Diese Arbeit wurde teilweise durch Zuschüsse zu WD von National Institutes of Health (RO1 NS059043 und RO1 ES015988), National Multiple Sclerosis Society, Roche Foundation for Anämie Research, Feldstein Medical Foundation und der Shriners Hospitals for Children unterstützt. Wir möchten Dr. David Pleasure und Jennifer Flugzeug für Anregungen bedanken. Wir erklären, keine konkurrierenden Interessen im Zusammenhang mit diesem Artikel.

Detaillierte Verfahren zur Erzeugung Oligodendrozyten-Vorläuferzellen (OPC) aus GFP-Olig2 embryonalen Stammzellen der Maus (mESCs): <ol><li> Maus ES-Zelllinie GFP-Olig2 (G-Olig2), wird von der American Type Culture Collection (ATCC) bezogen. Die mESCs werden routinemäßig alle 3 Tage passagiert auf einer bestrahlten Maus embryonale Fibroblasten (MEF) Feeder-Schicht. Die MEF-Zellen werden in 0,1% Gelatine beschichtete sechs gut Gewebekulturplatte mindestens einen Tag vor der Passage der WSR überzogen. Die MESC Kulturmedium Dulbecco modifiziertem Eagle-Medium (DMEM) (Gibco) mit 20% fötalem Rinderserum (GIBCO), 2 mM L-Glutamin, 1 mM Natriumpyruvat, 0,1 mM β-Mercaptoethanol /, 1% nicht-essentiellen Aminosäuren ergänzt ( NEAA), und 1.000 U / ml Leukämie hemmender Faktor. </li><li> mES Kolonien trypsiniert (TrypLE, 5 min, 37 ° C) in einzelne Zellen, suspendiert in Knockout-Serum-Ersatz (KSR) Medium, dann werden die Zellen zu einer Costar Ultra-Low-Anlage Sechs-Well-Platte (Corning) in einer Zelle übertragen Dichte von 50.000 Zellen / cm 2. Unter diesen Bedingungen bilden mESCs Embryoidkörpern (EBS). KSR Medium besteht aus-minimal essential medium (-MEM) mit 20% KSR, 1 mM Natriumpyruvat, 1% NEAA und 0,1 mM β-Mercaptoethanol / ergänzt. </li><li> Von Tag 4 bis Tag 7, Retinsäure (RA, 0,2 M) und purmorphamine (Pur, 1 M) enthalten, wie in Abb. gezeigt. 1A in KSR oder N2 Medium. Die N2 Medium-MEM mit 1X N2 zu ergänzen, 1 mM Natriumpyruvat, 1% NEAA und 0,1 mM β-Mercaptoethanol /. Das Medium wird täglich gewechselt. </li><li> Am Tag 8, EBs aufgeschlüsselte mit TrypLE (5min, 37 ° C) und vergoldet auf 0,01% Polyornithin-beschichtete Platten in OPC Medium, das von N2 mittel-und Fibroblasten besteht growth factor-2 (FGF-2, 20 ng / ml). Das Medium wird alle zwei Tage gewechselt. </li><li> Die Zellen werden trypsiniert (TrypLE, 3 min, 37 ° C) und replattiert etwa jede Woche, wenn sie konfluent geworden. Am Tag 30, zeigen mehr als 80% der Zellen GFP/Olig2 Ausdruck und sind NG2-positive, die charakteristisch für OPCs. </li><li> Um spiking OPCs zu generieren, die BacMam Na +-Kanal-Kit wird verwendet, um Na v 1.2-Untereinheit in diese MESC-derived OPCs einzuführen. Die BacMam Reagenz (1 ml, Komponente A) wird mit PBS auf ein Gesamtvolumen von 5 ml gemischt. Dann wird die gemischte BacMam Reagenz in MESC-derived OPCs Kultur in einer 60 mm Kulturschale gegeben (bei 50-70% Konfluenz) vor, mit PBS gespült. Nach 2 Stunden Inkubation wird die gemischte BacMam Reagenz entfernt und durch OPC Medium mit 1X-Enhancer ergänzt ersetzt. Der Verstärker ist die Komponente B rekonstituiert in DMSO (Komponente C). Als nächstes wird nach zusätzlichen 2-Stunden-Inkubation das Medium mit Enhancer wird entfernt und durch vollständige OPC-Medium ersetzt. Die Zellen werden 24 Stunden später untersucht. </li></ol> Repräsentative Ergebnisse: Nach der Differenzierung Protokoll in Abb.. 1A, G-Olig2 mESCs wurden zunächst in KSR Medium und für 4 Tage ausgesetzt, um Embryoidkörpern (EVG) (Abb. 1B) zu bilden. Dann haben wir nacheinander behandelt die EBs mit RA und Pur. Die EBs zeigten keine GFP-Fluoreszenz an Tag 4 und äußerte starke GFP-Fluoreszenz bei D8 (Abb. 1B). Zu diesem Zeitpunkt wurden die EBs trypsiniert und vernickelt in N2-Medium mit Wachstumsfaktoren FGF-2 ergänzt. Nach weiteren Kultur für 22 Tage (D30), erreichte der Anteil der GFP + Zellen 80,3 ± 0,6% nach unseren Durchflusszytometrie Ergebnisse. Wie in Abb.. 1C, GFP +-Zellen mit NG2 Färbung konsequent (96,4 ± 1,3% der GFP-positive Zellen wurden NG2 positiv, und 94,8 ± 1,5% vom NG2 positive Zellen wurden GFP positive) überlagert. So exprimieren diese Zellen GFP/Olig2 und NG2, definieren sie als OPC. Die MESC-derived OPCs (76 Zellen) nicht Aktionspotentiale auf Depolarisation (Abb. 2A) Feuer. Nach Transduktion mit einem Baculovirus Durchführung der Na v 1.2-Untereinheit, könnten diese MESC-derived OPCs (94,1%, 16 von 17 Zellen) als Spike (Abb. 2B) eingestuft werden. <img alt="Abbildung 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Abbildung 1. Differenzierung von GOlig-MESC in OPCs. (A) Schematische Darstellung des Protokolls der Embryoidkörper (EB)-basierte und Small Molecule-driven Differenzierung. Bei D8 wurden die EBs aufgeschlüsselt und vergoldet. Die Zellen wurden einmal pro Woche passagiert, wenn sie konfluent geworden. (B) D8, aber nicht D4 EBs, zeigte die GFP-Expression. (C) Immunfärbung von NG2 (rot) stand im Einklang mit GFP (grün) Expression auf D30. DAPI (blau) wurde verwendet, um die Kerne zu identifizieren. Maßstab: 20 mm. <img alt="Abbildung 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Abbildung 2. Generierung von spiking OPCs aus nonspiking MESC-derived OPCs durch Virus-vermittelten Na v-Kanal Ausdruck. (A) Die Membranpotential wurde auf -60 mV und MESC-derived OPCs hielt nicht Aktionspotentiale Feuer, auch wenn die Zelle depolarisiert auf 0 mV. (B) Ein Beispiel für MESC-derived OPCBrennen Aktionspotential (rot markiert) nach der viralen Transduktion.

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

Embryonale Stammzellen (ES-Zellen), aus einer Blastozyste Embryo isoliert, kann in allen Zelllinien des Organismus 3, 4 zu unterscheiden, bietet ein in vitro Modell für die Erforschung von frühen Entwicklung von Säugetieren, einschließlich Oligodendrozyten-Spezifikation. mESCs haben gezeigt, dass in Oligodendrozyten-Vorläuferzellen (OPC) mit der Behandlung von Sonic Hedgehog (SHH) 5 zu unterscheiden. Darüber hinaus behält sich das Shh-induzierten OPC Differenzierung von mESCs das richtige Timing bei der Embryonalentwicklung 6 beobachtet. Daher ist die Art der In-vitro-Differenzierung von OPC mESCs als im Einklang mit dem, was von in vivo Entwicklung gelernt. Hier mit dem kleinen Molekülen RA und die Shh-Agonisten Pur, haben wir erfolgreich die G-Olig2 mESCs in GFP + + Olig2 NG2 + OPCs mit hohem Wirkungsgrad unterschieden. OPCs, durch den Ausdruck der Proteoglykan NG2 7 und die Helix-Loop-Helix-Transkriptionsfaktor Olig2 8 gekennzeichnet, erzeugen Oligodendrozyten in den Entwicklungsländern und reifen ZNS, wo sie einen bedeutenden Prozentsatz (~ 5%) der gesamten Zellen und enthalten, sind die Haupt-proliferierenden Zelltyp 9. Seit fast zwei Jahrzehnten haben Forscher Na V-Kanäle gezeigt werden in einer Subpopulation von OPCs zum Ausdruck und kann auf Depolarisation 10, 11 aktiviert werden. Darüber hinaus wurde kürzlich bemerkenswerte Beobachtung 9, die in in situ Ratte CNS weißen Substanz, OPCs (~ 50%) Aktionspotenziale erzeugt, wenn depolarisiert abhängig von der Expression von spannungsabhängigen Natrium (Na V) Kanäle, und so konnte in spiking unterteilt werden und nonspiking Subpopulationen. Allerdings sind die Funktionen dieser spiking Eigenschaften noch weitgehend unbekannt. Wir fanden, dass, elektrophysiologisch, die MESC-derived OPCs mit dem Shh-abhängigen Protokoll differenzierte, nicht die gleiche wie die in situ Gehirn OPCs. Nach der Einführung Untereinheit Na V 1.2 wurden diese stille MESC-derived OPCs in der Lage spiking. So kann das Aufstocken / nonspiking MESC-derived OPCs und Differenzierung hier beschriebene Protokoll (1) Untersuchung der funktionellen Unterschiede zwischen Spike und nonspiking OPCs, (2) Screening neuer Faktoren, die Natrium-Kanal Ausdruck in der MESC-derived OPCs, fördern könnte zu erleichtern ( 3) Entwicklung und Optimierung der Differenzierung Protokoll der OPCs aus menschlichem ESC oder induzierten pluripotenten Stammzellen (IPSCs).

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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrozyten sind die myelinisierenden Zellen des zentralen Nervensystems. Für regenerative Zelltherapie in demyelinisierenden Erkrankungen, gibt es großes Interesse bei der Ableitung eines reinen Population von Lineage-committed Oligodendrozyten-Vorläuferzellen (OPC) für die Transplantation. OPCs werden durch die Aktivität des Transkriptionsfaktors Olig2 und Oberfläche Ausdruck eines Proteoglykan NG2 aus. Mit der GFP-Olig2 (G-Olig2) Maus embryonalen Stammzellen (MESC) Reporter Linie, optimierten wir die Bedingungen für die Differenzierung von mESCs in GFP + + Olig2 NG2 + OPCs. In unserem Protokoll, beschreiben wir zunächst die Generierung von Embryoidkörpern (EVG) aus mESCs. Zweitens beschreiben wir die Behandlung von MESC-derived EBs mit kleinen Molekülen: (1) Retinsäure (RA) und (2) ein Sonic Hedgehog (SHH)-Agonist purmorphamine (Pur) unter definierten Kulturbedingungen zu EB Differenzierung in die oligodendroglialen Linie zu lenken. Mit diesem Ansatz kann OPCs mit hohem Wirkungsgrad (&gt; 80%) in einem Zeitraum von 30 Tagen erreicht werden. Zellen aus mESCs in diesem Protokoll ergeben, sind phänotypisch ähnlich OPCs aus primärem Gewebe Kultur abgeleitet. Die MESC-derived OPCs zeigen nicht die Spikes-Eigenschaft für eine Subpopulation von Gehirn OPCs in situ beschrieben. Um dies zu untersuchen elektrophysiologische Eigenschaft beschreiben wir die Erzeugung von spiking MESC-derived OPCs von ektopisch auszudrücken Na<sub&gt; V</sub&gt; 1,2-Untereinheit. Der Spick und nonspiking Zellen aus diesem Protokoll erhalten wird vorab funktionelle Untersuchungen an den beiden Subpopulationen von OPCs zu helfen.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Wir beschreiben ein kleines Molekül-basiertes Protokoll zur Differenzierung von embryonalen Stammzellen der Maus in Oligodendrozyten-Vorläuferzellen (OPC). Dieses Protokoll erzeugt Olig2 + NG2 + OPCs mit hohem Wirkungsgrad von 30 Tagen nach Differenzierung. Wir beschreiben eine Methode, die auf &quot;Aufstocken&quot; OPCs, dass Aktionspotentiale Feuer erzeugen kann.

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 ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

Research

JoVE Journal

Biology

15.2K Aufrufe.  School of Medicine, University of California, Davis. Wir beschreiben ein kleines Molekül-basiertes Protokoll zur Differenzierung von embryonalen Stammzellen der Maus in Oligodendrozyten-Vorläuferzellen (OPC). Dieses Protokoll erzeugt Olig2 + NG2 + OPCs mit hohem Wirkungsgrad von 30 Tagen nach Differenzierung. Wir beschreiben eine Methode, die auf "Aufstocken" OPCs, dass Aktionspotentiale Feuer erzeugen kann.

Serie: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Vermehrung von humanen embryonalen Stammzellen (ES-Zellen)

Forschung

Biologie

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Kultur der Maus Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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 ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

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 ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Differenzierung von Newborn Mäusehaut Derived Stem Cells in Germ-like Cells<em&gt; In-vitro-</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...