Questo lavoro è stato in parte sostenuto dalle concessioni a WD dal National Institutes of Health (RO1 NS059043 e RO1 ES015988), la National Multiple Sclerosis Society, la Roche Fondazione per la ricerca anemia, Feldstein Medical Foundation, e la Shriners Hospitals for Children. Vorremmo ringraziare il Dr. Pleasure David e Jennifer aereo per i suggerimenti. Si dichiara non interessi contrastanti relativi a questo articolo.

Le procedure dettagliate di generare cellule precursori degli oligodendrociti (OPC) da GFP-Olig2 cellule staminali embrionali di topo (mESCs): <ol><li> Topo linea di cellule ES GFP-Olig2 (G-Olig2), viene acquistato dalla American Type Culture Collection (ATCC). Il mESCs sono diversi passaggi di routine ogni 3 giorni su un irradiato embrionale fibroblasti del mouse (MEF) strato alimentatore. Le cellule MEF sono placcati in 0,1% di gelatina rivestite sei pozzetti coltura tissutale almeno un giorno prima passaging il CES. Il terreno di coltura Mesc è medio Dulbecco modificato Eagle (DMEM) (GIBCO) integrato con il 20% fetale di siero bovino (GIBCO), 2 mM L-glutamina, 1 piruvato di sodio mM, 0,1 mM β-mercaptoetanolo /, 1% di acidi essenziali aminoacidi ( NEAA), e 1.000 U / ml fattore inibitorio leucemia. </li><li> colonie MES sono trypsinized (TrypLE, 5 min, 37 ° C) in celle singole e sospese nel siero sostituzione ad eliminazione diretta (KSR) medio, poi le cellule sono trasferiti ad un ultra-low attaccamento Costar sei pozzetti (Corning) in una cella densità di 50.000 cellule / cm 2. In queste condizioni, mESCs formare corpi embrionali (EBS). Medio KSR è costituito da-minimal mezzo fondamentale (-MEM) arricchito con 20% KSR, 1 mM sodio piruvato, 1% NEAA, e 0,1 mM β-mercaptoetanolo /. </li><li> Dal giorno 4 al giorno 7, l&#39;acido retinoico (RA, 0,2 M) e purmorphamine (Pur, 1 M) sono inclusi come mostrato in fig. 1A in KSR o medio N2. Il mezzo è N2-MEM contenente supplemento N2 1X, 1 mM di piruvato di sodio, 1% NEAA, e 0,1 mM β-mercaptoetanolo /. Il mezzo è cambiato ogni giorno. </li><li> Al giorno 8, EBS sono disaggregati utilizzando TrypLE (5 minuti, 37 ° C) e placcato su 0,01% polyornithine rivestite piatti medio OPC che consiste di media N2 e di crescita dei fibroblasti fattore-2 (FGF-2, 20 ng / ml). Il mezzo viene cambiata ogni due giorni. </li><li> Le cellule sono trypsinized (TrypLE, 3 min, 37 ° C) e saltuariamente circa ogni settimana quando diventano confluenti. Al giorno 30, oltre l&#39;80% delle cellule mostrano GFP/Olig2 espressione e sono NG2-positivi, caratteristici OPC. </li><li> Per generare OPC spiking, il Na + canale BacMam kit viene utilizzato per introdurre Na v 1.2 subunità in questi Mesc derivati ​​OPC. Il reagente BacMam (1 ml, componente A) viene mescolato con PBS per un volume finale di 5 ml. Poi, il reagente BacMam mista è aggiunto in Mesc derivati ​​cultura OPC in un piatto di cultura 60 mm (a 50-70 confluenza%) prima di risciacquo con PBS. Dopo 2 ore di incubazione, il reagente BacMam misto viene rimosso e sostituito con la media OPC integrato con 1X enhancer. Il potenziatore è il componente B ricostituito in DMSO (componente C). Poi, dopo ulteriori 2-ore di incubazione il mezzo con potenziatore viene rimosso e sostituito con il completo supporto OPC. Le cellule sono stati analizzati 24 ore più tardi. </li></ol> Rappresentante dei risultati: Seguendo il protocollo differenziazione mostrato in fig. 1A, G-Olig2 mESCs sono stati inizialmente sospesi in media KSR e per 4 giorni per formare corpi embrionali (EBS) (Fig. 1B). Poi, in sequenza trattati EBS con RA e Pur. L&#39;EBS non ha mostrato alcuna fluorescenza GFP al giorno 4 ed ha espresso una forte fluorescenza GFP a D8 (Fig. 1B). A questo punto il tempo, l&#39;EBS sono stati trypsinized e placcato in media N2 integrato con fattori di crescita FGF-2. Dopo la cultura ulteriore per 22 giorni (D30), la percentuale di cellule GFP + raggiunto 80,3 ± 0,6% secondo i risultati di citometria a flusso nostra. Come mostrato in fig. 1C, GFP + cellule sovrapposte con NG2 colorazione costantemente (96,4 ± 1,3% delle cellule GFP positive sono state positive NG2, e 94,8 ± 1,5% del NG2 cellule GFP positive sono state positive). Quindi, queste cellule esprimono GFP/Olig2 e NG2, definendoli come OPC. Il Mesc derivati ​​OPC (76 celle) non è riuscito a fuoco i potenziali d&#39;azione sulla depolarizzazione (Fig. 2A). Dopo trasduzione con un baculovirus porta la Na v 1.2 subunità, questi Mesc derivati ​​OPC (94,1,% 16 di 17 celle) può essere classificato come spiking (Fig. 2B). <img alt="Figura 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Figura 1. Differenziazione dei GOlig-Mesc in OPC. (A) Schema che mostra il protocollo del corpo embrionali (EB)-based e piccole molecole-driven differenziazione. Al D8, l&#39;EBS sono stati disaggregati e placcato. Le cellule sono state diversi passaggi una volta a settimana quando sono diventati confluenti. (B) D8, ma non D4 EBS, ha mostrato un&#39;espressione GFP. (C) immunocolorazione di NG2 (rosso) è stato coerente con GFP (verde) espressione a D30. DAPI (blu) è stato utilizzato per identificare i nuclei. Scala grafica: 20 mm. <img alt="Figura 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Figura 2. Generazione di OPC spiking da nonspiking Mesc derivati ​​OPC da virus-mediata Na v espressione canale. (A) Il potenziale di membrana si è tenuta a -60 mV e Mesc derivati ​​OPC non è riuscito a fuoco potenziali d&#39;azione, anche quando la cella è stata depolarizzata a 0 mV. (B) Un esempio di Mesc derivati ​​OPCcottura potenziale d&#39;azione (evidenziati in rosso) dopo la trasduzione virale.

<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>

Nessun conflitto di interessi dichiarati.

Le cellule staminali embrionali (ESC), isolate da un embrione blastocisti, possono differenziarsi in tutte le linee cellulari dell&#39;organismo 3, 4, fornendo un sistema di modello in vitro per lo studio precoce sviluppo dei mammiferi, compresa la specificazione degli oligodendrociti. mESCs hanno dimostrato di differenziarsi in cellule precursori degli oligodendrociti (OPC) con il trattamento di Sonic hedgehog (Shh) 5. Inoltre, il Shh differenziazione, indotta dal mESCs OPC conserva i tempi corretti osservata nello sviluppo embrionale 6. Quindi, la natura del differenziamento in vitro OPC da mESCs è considerato coerente con quanto si è appreso dal vivo in fase di sviluppo. Qui, utilizzando il piccolo molecole RA e l&#39;Pur agonista Shh, abbiamo differenziato con successo il G-Olig2 mESCs in Olig2 GFP + + + NG2 OPC con alta efficienza. OPC, caratterizzato dall&#39;espressione di proteoglicani NG2 7 e l&#39;elica-loop-elica fattore di trascrizione Olig2 8, generare oligodendrociti nel sistema nervoso centrale sviluppo e maturo, dove essi costituiscono una percentuale significativa (~ 5%) delle cellule totali e sono la principale tipo di cellule proliferanti 9. Per quasi due decenni, i ricercatori hanno dimostrato Na canali V sono espressi in una sottopopolazione di OPC e può essere attivato su depolarizzazione 10, 11. Inoltre, una recente osservazione sorprendente 9 è stato che nel sistema nervoso centrale di ratto in situ della sostanza bianca, OPC (~ 50%) ha generato un potenziale d&#39;azione quando depolarizzata seconda l&#39;espressione del sodio voltaggio-dipendenti (Na V) canali, e quindi potrebbe essere suddiviso in spiking e nonspiking sottopopolazioni. Tuttavia, le funzioni di queste proprietà spiking sono ancora largamente sconosciuti. Abbiamo scoperto che, elettrofisiologico, la Mesc derivati ​​OPC differenziato con il protocollo di Shh-dipendente, non erano la stessa in OPC cervello in situ. Dopo aver introdotto subunità Na V 1.2, questi silenziosi Mesc derivati ​​OPC sono state in grado di spike. Così, la spiking / nonspiking Mesc derivati ​​OPC e il protocollo di differenziazione descritto qui può facilitare (1) studiando le differenze funzionali tra spike e nonspiking OPC, (2) fattori di screening in grado di promuovere il canale di espressione di sodio nel Mesc derivati ​​OPC, ( 3) sviluppare e ottimizzare il protocollo di differenziazione delle OPC da ESC umane o cellule staminali pluripotenti indotte (iPSCs).

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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
<td>&nbsp;</td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
<td>&nbsp;</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
<td>&nbsp;</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
<td>&nbsp;</td>
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<td>FGF-2</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>GF003</td>
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<td>BacMam NaV 1.2</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrociti sono cellule mielinizzanti del sistema nervoso centrale. Per la terapia cellulare rigenerativa nelle malattie demielinizzanti, c&#39;è notevole interesse nel derivare una popolazione di stirpe pura-committed cellule precursori degli oligodendrociti (OPC) per il trapianto. OPC si distinguono per l&#39;attività del Olig2 fattore di trascrizione e l&#39;espressione superficiale di un NG2 proteoglicani. Utilizzando la GFP-Olig2 (G-Olig2) topo con cellule staminali embrionali (Mesc) linea reporter, abbiamo ottimizzato le condizioni per la differenziazione dei mESCs in Olig2 GFP + + + NG2 OPC. Nel nostro protocollo, per prima cosa descrivere la generazione dei corpi embrionali (EBS) da mESCs. In secondo luogo, si descrive il trattamento di Mesc derivati ​​EBS con piccole molecole: (1) acido retinoico (RA) e (2) un Sonic hedgehog (Shh) purmorphamine agonista (Pur) in condizioni cultura definita per dirigere la differenziazione EB nel lignaggio oligodendrogliali. Con questo approccio, OPC può essere ottenuto con alta efficienza (&gt; 80%) in un periodo di tempo di 30 giorni. Cellule derivate da mESCs in questo protocollo sono fenotipicamente simile a OPC derivati ​​da colture cellulari primarie. Il Mesc derivati ​​OPC non mostrano la proprietà spiking descritto per una sottopopolazione di OPC cervello in situ. Per studiare questa proprietà elettrofisiologiche, si descrive la generazione di spiking Mesc derivati ​​OPC da ectopica esprimendo Na<sub&gt; V</sub&gt; 1,2 subunità. Le cellule spiking e nonspiking ottenuti da questo protocollo contribuirà a studi funzionali anticipo sui due sottopopolazioni di OPC.

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

Descriviamo una piccola molecola a base di protocollo per la differenziazione delle cellule staminali embrionali di topo in cellule precursori degli oligodendrociti (OPC). Questo protocollo genera Olig2 + NG2 + OPC ad alta efficienza da 30 giorni di differenziazione. Abbiamo anche descrivere un metodo per generare &quot;chiodare&quot; OPC che può sparare potenziali d&#39;azione.

Differenziazione delle cellule staminali embrionali in precursori degli oligodendrociti

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 Views.  School of Medicine, University of California, Davis. Descriviamo una piccola molecola a base di protocollo per la differenziazione delle cellule staminali embrionali di topo in cellule precursori degli oligodendrociti (OPC). Questo protocollo genera Olig2 + NG2 + OPC ad alta efficienza da 30 giorni di differenziazione. Abbiamo anche descrivere un metodo per generare "chiodare" OPC che può sparare potenziali d'azione.

Video: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagazione di staminali embrionali umane (ES) Cellule

Ricerca

Biologia

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.

Cultura di cellule precursori staminali neurali del mouse

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.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

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.

Derivazione di cellule staminali embrionali umane da 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.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

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.

La differenziazione in vitro di staminali embrionali di topo (MES) Cellule con il metodo goccia

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.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

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.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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.

Differenziazione efficiente di cellule staminali embrionali di topo in neuroni motori

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.

Differenziazione del neonato mouse pelle cellule staminali derivate in cellule germinali-simili<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 ...