Este trabajo fue apoyado por la Fundación Alexander von Humboldt (5090371), el Centro de Kühne Christine para alérgicos Investigación y Educación (CK-CARE), y el Bayerischen Staatsministerium (para KD).

1. Aislamiento SKP de cultivos de fibroblastos primarios <ol><li> Cultivos de fibroblastos ya sea de bancos de células o directamente obtenido a partir de biopsias de la piel se mantienen en cultivo en un medio de crecimiento de fibroblastos DMEM que contenía 15% de suero de ternera fetal, 2 mM de glutamina, 10 mg / ml de penicilina, y 10 mg / ml de estreptomicina. </li></ol> Fibroblastos humanos GMO3349C y GMO8398A se obtuvieron del Instituto Coriell de Investigación Médica (Camden, Nueva Jersey) y se utilizaron en este estudio. <ol start="2"><li> Los cultivos de duplicaciones de la población (PPD) de 20 a 35 se utilizaron para culturas SKP a una confluencia de 80%. Un 10 cm placa de cultivo de tejidos (BD Falcon) contiene aproximadamente 1.5x10 6 células. </li><li> Lavar las células con PBS y se incuba con 2 ml de solución de tripsina (0,25%, Invitrogen) durante 1 hora a 37 ° C. </li><li> Recoger las células de la placa con 5 ml de PBS y transferir la suspensión celular a un tubo de 15 ml halcón. </li><li> Se incuban las células a 4 º C durante 24 h. </li><li> Preparar medio de crecimiento SKP que consiste de DMEM-F12, 03:01 (V / V) y 40 ng / ml de FGF2 (BD Biosciences, 20 ng / ml de EGF (BD Biosciences), suplemento libre de suero B27 2% (Invitrogen), 1 g / ml de Fungizone (Invitrogen) y 25 m / ml de gentamicina (Invitrogen). </li><li> Sedimentar las células a 1200 rpm durante 5 min y resuspender el sedimento de células directamente en 4 ml de medio de crecimiento SKP. Transferir la suspensión celular a un matraz de 25 cm 2 de cultivo de tejidos (BD Falcon). </li><li> Se incuba el matraz a 37 ° C y supervisar la cultura para la formación de la esfera al día durante 3 a 4 semanas. </li></ol> 2. Esfera Condiciones de cultivo <ol><li> Células de cultivo en un matraz de 25 cm 2 (BD Falcon) a 37 ° C, 5% de CO 2. </li><li> Agitar vigorosamente el matraz diariamente para evitar las células para adherirse a la parte inferior del matraz. Si es necesario, una pipeta de arriba abajo con una pipeta estéril 2 para separar las células adherentes y transferir la cultura a un nuevo frasco. </li><li> Primero esferas comienzan a construir dentro de 3 to 4 días. </li><li> Vamos esferas sedimento al fondo del matraz y el cambio de la mitad del medio cada 3 días. Para mantener la misma concentración final de factores de crecimiento 2X añadir factores de crecimiento al medio de crecimiento SKP recién preparado. </li><li> Mantenga el volumen constante de un máximo de 4 ml. </li><li> Cuando las esferas alcanzan un tamaño de ~ 200 mm deben ser pasados ​​por romper las esferas de menor tamaño por un vigoroso pipeta hacia arriba y hacia abajo con una pipeta de 2. </li><li> El cultivo se divide en dos 25 cm 2 frascos (BD Falcon) y esferas culturas son alimentación como se describe en el paso 2.4). </li><li> Las esferas se recogen para marcadores de células madre de cribado de día 16 al 21. </li></ol> El procedimiento de 2.6) y 2.7) describen la propagación de las esferas a (1) permite nutrientes y factores de crecimiento incluidos en medio SKP para acceder a todas las células dentro de cada esfera y (2) para ampliar la cultura esfera SKP antes de uso. Típicamente culturas Esfera bajo nuestros piescondiciones lture mantienen la capacidad de crecer en un período de tres meses y se pasan entre 3 y 4 veces. 3. La inmunocitoquímica para marcadores de células madre <ol><li> Culturas Esfera se cosechan en PBS el día 16 al 21. Los cultivos son sedimentaron a 1000 rpm durante 5 min. </li><li> Las esferas se volvieron a suspender en un pequeño volumen de PBS. </li><li> Dibuja dos círculos con un lápiz DAKO de aproximadamente 0,5 m de diámetro en el microscopio (marca Fisher). </li><li> Añadir 50 l de suspensión de esfera en los círculos. </li><li> Compruebe por microscopía de luz para detectar la presencia de esferas en cada gota. </li><li> Deje que las gotas secas bajo el capó. </li><li> Cualquiera de congelar los portaobjetos a -80 ° C o proceder con el protocolo de tinción de inmunofluorescencia. </li><li> Para la tinción de inmunofluorescencia, fijar las diapositivas con metanol previamente enfriado (100%) a -20 º C durante 10 min. </li><li> Lave los portaobjetos con PBS. </li><li> Bloquear las diapositivas con PBS/10% de suero fetal bovino / 0,02% de Triton X100durante al menos 1 hora. </li><li> Incubar con el anticuerpo primario (por ejemplo, anti-nestina, anti-Oct4, anti-TG30, anticuerpos anti-Nanog, Tabla 3) diluido en tampón de bloqueo: PBS/10% de FBS durante 2 horas a temperatura ambiente o durante la noche a 4 ° C. </li><li> Lavar 3 veces con tampón de bloqueo y se incuba con el anticuerpo secundario durante 1 hora a temperatura ambiente. </li><li> Lavar 3 veces con tampón de bloqueo y 3 veces con PBS. </li><li> Montaje diapositivas con medio de montaje Vectashield (Vector Inc.). </li><li> Analizar las muestras para la expresión de marcadores de células madre por microscopía de inmunofluorescencia. </li></ol> 4. Análisis de PCR en tiempo real de los niveles de expresión de marcadores de células madre <ol><li> Tome ml de cultivos de aproximadamente 2-3 días Esfera de 18 a 21. </li><li> Pellet las esferas de 1200 rpm durante 5 minutos y aspirar todo el medio. </li><li> Aislar ARN a partir de las esferas utilizando el Minikit RNeasy (Qiagen, Valencia, CA). </li><li> Evaluar la pureza de ARN y por espectrometría de gel de agarosa. &lt;/ Li&gt; <li> Sintetizar ADNc (transcriptasa inversa Omniscript (Qiagen)) utilizando los ARN celular total como plantilla. </li><li> Validar los marcadores de células madre por el tiempo real-PCR utilizando cebadores para marcadores de células madre (que se muestran en la Tabla 1) en un Poder SYBR Green PCR Mastermix (Applied Biosystems) con una concentración de 375 nM de cada cebador y 50 ng de plantilla en un 20 l volumen de reacción. GAPDH se utiliza como control endógeno. </li><li> Para la amplificación utilizar una desnaturalización inicial a 95 ° C durante 2,5 min seguido de 40 ciclos a 95 ° C durante 5 seg y 60 ° C durante 20 seg. </li><li> Analizar la ejecución con el (ΔΔCt) Método 2 8. </li></ol> 5. Diferenciación dirigida en células musculares lisas <ol><li> Esferas del día 21 al 26 se sembraron en placas de cultivo de 6 pocillos (BD Falcon) en medio SKP. </li><li> Las células se dejaron adherir y superar de las esferas en medio SKP durante 72 horas. </li><li> Las células musculares lisas (CML) differentiationes comenzó iniciado por reemplazó el medio con SMC medio de diferenciación que consta de alta glucosa medio de Eagle modificado de Dulbecco (Invitrogen) que contiene 5% de FBS, 5 ng / ml de PDGF-BB (Invitrogen), y 2,5 ng / ml de TGF-b1 (Invitrogen) . </li><li> El medio se cambió cada 3 días con medio de diferenciación SMC recién preparado durante un periodo de 3 a 4 semanas en las culturas. </li><li> La detección de marcadores de SMC se realizó después de 3 a 4 semanas por inmunohistoquímica. </li><li> Las células se fijaron con solución de paraformaldehído al 4% en PBS durante 10 min. </li><li> Las células se permeabilizaron con PBS que contenía 0,3% de Triton X-100 durante 30 min. </li><li> Las células se incubaron con anticuerpos contra αSMA (MO851, Dako, 1:100), o calponina (M3556, Dako, 1:100) durante 1 hora a temperatura ambiente y aún más como se describe en 3,12) a 3,15). </li></ol>

<ol>
	<li>Jahoda, C. A., Whitehouse, J., Reynolds, A. J., Hole, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+cells+differentiate+into+adipogenic+and+osteogenic+lineages.">Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages.</a> Exp. Dermatol. 12, 849 (2003).</li><li>Watt, F. M., Celso, L. o., C, V., Silva-Vargas, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells:+an+update.">Epidermal stem cells: an update.</a> Curr. Opin. Genet. Dev. 16, 518 (2006).</li><li>Blanpain, C., Horsley, V., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells:+turning+over+new+leaves.">Epithelial stem cells: turning over new leaves.</a> Cell. 128, 445 (2007).</li><li>Hunt, D. P., Jahoda, C., Chandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+skin-derived+precursors:+from+biology+to+clinical+translation.">Multipotent skin-derived precursors: from biology to clinical translation.</a> Vurr. Opin. Biotechnol. 20, 522 (2009).</li><li>Toma, J. G., et al. <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+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat. Cell Biol. 3, 778 (2001).</li><li>Fernandes, K. J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dermal+niche+for+multipotent+adult+skin-derived+precursor+cells.">A dermal niche for multipotent adult skin-derived precursor cells.</a> Nat. Cell Biol. 6, 1082 (2004).</li><li>Wenzel, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+adult+stem+cells+from+patients+with+Hutchinson-Gilford+progeria+syndrome+express+low+levels+of+progerin+in+vivo.">Na&#239;ve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo.</a> Bio. Open. 1, (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402 (2001).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. T., Miller, F. D. <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+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nature Protocol. 1, 2803 (2006).</li><li>Toma, J. G., McKenzie, I., Bagli, D., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23, 727 (2005).</li><li>Fernandes, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+neurogenic+potential+of+multipotent+skin-derived+precursors.">Analysis of the neurogenic potential of multipotent skin-derived precursors.</a> Electrophoresis. 201, 32 (2006).</li><li>Hill, l. K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+vascular+progenitor+cells+capable+of+endothelial+and+smooth+muscle+cell+function.">Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function.</a> Exp. Hematol. 38, 246 (2010).</li></ol>

No tenemos nada que revelar.

Utilizando el método descrito en este documento, las células madre dérmicas ingenuos se pueden aislar a partir de cultivos primarios de fibroblastos dérmicos. Con este enfoque, se informó recientemente, el aislamiento y caracterización de células madre adultas de cultivos de fibroblastos procedentes de pacientes con un síndrome genético raro, Hutchinson-Gilford progeria síndrome 7. Como se muestran en este documento esos precursores células expresan marcadores de células madre son capaces de auto-...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Invitrogen</td>
			<td>21885-108</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td>Final conc.: 200 mM</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Final conc.: 10 mg/ml /10 mg /ml</td>
		</tr>
		<tr>
			<td>trypsin solution (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture (Ham)</td>
			<td>Invitrogen</td>
			<td>21765-029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>BD Biosciences</td>
			<td>4114-TC-01M</td>
			<td>Final conc.: 40 ng/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>236-EG-200</td>
			<td>Final conc.: 20 ng/ml</td>
		</tr>
		<tr>
			<td>PDGFBB</td>
			<td>Invitrogen</td>
			<td>PHG0043</td>
			<td>Final conc.: 5 ng/ml</td>
		</tr>
		<tr>
			<td>TGF-b1</td>
			<td>Invitrogen</td>
			<td>PHG9204</td>
			<td>Final conc.: 2.5 ng/ml</td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Omnilab</td>
			<td>FALC353109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS w/o CaMg</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Roth</td>
			<td>8388.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Inc.</td>
			<td>H-1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy Minikit</td>
			<td>Qiagen, Valencia, CA</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Omniscript Reverse Transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>172-5201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td>Final conc.:1 mg/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Specific reagents and equipment.</td>
		</tr>
	</tbody>
</table>

na&iuml;ve adult stem cells isolation from primary human fibroblast cultures

Durante la última década, varias poblaciones de células madre adultas se han identificado en la piel humana 1-4. El aislamiento de los precursores multipotenciales dérmicos adultos se informó por primera vez por Miller F. D de laboratorio 5, 6. Estos primeros estudios describen una población de células precursoras multipotentes a partir de la dermis de mamífero adulto 5. Estas células - SKPs denominados precursores, para derivadas de la piel - se aislaron y expandieron a partir de roedores y de la piel humana y se diferencian tanto en la progenie neuronal y mesodérmico, incluyendo tipos de células nunca se encuentran en la piel, tales como las neuronas 5. Estudios inmunocitoquímicos en SKPs cultivadas revelaron que las células expresan vimentina y nestina, una proteína de filamento intermedio expresado en precursores neuronales y musculares del esqueleto, además de fibronectina y marcadores de células madre multipotentes 6. Hasta ahora, las células madre adultas SKPs población han sido aislados de recién recogidas biopsias de piel de mamífero. ve_content &quot;&gt; Recientemente, hemos establecido e informó que una población de células precursoras derivadas piel puede permanecer presente en cultivos de fibroblastos primarios establecidos a partir de biopsias de la piel 7. La suposición de que algunas células madre somáticas pueden residir en cultivos de fibroblastos primarios en duplicaciones de la población primitiva era basan en las siguientes observaciones: (1) SKPs y cultivos de fibroblastos primarios se derivan de la dermis, y por lo tanto, un pequeño número de células SKP podrían permanecer presente en cultivos de fibroblastos dérmicos primarios y (2) los cultivos primarios de fibroblastos cultivados a partir de alícuotas congelados que han sido sometidos a temperatura desfavorable durante el almacenamiento o transferencia contenía un pequeño número de células que permanecieron viables 7. Estas células raras fueron capaces de ampliar y se pudo pasar varias veces. Esta observación sugiere que un pequeño número de células con alta potencia de proliferación y la resistencia al estrés estuvieron presentes en cultivos de fibroblastos humanos 7. Nos aprovechamos de estos resultados para establecer un protocolo para el aislamiento rápido de las células madre adultas a partir de cultivos primarios de fibroblastos que son fácilmente disponibles en los bancos de tejidos de todo el mundo (Figura 1). Este método tiene un significado importante, ya que permite el aislamiento de las células precursoras cuando las muestras de piel no son accesibles mientras que cultivos de fibroblastos pueden estar disponibles en los bancos de tejidos, por lo tanto, la apertura de nuevas oportunidades para diseccionar los mecanismos moleculares que subyacen a enfermedades genéticas raras, así como enfermedades de modelado en un plato.

Se demuestra que una población de células que se expanden selectivamente para generar esferas SKP bajo condiciones de crecimiento controlado que consisten en EGF y FGF2 están presentes en cultivos de fibroblastos dérmicos primarios (Figura 1) como se informó recientemente 7. Cultivos de fibroblastos de PPD 15 a 25 que normalmente se corresponden con las cepas de fibroblastos primarios disponibles a partir de bancos de células se utilizaron en este estudio. C...

Watch this Scientific Journal Video about NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures at JoVE.com

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Se presenta un método para aislar precursoras derivadas de la piel (SKP) células multipotentes ingenuos de cultivos de fibroblastos humanos primarios. Se demuestra que estos SKPs derivados de cultivos de fibroblastos comparten propiedades de células madre similares a los obtenidos directamente a partir de biopsias de piel humana. Estas células expresan el marcador de la cresta neural, nestina, además de los marcadores multipotentes, tales como Oct4 y Nanog.

Naïve Adult Stem Cells Aislamiento de Primaria cultivos de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

nave-adult-stem-cells-isolation-from-primary-human-fibroblast-cultures

Research

JoVE Journal

Biology

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

14.0K vistas.  Technische Universität München. Se presenta un método para aislar precursoras derivadas de la piel (SKP) células multipotentes ingenuos de cultivos de fibroblastos humanos primarios. Se demuestra que estos SKPs derivados de cultivos de fibroblastos comparten propiedades de células madre similares a los obtenidos directamente a partir de biopsias de piel humana. Estas células expresan el marcador de la cresta neural, nestina, además de los marcadores multipotentes, tales como Oct4 y Nanog.

Video: Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Investigación

Biología

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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 and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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

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

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

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

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

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

La eliminación rápida de fibroblastos de alta densidad Humanos culturas de células madre embrionarias

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Aislamiento Manual de células madre derivadas de tejido adiposo de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...