Los autores están muy agradecidos de la Sra. Haiying Pan y el Sr. Ian Bellayr por su ayuda técnica en el aislamiento de células de las películas actuales, y el Sr. Kyle Holden y el Sr. Jonathan Lucas, por su ayuda en la edición de diapositivas. Muchas gracias a la siguiente lista de personas que han estado involucrados con el desarrollo de las técnicas preplate modificado en los últimos años: los Dres. Burhan Gharaibeh, Cao Baohong, Bridget Deasy, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, Kimimasa Tobita, Ron Jankowski, Makato Ikezawa. Estamos muy agradecidos por la asistencia técnica de James H. Cummins, Pruchnic Ryan, Feduska José, Rebecca C. Schugar, Pan y Haiying Tebbets Jessica. El autor también desea agradecer el apoyo financiero para este trabajo de la Asociación de Distrofia Muscular (MDA), los Institutos Nacionales de Salud (NIH), el Departamento de Defensa (DOD), el Hospital de Niños de Pittsburgh de UPMC, el Departamento de Cirugía Ortopédica y Traumatología, y la Universidad de Pittsburgh.

En general, la completa modificación del procedimiento preplate requiere 1-2 días de preparación, una semana de realizar el procedimiento técnico, de 2-3 días de la identificación de células con inmunocitoquímica, y un adicional de 2-3 semanas de la expansión de la población de células madre. El equipo necesario para el cultivo de células madre es similar a la de otros sistemas de cultivo celular que incluye un sobremesa centrífuga, incubadora de CO 2, campana de flujo laminar de cultivo de tejidos y un microscopio invertido equipado con fluorescencia y una cámara digital. Las células madre aisladas también se pueden clonar y se pueden almacenar a largo plazo en nitrógeno líquido para el 1,2 estudios actuales o futuros. Todos los reactivos y materiales utilizados para este procedimiento deben ser estériles y manipularse asépticamente. Paso 1: Preparación <ol><li> Colágeno capa placas de cultivo de tejidos y frascos: colágeno de tipo I derivado de piel de ternera (0,1 g en 1 litro, Sigma-Aldrich). Los recipientes de plástico para cubrir incluye: T-25 frascos, 6 o 12 pocillos, platos (60 y 100 mm, BD Falcon) como se describió anteriormente 1,2. Agite con cuidado el utensilios de plástico cada media hora, durante 4 horas para asegurar una capa de colágeno. La solución de colágeno se extrae. Y los utensilios de plástico recubierto luego se deja secar en la campana de cultivo de tejidos con la luz UV y el capó tanto, para asegurar la esterilidad y, finalmente, volver a tapar o cubrir para su uso posterior. </li><li> Preparar medio de cultivo celular: 400 ml DMEM (DMEM, la glucosa alta, Invitrogen), 50 ml inactivado por calor suero fetal bovino (FBS, Invitrogen), 50 ml de suero de caballo (HS, Invitrogen), y 5 ml de extracto de embrión del polluelo (CEE, precisa Chemical Co. ) se filtró al vacío con un disponible de 0,22 micras de 500 ml sistema de filtro estéril (Corning). Estéril a la penicilina / estreptomicina solución (Invitrogen) se añadirá finalmente de 100 unidades / ml. Las soluciones preparadas se pueden almacenar a 4 ° C durante al menos un mes. Esta solución se utiliza para separar las células del frasco cuando los cultivos de la división o la preparación de las células para el trasplante. </li><li> Calentamiento de soluciones para el aislamiento de células: Los siguientes reactivos deben ser calentado a 37 ° C antes de su uso para el aislamiento de células: buffer de Hank solución de sal (HBSS, Invitrogen), 0,2% (wt / vol) de colagenasa tipo XI (Sigma -Aldrich), con un promedio de 3.200 unidades de colágeno digestión por ml HBSS; Dispasa 2,4 units/1mL (Invitrogen), 0,5% (wt / vol) de tripsina (Invitrogen). </li><li> Esterilizar el equipo quirúrgico en la preparación: Procedimiento quirúrgico estéril requieren instrumentos quirúrgicos, incluyendo: diferentes tamaños pinzas, tijeras micro, tijeras iris y / u hojas de bisturí. Además, el 15 y 50 ml tubos de centrífuga de polipropileno (BD Falcon); células colador (tamaño de poro 70 m) (BD Falcon), 18 -, 23 - y 27 agujas de calibre (BD PrecisionGlide) y 10 - o 20 ml- jeringas estériles (BD). </li></ol> Paso 2: biopsias de tejidos, el aislamiento y la disociación mecánica 4.1 Las biopsias de los tejidos se llevan a cabo bajo una campana de cultivo estéril e incluyen recién cosechado los tendones y los músculos esqueléticos se obtienen de la tibia o el sóleo músculos de la extremidad posterior de ratones de tipo salvaje (Mujer C57BL/6J, 4-5 semanas de edad o más joven Jackson, de laboratorio). Cualquier tejido visible conectivo, vasos sanguíneos y el tejido adiposo se elimina de las muestras de biopsia. Los tejidos de los tendones y los músculos son cuidadosamente identificadas bajo un microscopio de disección quirúrgica para eliminar los restos de piel y hueso y se coloca en un recipiente lleno de frío (4 ° C) HBSS (complementado con añadir un 5% de FBS). Los tejidos aislados luego se coloca en los platos por separado colágeno recubierto contiene HBSS frío y se procederá al picado (&lt;1x1 mm 3) en una pasta gruesa con micro-tijeras y / u hojas de bisturí. Paso 3: La digestión enzimática de los tejidos: El tejido picado luego disociadas enzimáticamente a través de una serie de pasos 2,4,5 <ol><li> Transferencia de las suspensiones de tejido picado en distintos tubos de 15 ml y centrifugar a ~ 3500 rpm a 4 ° C durante 5 minutos. </li><li> Eliminar el sobrenadante, lavar con HBSS y repetir la centrifugación de nuevo. </li><li> Eliminar el sobrenadante, y digerir estos lodos mediante la adición de 10 ml de precalentado 0,2% de colagenasa tipo XI (Sigma). Incubar durante 60 minutos a 37 ° C, mientras que continuamente moviendo suavemente los tubos. Por otra parte, estrechar la mano cada 10 minutos. </li><li> Centrifugar y resuspender estos lodos en 10 ml dispasa (2,4 unidades / ml, Gibco) solución. Incubar durante 45 minutos a 37 ° C, agitando con las manos o mecerse suavemente cada 10 minutos. </li><li> Centrifugar y resuspender estos lodos en 10 ml de solución HBSS 0,2% de tripsina. De incubación puede variar en función de la medida en que las células solo se liberan de los tejidos que se pueden realizar mediante la observación de la suspensión bajo el microscopio. No se recomienda exceder de 30 minutos a 37 ° C, con inversión de los tubos o agitando suavemente cada 10 minutos. </li><li> Centrifugar la célula resultantes y vuelva a suspender el sedimento celular en 10 ml de la proliferación Medio (PM). </li><li> Disociar la suspensión de células al pasar el extracto a través de una serie de agujas. Las células se pasa dos veces a través de una 18G, 23G entonces, y, finalmente, una aguja 27G. </li><li> Pasar el extracto de células a través de un filtro de 70 micras de células. </li></ol> Paso 4: Preplating para aislar diferentes poblaciones de células basadas en células tasa de 3.1 adhesión <ol><li> Centrifugar y resuspender los sedimentos celulares en medio de proliferación. </li><li> Placa de mezclas de la célula en un colágeno recubiertas frasco T-25 (o 60 mm plato) y se marca como PP1. La suspensión celular debe ser observada como poseedores de un gran número de núcleos de refracción y otros restos bajo el microscopio. </li><li> Se incuba a 37 ° C en un humidificado 5% incubadora de CO 2 durante 2 horas. </li><li> La transferencia de células no adherentes a un nuevo colágeno recubiertas frasco T-25 (o 60 mm plato) y la marca es tan PP2. Añadir 5 ml de PM en la placa de la etiqueta PP1. Las primeras células que se unen a la adhesión del frasco son en su mayoría miofibroblastos 3. </li><li> Retorno de los matraces a la incubadora durante 24 horas más, la transferencia de células no adherentes a un nuevo colágeno recubiertas frasco T-25 (o plato) y marcarlo como PP3. Añadir 5 ml de medio de proliferación en la placa de la etiqueta PP2. Estas células que se adhieren, que se adhieren a este frasco son en su mayoría fibroblastos 2,3. </li><li> Volver frascos a la incubadora y repita el procedimiento en otras 24 horas hasta PP6 población se crea. Mantener a cada grupo de células en suspensión y permite que proliferen y examinar con regularidad utilizando un microscopio invertido. PP3-PP4 mioblastos son en su mayoría, mientras que las células musculares satélite a menudo se observa sobre todo en PP5 2. </li><li> La mayoría de las células poco a poco la adhesión típica, se adhieren al PP6. En esta etapa, las células parecen pequeñas, redondas de refracción, la luz y son muy escasos en número y son considerados como una célula madre 1,2 de la población. </li></ol> Paso 5: Identificación y expansión de las células madre aisladas <ol><li> El aislado PP6 células son muy pocos y deben ser mantenidos en los países ricos FBS medio de proliferación (20% de SFB en DMEM) que se cambia a diario. La mayoría de las células presentes en la matriz de la cultura PP6 durante las siguientes 1-2 semanas de cultivo, pero una gran y saludable PP6 población se crea normalmente después de un 1-2 semanas adicionales de expansión. Las células madre aisladas de los ratones altamente expresan el antígeno de células madre 1 (SCA1), CD34, hígado fetal kinasa 1 (Flk1), y otros marcadores 1,2,6,7 madre que puede ser visualizado a través de tinción inmunocitoquímica. Estas células madre también presentan múltiples capacidades de diferenciación 3,6,8. </li><li> Las células aisladas se mantienen y se expandió a una baja densidad celular en cultivo (de 12 y platos a 50-100 células / o confluencia &lt;20-30% en el frasco o plato) para evitar la diferenciación de 1,2. Muy pocas células forman los clones durante la expansión, y estas células madre clonadas pueden someterse a la proliferación de tiempo (LTP) en materia de estudios de otra competencia. Las células madre clonadas también se puede almacenar en nitrógeno líquido con los procedimientos generales de almacenamiento de células pe un PM con un 10% DMSO. Hacer una serie de poblaciones de paso bajo de las células madre de todos los estudios al otro hacia atrás. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

Se ha reconocido que algunos tejidos adultos hay células madre múltiples. En los últimos años de estudio, las células madre se han detectado en los tejidos blandos musculoesqueléticos, incluyendo el músculo esquelético y el tendón. Este protocolo actual de los detalles de un procedimiento, conocido como la técnica preplate modificado, que ha sido utilizado con éxito en nuestro laboratorio para aislar las células madre adultas de los tendones y los músculos esqueléticos. Utilizando la técnica de preplate m...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
<tr>
<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
<tr>
<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
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<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type XI 0.2%(wt/vol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C7657</td>
<td>&nbsp;</td>
<tr>
<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
<tr>
<td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>#D-2650</td>
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isolating stem cells from soft musculoskeletal tissues

Las células madre adultas han sido discutidos en lo que respecta a su aplicación en medicina regenerativa. Las células madre adultas han generado un gran entusiasmo para el tratamiento de los tejidos lesionados y enfermos debido a su impresionante capacidad de someterse a multi-linaje diferenciación de las células y su capacidad de auto-renovación. Más importante aún, estas cualidades han hecho ventajoso para su uso en terapias de células autólogas de trasplante. El protocolo actual se introduce a los lectores a la técnica preplate modificado en los tejidos blandos del aparato locomotor, por ejemplo, los tendones y los músculos, son un<sup&gt; St</sup&gt; Disociadas enzimáticamente y luego se colocan en frascos de colágeno recubiertas con un medio. El sobrenadante, que se compone de medio y las células restantes flotante, es de serie transferidos diariamente a nuevos frascos. Las células madre son los más lentos en cumplir con los frascos que se tarda generalmente 5-7 días (transferencias de serie o preplates). Mediante el uso de esta técnica, las células madre adultas presentes en estos tejidos pueden ser fácilmente recolectadas a través de bastante los procedimientos no invasivos.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

Aislar las células madre adultas de músculo esquelético tejidos blandos sobre la base de la velocidad de la adhesión de las células para matraz.

Aislamiento de células madre de tejidos blandos musculoesqueléticos

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Research

JoVE Journal

Biology

13.3K vistas.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. Aislar las células madre adultas de músculo esquelético tejidos blandos sobre la base de la velocidad de la adhesión de las células para matraz.

Video: Isolating Stem Cells from Soft Musculoskeletal Tissues

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 ...

Investigación

Biología

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivación de células madre de ratón a partir de blastocistos Trofoblasto

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

El aislamiento de células madre de cáncer de páncreas xenoinjertos

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Aislamiento de células madre de la retina del ojo del ratón

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selección y aislamiento de las colonias de humanos células madre pluripotentes inducidas a partir de fibroblastos adultos Reprogramado

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Generación de ratones derivados de células madre pluripotentes inducidas

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

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Aislar las células melanocitos como primarios del corazón alfombrillas

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

El aislamiento de células madre intestinales de Adultos<em&gt; Drosophila</em&gt; Midguts por FACS para estudiar el comportamiento de la célula de vástago durante el envejecimiento

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...