Este trabajo fue apoyado en parte por subvenciones de WD de los Institutos Nacionales de Salud (SR1 NS059043 y ES015988 SR1), la Sociedad Nacional de Esclerosis Múltiple, Fundación Roche para la Investigación de la Anemia, Feldstein Medical Foundation, y los Hospitales Shriners para niños. Nos gustaría agradecer al Dr. David Placer y el plano de Jennifer sugerencias. Declaramos que no hay intereses contrapuestos en relación con este artículo.

Los procedimientos detallados de la generación de oligodendrocitos precursor (OPC) de la GFP-Olig2 células madre embrionarias de ratón (mESCs): <ol><li> Línea de células ES de ratón GFP-Olig2 (G-Olig2), se compra a la American Type Culture Collection (ATCC). El mESCs son rutinariamente pasajes cada 3 días sobre una capa de ratón irradiados alimentador de fibroblastos embrionarios (MEF). Las células MEF se colocan en el 0,1% de gelatina con cubierta de seis pocillos de cultivo de tejidos por lo menos un día antes de los pases de la CES. El medio de cultivo MESC es Dulbecco modificado de Eagle (DMEM) (Gibco) suplementado con 20% de suero fetal bovino (GIBCO), 2 mM L-glutamina, 1 mM piruvato de sodio, 0,1 mM mercaptoetanol β-/, 1% aminoácidos no esenciales ( AANE), y 1.000 U / ml de factor inhibidor de la leucemia. </li><li> colonias se MES trypsinized (TrypLE, 5 min, 37 ° C) en las células individuales y en suspensión en octavos de final de reemplazo de suero (KSR) medio, entonces las células se transfieren a un Costar muy bajo apego de seis pocillos (Corning) en una celda densidad de 50.000 células / cm 2. En estas condiciones, mESCs formar cuerpos embrioides (EBS). Medio KSR consta de medio esencial mínimo (MEM-), complementado con un 20% KSR, 1 mM piruvato de sodio, 1% AANE, y 0,1 mM β-mercaptoetanol /. </li><li> Del día 4 al día 7, el ácido retinoico (RA, 0,2 M) y purmorphamine (Pur, 1 M) se incluyen, como se muestra en la figura. 1A en KSR o medio N2. El medio es N2-MEM con suplemento 1X N2, 1 mM piruvato sódico, 1% AANE, y 0,1 mM β-mercaptoetanol /. El medio se cambia todos los días. </li><li> En el día 8, EBS se desglosan con TrypLE (5 minutos, 37 º C) y se colocaron en poliornitina recubierto 0,01% los platos en medio de OPC, que consiste en medio N2 y factor de crecimiento fibroblástico-2 (FGF-2, 20 ng / ml). El medio se cambia cada dos días. </li><li> Las células se trypsinized (TrypLE, 3 min, 37 ° C) y replated aproximadamente cada semana, cuando se hacen confluentes. El día 30, más del 80% de las células muestran GFP/Olig2 expresión y NG2 positivo, característico de los OPC. </li><li> Para generar los OPC remate, el Na + BacMam canal kit se utiliza para introducir Na v 1.2 subunidad en estos OPC MESC derivados. El reactivo BacMam (1 ml, el componente A) se mezcla con PBS hasta un volumen final de 5 ml. Luego, el reactivo BacMam mezclado se añade en MESC derivados de la cultura OPC en una placa de cultivo de 60 mm (en la confluencia% 50-70) antes de lavar con PBS. Después de 2 horas de incubación, el reactivo BacMam mixto es eliminado y reemplazado con medio suplementado con 1X OPC potenciador. El potenciador es el componente B reconstituido en DMSO (componente C). A continuación, después de más de 2 horas de incubación con el medio potenciador es eliminado y reemplazado con medio completo OPC. Las células se analizaron 24 horas más tarde. </li></ol> Los resultados representativos: Siguiendo el protocolo de diferenciación de la figura. 1A, G-Olig2 mESCs fueron inicialmente suspendidos en un medio de KSR y durante 4 días para formar cuerpos embrioides (EBs) (Fig. 1B). Luego, sucesivamente tratados de la EBS con AR y Pur. El EBS no mostró fluorescencia de GFP en el Día 4 y expresó una fuerte fluorescencia de GFP en D8 (Fig. 1B). En este punto del tiempo, el EBS se trypsinized y chapada en medio N2 complementado con factores de crecimiento FGF-2. Después de la cultura más de 22 días (D30), el porcentaje de células GFP + llegó a 80,3 ± 0,6%, según los resultados de citometría de flujo. Como se muestra en la figura. 1C, GFP + células se superponen con NG2 coloración consistente (96,4 ± 1,3% de las células GFP positivos fueron positivos NG2, y 94,8 ± 1,5% de NG2 células positivas fueron positivas GFP). Por lo tanto, estas células expresan GFP/Olig2 y NG2, definiéndolos como OPC. El OPC MESC derivados (76 células) no disparó los potenciales de acción en la despolarización (Fig. 2A). Después de transducción con un baculovirus que lleva el Na v 1.2 subunidad, estas OPCs MESC derivados (94,1%, 16 de 17 células) pueden ser clasificados como picos (Fig. 2B). <img alt="Figura 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Figura 1. La diferenciación de GOlig-MESC en OPC. (A) Esquema que muestra el protocolo del cuerpo embrioides (EB) y basado en moléculas pequeñas impulsada por la diferenciación. En D8, el EBS se desagregaron y plateado. Las células fueron pasadas una vez por semana cuando se convirtió en confluente. (B) D8, pero no D4 EBS, mostró la expresión de GFP. (C) La inmunotinción de NG2 (rojo) fue consistente con las buenas prácticas agrarias (verde) en la expresión D30. DAPI (azul) se utilizó para identificar los núcleos. Barra de escala: 20 mm. <img alt="Figura 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Figura 2. Generación de OPC picos de nonspiking MESC derivados de OPC por el virus mediada por Na v canal de expresión. (A) El potencial de membrana se llevó a cabo a -60 mV y OPC MESC derivados de no despedir a los potenciales de acción, incluso cuando la célula se despolariza a 0 mV. (B) Un ejemplo de MESC derivados de OPCdisparar potenciales de acción (en rojo) después de la transducción viral.

<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>

Las células madre embrionarias (CME), aisladas de un embrión blastocisto, pueden diferenciarse en todos los linajes de las células del organismo 3, 4, proporcionando un sistema en el modelo in vitro para el estudio de desarrollo de los mamíferos tempranos, incluyendo la especificación de oligodendrocitos. mESCs se ha demostrado que se diferencian en células precursoras de oligodendrocitos (OPC) con el tratamiento de sonic hedgehog (Shh) 5. Por otra parte, la diferenciación inducida por Shh OPC de mESCs mantiene la sincronización correcta observado en el desarrollo embrionario 6. Por lo tanto, la naturaleza de la diferenciación in vitro de OPC mESCs se considera que es consistente con lo que se ha aprendido en el desarrollo in vivo. En este caso, utilizando el pequeño RA moléculas y el Pur agonista Shh, hemos podido diferenciar el mESCs G-Olig2 en GFP + + Olig2 NG2 + OPC con alta eficiencia. OPC, que se caracteriza por la expresión de los proteoglicanos NG2 7 y el factor de transcripción hélice-lazo-hélice Olig2 8, generar oligodendrocitos en el SNC en desarrollo y madurez, donde representan un porcentaje importante (~ 5%) del total de células y son las principal tipo de células proliferantes 9. Durante casi dos décadas los investigadores han demostrado los canales de Na V se expresan en una subpoblación de los OPC y se puede activar a la despolarización de 10, 11. Por otra parte, una reciente observación sorprendente fue que en nueve en el SNC de rata in situ la materia blanca, los OPC (~ 50%) generaron los potenciales de acción cuando se despolariza dependiendo de la expresión del voltaje de sodio (Na V) canales, y por lo tanto podría subdividirse en remate y nonspiking subpoblaciones. Sin embargo, las funciones de estas propiedades de picos aún no se conocen. Hemos encontrado que, electrofisiológica, la OPC MESC derivados diferenciados con el protocolo de Shh-dependiente, no eran las mismas que las OPC en el cerebro in situ. Después de la introducción de la subunidad Na V 1.2, estos silenciosos MESC derivados de los OPC eran capaces de clavar. Por lo tanto, la adición / nonspiking MESC derivados de los OPC y protocolo de diferenciación descrito aquí puede facilitar (1) el estudio de las diferencias funcionales entre los picos y nonspiking OPC, (2) los factores de selección nueva que podría promover la expresión de los canales de sodio en el OPC MESC derivados, ( 3) el desarrollo y la optimización del protocolo de diferenciación de los OPC de la ESC humanos o células madre pluripotentes inducidas (CMPi).

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<td>MEFs</td>
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<td>GlobalStem </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>
<td>10009634</td>
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<td>&nbsp;</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

Los oligodendrocitos son las células myelinating del sistema nervioso central. Para la terapia celular regenerativa en enfermedades desmielinizantes, existe un interés significativo en la obtención de una población pura de linaje comprometido precursores de oligodendrocitos (OPC) para el trasplante. OPC se caracterizan por la actividad de la Olig2 factor de transcripción y expresión en la superficie de un proteoglicano NG2. Uso de la GFP-Olig2 (G-Olig2) de ratón con células madre embrionarias (MESC) línea de reportero, que optimiza las condiciones para la diferenciación de las buenas prácticas agrarias en mESCs + + Olig2 NG2 + OPC. En nuestro protocolo, lo primero que describe la generación de cuerpos embrioides (EBS) de mESCs. En segundo lugar, se describe el tratamiento de la MESC derivados de EBS con pequeñas moléculas: (1) el ácido retinoico (RA) y (2) un erizo sónico (Shh) purmorphamine agonista (Pur) bajo condiciones de cultivo definido para dirigir la diferenciación EB en el linaje oligodendroglial. Con este enfoque, los OPC se puede obtener con una alta eficiencia (&gt; 80%) en un plazo de 30 días. Las células derivadas de mESCs en este protocolo son fenotípicamente similares a los OPC derivadas del cultivo de tejidos primarios. El OPC MESC derivados no muestran la propiedad de adición se describe de una subpoblación de OPC cerebro in situ. Para el estudio de esta propiedad electrofisiológicos, se describe la generación de picos MESC derivados de OPC por ectópica expresando Na<sub&gt; V</sub&gt; 1.2 subunidad. Las células enriquecidas y nonspiking obtenidos a partir de este protocolo ayudará a avanzar los estudios funcionales en las dos subpoblaciones de 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

Se describe una molécula basada en pequeñas protocolo para la diferenciación de las células madre embrionarias de ratón en células precursoras de oligodendrocitos (OPC). Este protocolo genera Olig2 + NG2 + OPC con alta eficiencia de 30 días de diferenciación. También describe un método para generar &quot;picos&quot; OPC que puede disparar potenciales de acción.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

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

Nanyang Technological University

Research

JoVE Journal

Biology

15.3K vistas.  School of Medicine, University of California, Davis. Se describe una molécula basada en pequeñas protocolo para la diferenciación de las células madre embrionarias de ratón en células precursoras de oligodendrocitos (OPC). Este protocolo genera Olig2 + NG2 + OPC con alta eficiencia de 30 días de diferenciación. También describe un método para generar "picos" OPC que puede disparar potenciales de acción.

Video: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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.

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

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 de ratón con células madre neurales Precursores

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.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

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.

Derivación de células estaminales embrionarias humanas por 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.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

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.

En la diferenciación in vitro de la madre embrionarias de ratón (MES), las células con el método de gota

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 ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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.

El aislamiento y la derivación de células embrionarias de ratón Germinal

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

El destino de la asignación de células estaminales embrionarias humanas mediante la formación de teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

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.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas