bereitstellung der HBG3 mES Zellen in dieser Studie verwendet. Diese Arbeit wurde von Nemours finanziert wird, ein Zuschuss (2 RR016472-10) unter der INBRE Programm des National Center for Research Resources (NCRR) und einem Cobre Gewährung von Finanzhilfen aus dem NCRR (5 RR020173 P20-05) an das Zentrum für Unterstützung Pädiatrische Forschung an der Alfred I. DuPont Hospital for Children, Wilmington, Delaware, USA.

1. Schritt 1: embryonalen Stammzellen der Maus (mES) Cell Culture (Timing: 3 Tage) 1. Ausplattieren primäre embryonale Maus-Fibroblasten (PMEF) <ol><li> Coat vier 100-mm Zellkulturschalen mit Gelatine für PMEF Haftung. 8 ml von 0,1% Gelatine-Lösung (StemCell Technologies) zu jeder Schale und Inkubation für 30 min bei Raumtemperatur. </li><li> Entfernen Sie das überschüssige Gelatine von Gerichten. Nicht spülen das Geschirr. </li><li> Verdünnen Sie ein Fläschchen mit Mitomycin C-inaktivierten PMEF (Millipore), die ca. enthält. 5 x 10 6 Zellen in 40 ml PMEF Medien (siehe Rezept in Tabelle 1) und Platte auf vier 100-mm Zellkulturschalen. Die PMEF eine Feeder-Schicht für MES-Zellen. </li><li> PMEF sollte mindestens einen Tag vor der Zugabe mES Zellkulturen plattiert werden. PMEF kann bis zu 1 Woche nach der Beschichtung verwendet werden. </li></ol> 2. Plating mES Cells Um die Effizienz von MES-Zell-Differenzierung in Motoneuronen zu überwachen, Verwendeten wir HBG3 kann eine ES-Zelllinie, die von einer transgenen Maus exprimiert eGFP unter der Kontrolle des Promotors Motoneuronen Hb9 abgeleitet. <ol><li> Defrost 1 Fläschchen HBG3 mES Zellen (ca. 1 x 10 7, beigetragen von Dr. Douglas Kerr, Johns Hopkins University) in einem 37 ° C warmes Wasserbad. 5 ml ES Medien (siehe Rezept in Tabelle 1) in einem 15 ml-Tube und Spin-down-Zellen bei 800 rpm für 5 min. </li><li> Saugen Sie den Überstand und resuspendieren mES Zellen in 20 ml ES Medien mit frisch zugegebenem Leukämiehemmfaktor (LIF) bei einer Endkonzentration von 10 ng / ml. HINWEIS: LIF sollte am Tag der Verwendung hinzugefügt werden und wird benötigt, um die MES-Zellen in einem undifferenzierten Zustand zu halten. </li><li> Nehmen Sie 10 ml PMEF Medien aus dem PMEF in jeder Schale und mit 10 ml des HBG3 ES Zell-Suspension zu jedem Gericht. </li><li> 24 h nach der Beschichtung, bilden mES Zellen kleine Kolonien, ähnlich groß wie der Kolonie durch die Pfeilspitze in 1b dargestellt. 72 h nach dem Ausplattieren, coloniES Zunahme der Größe, da viele etwa 100 um im Durchmesser (Abbildung 1B, durch Pfeile angedeutet). Diese Zellen sind bereit für Differenzierung. Die Medien sollten täglich ausgetauscht werden, um die Proliferation von Zellen aufrecht zu erhalten. </li></ol> 2. Schritt 2: neurale Induktion (Timing: 2 Tage) Zur Differenzierung von MES-Zellen in motorischen Neuronen zu induzieren, müssen mES Zellen aus PMEF getrennt werden und kultiviert in einer Suspension Umwelt. Gelatine beschichtete Kolben werden verwendet, um PMEF von MES-Zellen zu trennen. <ol><li> Wenn mES Kolonien bereit sind für neurale Induktion (definiert als Differenzierung Tag 0), Mantel 2 T150 Flaschen mit 0,1% Gelatine (18 ml / Kolben) für 30 min bei Raumtemperatur und anschließend zu entfernen überschüssige Gelatine und waschen mit 1x PBS dreimal. HINWEIS: Jeder 100-mm-Schale Kultur wird ein T150-Gelatine beschichtete Kolben müssen PMEF trennen. </li><li> Entfernen Sie vorsichtig Medien durch Aspiration darauf achten, nicht zu lose befestigten Kolonien zu vertreiben. 10 ml PBS kurzly und entfernen Sie dann durch Absaugen zu Waschschritt abzuschließen. </li><li> MES-Zellen aus PMEF trennen, fügen 0,25% Trypsin / EDTA (3 ml / Schale, StemCell Technologies) und Inkubation für 3-5 min bei 37 ° C, bis die Stammzellen und PMEF von der Platte zu lösen. 15 ml frisches ES-Medien ohne LIF und Pipette Zellen nach oben und unten, um Kolonien (ca. 15-20 mal) zu brechen. Dann wurde zu einem 0,1% Gelatine beschichtete T150 Destillierkolben und Inkubation für 30 min bei 37 ° C Nach der Inkubation sollten PMEF auf die Oberfläche legen, während gelatinierte ES-Zellen zu schweben sollte. Sammeln Sie die schwimmenden mES Zellen in eine 50 ml Tube. </li><li> Spin-Down bei 800 UpM und Resuspendieren Sie die Zellen in 10 ml Medium neurale Differenzierung (siehe Rezept in Tabelle 1). </li><li> Zählen der Zellen unter Verwendung eines Hämozytometers und dann Platte ungefähr 2 x 10 6 Zellen mES auf einer 100 mm bakteriellen oder Suspension Kulturschale. Diese Platten erlauben Wachstum von Suspensionskulturen und fördern Bildung von Embryoidkörpern (EBS). </li><li> Auf differentiation Tag 1, bilden mES Zellen kleine schwimmende Aggregate (EVG), die sichtbar unter einem Lichtmikroskop sind. Jede Verschleppung PMEF in die Schale legen. Übertragen Sie die Suspensionszellen und Medien in ein 15 ml Tube und zentrifugieren bei niedriger Drehzahl (500 rpm) für 3 min. Medien sorgfältig absaugen, 10 ml frisches Medium neurale Differenzierung der EBs pelletiert und anschließend Platte Zellen in einem neuen bakteriellen Gericht. Zusätzlich zur Aufstockung der Medien, entfernt dieser Schritt keine PMEF tragen, die über aus dem Stand Schritt. </li><li> Auf Differenzierung Tag 2 sind EBs bereit, in Motoneuronen induziert werden. </li></ol> 3. Schritt 3: Motor Neuron-Spezifikation (Timing: 5 Tage) <ol><li> Am Tag 2 Differenzierung, wirbeln die Kulturschale und überweisen Sie den Medien-und EBS in einem 15 ml-Tube. Lassen EBs durch die Schwerkraft (~ 10 min) oder durch Zentrifugation bei niedriger Geschwindigkeit (500 rpm) für 3 min zu begleichen. </li><li> Aspirieren Medium vorsichtig resuspendieren und EBs in 10 ml Motor Neuron Differenzierung (MND) Medium (siehe Rezept in <strong&gt; Tabelle 1). Platte EBs in einem neuen bakteriellen Schüssel und ändern Sie die Medien täglich über 5 Tage. </li><li> 200 um im Durchmesser und sollte stark GFP-Signale (Abbildung 1C) zum Ausdruck bringen - Am Tag 7 Differenzierung sollte EBs etwa 150 sein. Diese EBs sind bereit, voneinander zu trennen und plattiert auf poly-DL-ornithine/laminin beschichteten Schalen oder Deckgläser. </li></ol> 4. Schritt 4: Herstellung von Poly-DL-ornithine/laminin beschichtete Deckgläser (Timing: 2 Tage) Zwei Tage vor distanziert EBs (dh auf die Differenzierung Tag 5), bereiten poly-DL-ornithine/laminin-coated Deckgläser. <ol><li> Platz 12-mm-Deckgläschen jähriger (Warner Instruments) an der Unterseite eines 24-Well-Platte. 25 mg Poly-DL-Ornithin (Sigma-Aldrich) in 25 ml sterilem, doppelt destilliertem Wasser (d 2 H 2 O), um ein 10X-Stammlösung (1 mg / ml) zu erhalten. Wenn Sie bereit sind zu verwenden, machen Sie eine 1/10 Verdünnung von Poly-DL-Ornithin mit D 2 H 2 O zu erhalten concentRation von 100 g / ml. 0,5 ml zu jeder Vertiefung der Platte mit 24 Vertiefungen, Inkubation bei 4 ° C über Nacht. </li><li> Am folgenden Tag (Differenzierung Tag 6), absaugen Poly-DL-Ornithin und lassen die Teller und Deckgläser an der Luft trocknen in einer Gewebekultur Kapuze. Spülen Sie die Platte Brunnen mit D 2 H 2 O dreimal. Der Luft trocknen lassen für eine weitere Stunde. </li><li> 1 mg Mauslaminin (Millipore) in 5 ml kaltem Eis-1X PBS 100x Stocklösung (200 pg / ml) zu machen. Wenn Sie bereit sind zu verwenden, stellen Sie 1/100 Verdünnung in vereisten kalten 1X PBS (2 ug Laminin / ml). 0,5 ml / Well in einer 24-Well-Platte und Inkubation 4 ° C über Nacht. Vor der Aussaat Zellen, sollten überschüssige Laminin entnommen und Vertiefungen mit 1X PBS zweimal gespült. Deckgläser in MND-Medium (0,5 ml / Vertiefung) gespeichert werden. </li></ol> 5. Schritt 5: Axon Dehnung (Timing: 2 Tage) <ol><li> Am Tag 7 Differenzierung, sammeln EBs in ein 15 ml Tube und lassen sich absetzen EBs (ca. 3 min). Aspirieren Medien und Re-Suspend EBs in PBS. Erlauben Sie EBs, sich niederzulassen und dann absaugen PBS. Wiederholen Sie diesen Schritt 3 mal. 1 ml der Accumax (Millipore) der EBS, vorsichtig mischen und Inkubation für 5 min bei Raumtemperatur. Dann saugen überschüssiges Accumax für den EBs. </li><li> Um EBs distanzieren, 3 ml Medium MND. Pipettieren nach oben und unten 20 Mal unter Verwendung einer 1 ml-Eppendorf-Mikropipette (blaue Spitze), um EBs stören. Inkubieren für 2 min bei Raumtemperatur. Übertragen Zellsuspension auf eine 12x75 mm-Rohr mit Zellsieb Kappe (BD Falcon), um eine Einzelzellen-Suspension zu erhalten. Wiederholen diese Trennung Schritt mit den restlichen Klumpen und Zellen durch den Filter, so dass die Ausbeute von einzelnen Zellen anzureichern erhalten. Die endgültige Einzelzellsuspension werden 6 ml sein. </li><li> Vorsichtig mischen diese einzelne Zellsuspension und Zählen von Zellen mit einer Zählkammer. Verdünnte Zellen in MND Medium (ergänzt mit 10 ng / ml von jeder BDNF, GDNF, CNTF und NT-3) auf eine Dichte von 2x10 5 Zellen pro ml. Zellsuspension (0,5 ml / Vertiefung) auf 24-Well-Platten, die poly-DL-ornithine/laminin beschichtete Deckgläser (zuvor hergestellt wie oben beschrieben). Die differenzierten Zellen verlängern lange Neuriten nach 2 Tagen in Kultur (1D). </li></ol> 6. Repräsentative Ergebnisse 1A zeigt einen Umriss des Protokolls. In Schritt 1 sind MES-Zellen auf PMEF kultiviert und ergänzt mit LIF eine spontane Differenzierung zu verhindern. Im Allgemeinen sind undifferenzierte Kolonien mES rund und kompakt und haben klar definierte Ränder auf. Die Kolonien sind nicht in Kontakt miteinander. In der Regel sollte mES Zellen bei einer 1:4-Verhältnis aufgeteilt werden alle 2 bis 3 Tagen. Jedoch wird jede einzelne Zelle Linie mit einer anderen Rate wachsen und das Split-Verhältnis muß empirisch ermittelt werden. Pfeile in Abbildung 1B zeigt das typische Erscheinungsbild von MES-Zellen nach Kultivierung auf einem PMEF Feeder-Schicht für 3 Tage. Diese Kolonien sind groß (50-100 um Durchmesser), aber immer noch rund und halten definierte Kantes. In diesem Stadium die Kolonien sind bereit für Differenzierung oder Subkultur. Eine Pfeilspitze in Abbildung 1B zeigt eine kleine Kolonie mES, dass Sie in der Regel finden 24 h nach der Beschichtung. Vier Tage nach dem Ausplattieren, werden mES Zellen überwachsen. Sie zeigen eine abgeflachte Aussehen und den Verlust der definierten Grenzen, was darauf hinweist, dass sie beginnen sich zu differenzieren. Solche Zellen sind nicht optimal für die Differenzierung in neuronale Zellen. MES-Zellen in Motoneuronen zu differenzieren, müssen mES Zellen in Suspension kultiviert werden, um EB Bildung zu ermöglichen. In Schritt 2 werden ES-Zellen auf einer Kulturoberfläche ohne Feeder-Schicht in EB Bildung zu fördern übertragen. In diesem Schritt werden sie in neuronalen Differenzierung Medium mit Noggin, bFGF und FGF-8 für 2 Tage in direkter Zellen eines neuronalen Linie ergänzt inkubiert. Kleine EBs können unter dem Mikroskop an Tag 1 der Differenzierung beobachtet werden. Sie sollten in dem Medium zu schweben. Verschleppung PMEF kann auch am Tag 1 gefunden werdender Differenzierung und müssen entfernt werden. Diese Zellen, um bakterielle Gerichte so beseitigt werden, wenn EBs auf eine neue bakterielle Gericht sind passagiert haften. So, am Tag 2 der Differenzierung, sollte wenige oder keine PMEF in der Kulturschale zu sehen. In Schritt 3 fort Übertragung von EBs zu neuen Gerichten sollte Entfernung jeglichen verbleibenden PMEF zu gewährleisten. EBs sind im Motor Neuron Differenzierung (MND) Medien mit RA ergänzt SAG kultiviert und für 5 Tage, um die Zellen in motorischen Neuronen zu differenzieren. Während der Kultur, weiterhin EBs in der Größe wachsen und kann mit dem bloßen Auge eine Differenzierung Tag 3 ersichtlich ~ 4. 1C zeigt das mikroskopische Bild von EBs auf die Differenzierung Tag 7. Im Gegensatz zu den undifferenzierten Zellen, für EBs starke Fluoreszenz aufgrund der Expression von GFP. Diese EBS sind optimal differenziert und sind bereit für die Dissoziation. Durchflusszytometrie zeigte, dass 51% ± 0,8% der Zellen von der dissoziierten EBs hatte in GFP exprimierenden Zellen differenziert 5. In Schritt 5 Kultur these Motoneuronen in Gegenwart von GDNF, CNTF, BDNF, NT3 und Ergebnisse in Verlängerung der langen Vorsprünge axonalen. 1D zeigt das Aussehen des differenzierten Zellen 2 Tage nach dem Ausplattieren von dissoziierten EBs. Beachten Sie lange Neuriten sich von den Zellkörper der plattierten Zellen. Immunfluoreszenzfärbung zeigt, dass die GFP +-Zellen die pan-neuronalen Marker (Neurofilament-mittlerer Kettenlänge) und zwei Motoneuronen spezifische Marker, (Islet-1 und Cholinacetyltransferase, 2) zu exprimieren. <img alt="1" src="/files/ftp_upload/3813/3813fig1.jpg" /> Abbildung 1. Differenzierung von MES-Zellen in Motoneuronen (Bild von Wu et al modifiziert. 5). A) Schema der Differenzierung Protokoll von MES-Zellen zu Motoneuronen. B) Undifferenzierte Zellen bilden mES runde Kolonien auf der Oberseite eines Fibroblasten-Feeder-Schicht. Sie haben schwache GFP-Expression. C) MES-Zellen wurden von Feeder-Schichten getrennt und KulturvereineRot auf Low-Befestigung Gerichte zu EBs zu bilden. In den ersten beiden Tagen nach der Differenzierung wurden die Zellen auf 50 ng / ml Noggin, 20 ng / ml bFGF und 20 ng / ml FGF-8 ausgesetzt. Anschließend wurden sie bewogen, mit 1 uM Retinsäure und 1 uM Sonic-Hedgehog-Agonist SAG für 5 Tage zu unterscheiden. Differenzierte Zellen exprimiert mES starke GFP-Fluoreszenz. A) Nach dem 7-tägigen Differenzierung wurden EBs dissoziiert und vergoldet auf poly-DL-ornithine/laminin beschichteten Platten. Die differenzierten Zellen verlängert langen neuronalen Prozesse nach 2 Tagen in Kultur. Maßstab = 200 um. MN = motorischen Neurons. B, C und D sind Hellfeld-Bilder und B &#39;, C&#39; und D &#39;sind entsprechende Fluoreszenz-Bildern. <img alt="2" src="/files/ftp_upload/3813/3813fig2.jpg" /> Abbildung 2. Charakterisierung von MES-cell-derived Motoneuronen. Immunfluoreszenzfärbung für Neurofilament mittlerer Kettenlänge (NF-M, rot), ChAT (rot) und Islet-1 (rot) fiel mit GFP (grün) SuchbegriffMultisession. DAPI (blau) wurde verwendet, um die Kerne zu identifizieren. Balken = 50 um.

<ol>
	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Miles, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+motoneurons+derived+from+mouse+embryonic+stem+cells.">Functional properties of motoneurons derived from mouse embryonic stem cells.</a> J. Neurosci. 24, 7848-7858 (2004).</li><li>Wichterle, H., Peljto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+embryonic+stem+cells+to+spinal+motor+neurons.">Differentiation of mouse embryonic stem cells to spinal motor neurons.</a> Curr. Protoc. Stem Cell. Biol. Chapter 1, Unit 1H.1.1-Unit 1H.1.9 (2008).</li><li>Kiris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem Cell Res. 6, 195-205 (2011).</li><li>Wu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+assessment+of+a+cell+model+of+spinal+muscular+atrophy.">Proteomic assessment of a cell model of spinal muscular atrophy.</a> BMC Neurosci. 12, 25 (2011).</li><li>McMahon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin-mediated+antagonism+of+BMP+signaling+is+required+for+growth+and+patterning+of+the+neural+tube+and+somite.">Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite.</a> Genes Dev. 12, 1438-1452 (1998).</li><li>Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+posterior+neural+tissue+is+induced+by+FGF+in+the+chick+embryo.">Early posterior neural tissue is induced by FGF in the chick embryo.</a> Development. 125, 473-484 (1998).</li><li>Launay, C., Fromentoux, V., Shi, D. L., Boucaut, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+FGF+receptor+blocks+neural+induction+by+endogenous+Xenopus+inducers.">A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.</a> Development. 122, 869-880 (1996).</li><li>Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purmorphamine+activates+the+Hedgehog+pathway+by+targeting+Smoothened.">Purmorphamine activates the Hedgehog pathway by targeting Smoothened.</a> Nat. Chem. Biol. 2, 29-30 (2006).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 14071-14076 (2002).</li><li>Lee, S. M., Danielian, P. S., Fritzsch, B., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+FGF8+signaling+from+the+midbrain-hindbrain+junction+regulates+growth+and+polarity+in+the+developing+midbrain.">Evidence that FGF8 signaling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.</a> Development. 124, 959-969 (1997).</li></ol>

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Die Qualität der mES Zellen ist die kritischen Parameter zur effizienten Erzeugung von Motoneuronen. mES Zellen müssen auf PMEF kultiviert werden, um spontane Differenzierung zu verhindern. Die Zugabe von LIF hilft bei der Erhaltung der die MES-Zellen in einem undifferenzierten Zustand. Als MES-Zellen teilen sich alle 12 bis 15 h wurde das Kulturmedium verarmt rasch und muss täglich ersetzt werden. Effiziente Aktivierung des Shh-Weg ist ein kritischer Parameter benötigt, um Motoneuronen ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Endkonzentration </td></tr><tr><td> PMEF </td><td> Millipore </td><td> PMEF-H </td><td> NA </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> PMEF Medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965-118 </td><td> NA </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-071 </td><td> 10% </td></tr><tr><td> L-Glutamin </td><td> Invitrogen </td><td> 25030-081 </td><td> 1% </td></tr><tr><td> Penicillin / Streptomycin </td><td> Invitrogen </td><td> 15240-063 </td><td> 1% </td></tr><tr><td></td><td></td&gt; <td></td><td></td></tr><tr><td> mES Medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES-Zell-Qualifizierte FBS </td><td> Invitrogen </td><td> 16141-079 </td><td> 15% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Nucleosides </td><td> Millipore </td><td> ES-008-D </td><td> 1% </td></tr><tr><td> β-Mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Penicillin / Streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> LIF </td><td> Millipore </td><td&gt; LIF2010 </td><td> 10 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Neuronale Differenzierung Medium </td><td></td><td></td><td></td></tr><tr><td> DMEM </td><td> StemCell Technologies </td><td> 36250 </td><td> NA </td></tr><tr><td> ES Cult FBS </td><td> StemCell Technologies </td><td> 06905 </td><td> 15% </td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> Invitrogen </td><td> 11140-050 </td><td> 1% </td></tr><tr><td> Mono-thio Glycerin </td><td> Sigma-Aldrich </td><td> M-6145 </td><td> 1mM </td></tr><tr><td> Birne </td><td> Invitrogen </td><td> PHC1506 </td><td> 50 ng / ml </td></tr><tr><td> FGF-8 </td><td> Invitrogen </td><td> PHG0274 </td><td> 20 ng / ml </td></tr><tr><td> bFGF </td><td> Invitrogen </td><td> PHG0024 </td><td> 20 ng / ml </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND Medium (Differenzierung) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout Serumersatz </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 Ergänzung </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Askorbinsäure </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillin / Streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td>Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-Glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% in D 2 H 2 O </td></tr><tr><td> Fibronektin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 pg / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml in D 2 H 2 O </td></tr><tr><td> β-Mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> Retinsäure </td><td> Sigma-Aldrich </td><td> R-2625 </td><td> 1 uM </td></tr><tr><td> SAG </td><td> EMD Chemicals </td><td> 566660 </td><td> 1 uM </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> MND Medium (Motor Neuron Kultur) </td><td></td><td></td><td></td></tr><tr><td> ES-Cult Basal Medium-A </td><td> StemCell Technologies </td><td> 5801 </td><td> NA </td></tr><tr><td> Knockout Serumersatz </td><td> Invitrogen </td><td> 10828-028 </td><td> 10% </td></tr><tr><td> N-2 Ergänzung </td><td> Invitrogen </td><td> 17502-048 </td><td> 1% </td></tr><tr><td> ITS Supplement-B </td><td> StemCell Technologies </td><td> 07155 </td><td> 1% </td></tr><tr><td> Askorbinsäure </td><td> StemCell Technologies </td><td> 07157 </td><td> 1% </td></tr><tr><td> Penicillin / Streptomycin </td><td> Millipore </td><td> TMS-AB2-C </td><td> 1% </td></tr><tr><td> Glutamax-I </td><td> Invitrogen </td><td> 35050-061 </td><td> 1% </td></tr><tr><td> D-Glucose </td><td> Sigma-Aldrich </td><td> G-8270 </td><td> 0,15% in D 2 H 2 O </td></tr><tr><td> Fibronektin </td><td> StemCell Technologies </td><td> 07159 </td><td> 5 pg / ml </td></tr><tr><td> Heparin </td><td> Sigma-Aldrich </td><td> H3149 </td><td> 20 ug / ml in D 2 H 2 O </td></tr><tr><td> β-Mercaptoethanol </td><td> Millipore </td><td> ES-007-E </td><td> 0,1 mM </td></tr><tr><td> BDNF </td><td> R &amp; D Systems </td><td> 248-BD-005/CF </td><td> 10 ng / ml </td></tr><tr><td> CNTF </td><td> R &amp; D Systems </td><td> 257-NY-010/CF </td><td> 10 ng / ml </td></tr><tr><td> GDNF </td><td> R &amp; D Systems </td><td> 212-GD-010/CF </td><td> 10 ng / ml </td></tr><tr><td> NT-3 </td><td> R &amp; D Systems </td><td> 267-N3-005/CF </td><td> 10 ng / ml </td></tr></tbody></table> NA = Nicht anwendbar. Tabelle 1. PMEF und Medien für MES-Zellkultur oder Differenzierung. <table border="1"><tbody><tr><td> &lt;strong&gt; Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Endkonzentration </td></tr><tr><td> 0,1% Gelatine </td><td> StemCell Technologies </td><td> 07903 </td><td> NA </td></tr><tr><td> Poly-DL-ornithin </td><td> Sigma-Aldrich </td><td> P0421 </td><td> 0,1 mg / ml in D 2 H 2 O </td></tr><tr><td> Mauslaminin </td><td> Millipore </td><td> CC095 </td><td> 2 pg / ml in PBS </td></tr><tr><td> 0,25% Trypsin / EDTA </td><td> StemCell Technologies </td><td> 07901 </td><td> NA </td></tr><tr><td> Accumax </td><td> Millipore </td><td> SCR006 </td><td> NA </td></tr></tbody></table> NA = Nicht anwendbar. Tabelle 2. Reagenzien für die Beschichtung und Dissoziation.

efficient differentiation of mouse embryonic stem cells into motor neurons

Direkte Differenzierung von embryonalen Stammzellen (ES)-Zellen in funktionelle Motoneuronen stellt eine viel versprechende Quelle für Krankheiten Mechanismen zu studieren, zu neuer Wirkstoffe zu screenen und zu neuen Therapien für Motoneuronen-Krankheiten wie zum Beispiel Spinale Muskelatrophie (SMA) und Amyotrophe Lateralsklerose entwickeln ( ALS). Viele aktuelle Protokolle verwenden eine Kombination von Retinsäure (RA) und Sonic Hedgehog (Shh) an embryonalen Stammzellen der Maus (mES) Zellen in Motoneuronen 4.1 differenzieren. Allerdings ist die Effizienz der Differenzierung mES Zellen in Motoneuronen nur mit mäßigem Erfolg erfüllt. Wir haben eine zweistufige Differenzierung Protokoll Nr. 5, das verbessert die Differenzierung Effizienz im Vergleich zu derzeit etablierten Protokollen entwickelt. Der erste Schritt besteht darin, die neuralization durch Zugabe von Noggin und Fibroblasten-Wachstumsfaktoren (FGFs) erweitern. Noggin ist ein knochenmorphogenetisches Protein (BMP)-Antagonisten und in Neuralinduktion gebracht einach der Standard-Modell der Neurogenese und führt zur Bildung der vorderen neuronalen Strukturieren 6. FGF wirkt synergistisch mit Noggin bei der Induktion von Nervengewebe Bildung durch die Förderung einer hinteren neuronalen Identität 9.7. In diesem Schritt wurden mES Zellen mit Noggin, bFGF und FGF-8 für zwei Tage grundiert werden, um neuronale Differenzierung in Richtung Linien zu präsentieren. Der zweite Schritt besteht darin, Motoneuronen-Spezifikation zu induzieren. Noggin / FGFs ausgesetzt mES Zellen wurden mit RA und einer Shh-Agonisten, Agonisten Smoothened (SAG), für weitere 5 Tage inkubiert, um Motoneuronen Generation zu erleichtern. Um die Differenzierung von MES in die Motor-Neuronen zu beobachten, verwendeten wir eine ES-Zelllinie von einer transgenen Maus exprimiert eGFP unter der Kontrolle des Promotors Motoneuronen Hb9 1 abgeleitet. Mit diesem robusten Protokoll erzielten wir 51 ± 0,8% der Differenzierung Effizienz (n = 3, p &lt;0,01, Student t-Test) 5. Die Ergebnisse der ImmunfluoreszenzFärbung zeigte, dass GFP +-Zellen die Motoneuronen spezifische Marker, Islet-1 und Cholin Acetyltransferase (ChAT) auszudrücken. Unsere Zwei-Schritt-Protokoll Differenzierung bietet eine effiziente Möglichkeit an MES-Zellen in spinalen motorischen Neuronen zu differenzieren.

Watch this Scientific Journal Video about Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons at JoVE.com

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Wir entwickelten ein neues Protokoll, um die Effizienz der in vitro Differenzierung von embryonalen Stammzellen der Maus in Motoneuronen zu verbessern. Die differenzierten ES-Zellen erworbenen motorischen Neuronen verfügt wie durch die Expression von neuronalen und motorischen Neurons Marker mit Hilfe von immunhistochemischen Techniken nachgewiesen.

Effiziente Differenzierung von embryonalen Stammzellen der Maus in Motoneuronen

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

efficient-differentiation-mouse-embryonic-stem-cells-into-motor

Universidad de Cartagena UDEC

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JoVE Journal

Biology

22.2K Aufrufe.  Alfred I. duPont Hospital for Children. Wir entwickelten ein neues Protokoll, um die Effizienz der in vitro Differenzierung von embryonalen Stammzellen der Maus in Motoneuronen zu verbessern. Die differenzierten ES-Zellen erworbenen motorischen Neuronen verfügt wie durch die Expression von neuronalen und motorischen Neurons Marker mit Hilfe von immunhistochemischen Techniken nachgewiesen.

Serie: Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Vermehrung von humanen embryonalen Stammzellen (ES-Zellen)

Forschung

Biologie

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Gewinnung menschlicher embryonaler Stammzellen durch Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Chromatin Immunopräzipitation von humanen embryonalen Stammzellen

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

In-vitro-Differenzierung von embryonalen Stammzellen der Maus (mES) Zellen unter Verwendung des Hanging-Drop-Methode

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolation und Ableitung von embryonalen Maus-Keimzellen

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Differenzierung von embryonalen Stammzellen in Oligodendrozyten Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...