Dr. Rountree reconoce el apoyo actual de la red del milagro de la Infancia, Instituto Nacional de Salud, K08DK080928 y R03DK088013, y la Sociedad Americana del Cáncer Premio Académico de Investigación, MGO-11651. Dr. Rountree reconoce que este procedimiento fue desarrollado inicialmente y refinado al mismo tiempo financiado por el Programa de Desarrollo Científico Pediátrico (NICHD concesión de una subvención K12-HD00850).

<ol>
	<li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD133-expressing+murine+liver+oval+cell+population+with+bilineage+potential.">A CD133-expressing murine liver oval cell population with bilineage potential.</a> Stem Cells. 25, 2419-2429 (2007).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++liver+cancer+stem+cells+from+methionine+adenosyl+transferase+1A-deficient+mice+demonstrate+resistance+to+transforming+growth+factor+(TGF)-beta-induced+apoptosis.">CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis.</a> Hepatology. 49, 1277-1286 (2009).</li><li>Rountree, C. B., Ding, W., He, L., Stiles, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+CD133-expressing+liver+cancer+stem+cells+in+liver-specific+phosphatase+and+tensin+homolog+deleted+on+chromosome+10-deleted+mice.">Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice.</a> Stem Cells. 27, 290-299 (2009).</li><li>Rountree, C. B., Senadheera, S., Mato, J. M., Crooks, G. M., Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+liver+cancer+stem+cells+during+aging+in+methionine+adenosyltransferase+1A-deficient+mice.">Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice.</a> Hepatology. 47, 1288-1297 (2008).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J. Cell. Physiol. 213, 286-300 (2007).</li><li>Fausto, N., Campbell, J. S., Riehle, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Hepatology. 43, 45-53 (2006).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+Stem+Cells.+III.+Emergent+themes+of+liver+stem+cell+biology:+niche,+quiescence,+self-renewal,+and+plasticity.">Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 290, 189-193 (2006).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+liver+repopulating+capacity+of+murine+oval+cells.">The origin and liver repopulating capacity of murine oval cells.</a> Proc Natl Acad Sci U. S. A. 100, 11881-11888 (2003).</li><li>Greenbaum, L. E., Wells, R. G. <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+stem+cells+in+liver+repair+and+fibrosis.">The role of stem cells in liver repair and fibrosis.</a> Int. J. Biochem. Cell Biol. 43, 222-229 (2011).</li><li>Choi, S. S., Omenetti, A., Syn, W. K., Diehl, A. M. <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+Hedgehog+signaling+in+fibrogenic+liver+repair.">The role of Hedgehog signaling in fibrogenic liver repair.</a> Int. J. Biochem. Cell. Biol. 43, 238-244 (2011).</li><li>Khurana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scopolamine+treatment+and+muscarinic+receptor+subtype-3+gene+ablation+augment+azoxymethane-induced+murine+liver+injury.">Scopolamine treatment and muscarinic receptor subtype-3 gene ablation augment azoxymethane-induced murine liver injury.</a> J. Pharmacol. Exp. Ther. 333, 639-649 (2010).</li><li>Sell, S., Leffert, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cancer+stem+cells.">Liver cancer stem cells.</a> J. Clin. Oncol. 26, 2800-2805 (2008).</li><li>Lee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+prognostic+subtype+of+human+hepatocellular+carcinoma+derived+from+hepatic+progenitor+cells.">A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.</a> Nat. Med. 12, 410-416 (2006).</li><li>Sell, S., Dunsford, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+stem+cell+origin+of+hepatocellular+carcinoma+and+cholangiocarcinoma.">Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma.</a> Am. J. Pathol. 134, 1347-1363 (1989).</li><li>Amin, R., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+tgf-Beta+in+hepatic+carcinogenesis.">Liver stem cells and tgf-Beta in hepatic carcinogenesis.</a> Gastrointest Cancer Res. 2, 27-30 (2008).</li><li>Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor/stem+cells+give+rise+to+liver+cancer+due+to+aberrant+TGF-beta+and+IL-6+signaling.">Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.</a> Proc Natl Acad Sci. 105, 2445-2450 (2008).</li><li>Kitisin, K., Pishvaian, M. J., Johnson, L. B., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+molecular+signaling+pathways+in+hepatocellular+carcinoma.">Liver stem cells and molecular signaling pathways in hepatocellular carcinoma.</a> Gastrointest Cancer Res. 1, 13-21 (2007).</li><li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+fails+to+differentiate+into+liver+epithelium+during+murine+development+and+regeneration.">Bone marrow fails to differentiate into liver epithelium during murine development and regeneration.</a> Hepatology. 45, 1250-1260 (2007).</li><li>Shimano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+oval+cells+have+the+side+population+phenotype+defined+by+expression+of+ATP-binding+cassette+transporter+ABCG2/BCRP1.">Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1.</a> Am J Pathol. 163, 3-9 (2003).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+isolation+and+clonal+identification+of+self-renewing+bipotent+hepatic+progenitor+cells+in+adult+mouse+liver.">Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.</a> Hepatology. , (2008).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+and+characterization+of+self-renewing+pluripotent+stem+cells+in+the+developing+liver.">Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.</a> J Cell Biol. 156, 173-184 (2002).</li><li>Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpCAM-positive+hepatocellular+carcinoma+cells+are+tumor-initiating+cells+with+stem/progenitor+cell+features.">EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.</a> Gastroenterology. 136, 1012-1024 (2009).</li><li>Yamashita, T., Budhu, A., Forgues, M., Wang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hepatic+stem+cell+marker+EpCAM+by+Wnt-beta-catenin+signaling+in+hepatocellular+carcinoma.">Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.</a> Cancer Res. 67, 10831-10839 (2007).</li><li>Furuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li><li>Kordes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++hepatic+stellate+cells+are+progenitor+cells.">CD133+ hepatic stellate cells are progenitor cells.</a> Biochem Biophys Res Commun. 352, 410-417 (2007).</li><li>Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Growth+Factor+10+is+critical+for+liver+growth+during+embryogenesis+and+controls+of+hepatoblast+survival+via+b-catenin+activation.">Fibroblast Growth Factor 10 is critical for liver growth during embryogenesis and controls of hepatoblast survival via b-catenin activation.</a> Hepatology. , (2007).</li><li>Turner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+stem+cell+and+maturational+liver+lineage+biology.">Human hepatic stem cell and maturational liver lineage biology.</a> Hepatology. 53, 1035-1045 (2011).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Galicia, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+hepatic+tumor+progenitor+cells+in+Pten-null+mice+requires+liver+injury+and+is+reversed+by+loss+of+AKT2.">Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.</a> Gastroenterology. 139, 2170-2182 (2010).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+of+murine+liver+tumor+cells+promotes+invasion.">Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion.</a> Hepatology. 52, 945-953 (2010).</li></ol>

Dr. Rountree cuenta con el apoyo de investigación de Bayer Farmacia. Los Dres. Ding y Maleantes y la Sra. Dang y VanKirk señora no tiene revelaciones que informar.

A diferencia del sistema hematopoyético, en la que las células madre hematopoyéticas son responsables de mantener un sistema de diferenciación celular que replacesnormal fisiológicos turn-over de los leucocitos, los glóbulos rojos y las plaquetas, las células madre del hígado, o un adulto las células progenitoras del hígado, no participan en el hígado normal homeostasis. 5,6 Después de la lesión hepática aguda o hepatectomía parcial, los hepatocitos, el epitelio del hígado diferenciada, someterse a varias sesiones de la proliferación de sustituir la masa hepática perdida. 5,6 Sólo en una lesión crónica son células madre del hígado observado la proliferación de 1,5. Estos -11 adultos, las células madre órgano específico se propone diferenciarse en hepatocitos y cholangiocytes. 1,5-8 Curiosamente, la gran mayoría de los cáncer de hígado se desarrolla en el fondo de una lesión crónica, y por lo tanto las células madre del hígado son también la hipótesis de que se los precursores de un subgrupo de cáncer de hígado. 2-4,12-17 Uno de los retos más importantes para detener el trabajo de células en el hígado es el aislamiento de poblaciones raras de células para el análisis funcional. Por ejemplo, la gran mayoría de las células en el hígado son los hepatocitos, que son significativamente mayores que cholangiocytes y otras más pequeñas células no parenquimatosas. Al romper el hígado entero en compartimentos celulares (hepatocitos grandes - fracción del parénquima y células más pequeñas - no parenquimatosas fracción) y, además, la selección de células CD45 negativas (no células hematopoyéticas), somos capaces de enriquecer a la población a partir de células madre por más de 600 veces. 1,18-21 Cabe destacar, que no son en absoluto lo que indica que la población CD133 + es un 100% de la población pura de células madre, pero es evidente que representa una población heterogénea de células, con un linaje diferente y el potencial de repoblación. Una de las principales limitaciones del campo es la definición de las células madre y células progenitoras. Usamos el término &quot;célula madre&quot; de manera más amplia en este trabajo, pero en la definición estricta, CD133 + células no parenquimatosas representan una población de progenitores bi-linaje. Dado el estado de la madre y la investigación actuales progenitor en el hígado, este informe proporciona un punto de partida para los investigadores que estén interesados ​​en este campo. Como marcadores surgen nuevas, como EpCAM 22,23, o factores de transcripción, como Sox9, 24 de ellos se pueden incorporar. Por ejemplo, hemos encontrado un índice bastante alto de solapamiento entre las células EpCAM + y células CD133 +. En este informe, se detalla un proceso de aislamiento de células madre de hígado normal murino, así como la lesión hepática crónica modelos, que demuestran los tallos aumentó la proliferación de células del hígado. Este método tiene claras ventajas sobre técnicas de inmunohistoquímica estándar de hígado congelado o fijado en formol como los estudios funcionales utilizando células vivas se puede realizar después de su aislamiento. 1,3,4 El objetivo específico de este proceso consiste en aislar a una población relativamente pura de células madre del hígado que puede ser ampliado clonal in vitro. La principal limitación de este procedimiento es que la mayoría de las células aisladas no será viable después de la citometría de flujo (véase la sección Solución de problemas). Este es el resultado de las horas necesarias para preparar las células, y los numerosos procedimientos necesarios para perfeccionar la población antes de su aislamiento. Si el hígado se digiere en suspensión de células individuales para el análisis de FACS inmediata, la diferencia de tamaño entre los hepatocitos y otras células no parenquimatosas hará efectiva la creación de la puerta FACS imposible. Si el hígado no parenquimatosas células se utilizan, sin la eliminación de las células hematopoyéticas, se corre el riesgo de que un número significativo de células CD133 + puede ser de origen hematopoyético pueden contaminar la fracción. Además, mediante el procesamiento de las células a través del filtro Miltenyi, sólo las células individuales y grupos pequeños de células se extraen. Esto asegura que la muestra no va a tapar la aguja consumo de FACS. Alternativas para este procedimiento incluyen la modificación de cualquier marcador de superficie celular alternativos, tales como CD49f, EpCAM, o una combinación de marcadores, como CD133 + + EpCAM Un segundo informe publicado utiliza un gradiente de densidad para aislar las células madre del hígado de una fracción no parenquimatosas. Este procedimiento requiere una ultra-centrífuga (8000 xg), añade un tiempo considerable en el procedimiento, y en nuestra experiencia, redujo significativamente la pre-FACS rendimiento y la viabilidad celular después de la FACS. 8 Experimentos futuros incluyen una amplia gama de análisis de expresión génica, incluidos los genes del hígado del feto, tales como HNF3, HNF4α, y además αfp.In, Western blot e inmunocitoquímica de proteínas que se expresan pueden ser utilizadas para confirmar los resultados de RT-PCR de células en cultivo. Una cuestión a destacar es el trabajo reciente que identifican CD133 expresión en las células estrelladas hepáticas. 25 Ahora examinan habitualmente a nuestras muestras de los marcadores de las células estrelladas (véase la sección Solución de problemas), y no han identified contaminación significativa en nuestras fracciones. Esto puede estar relacionado con las diferentes técnicas de la digestión y el aislamiento de células del hígado. 2,3,26 Con base en el hecho de que la mayoría de las células no será viable inmediatamente después de su aislamiento FACS, se recomienda que las células se chapada, ya sea a granel o de células CD133 + células individuales, antes de su uso en animales. 5-7 días in vitro de manera significativa a mejorar los resultados de los análisis del tumor. Además, debido a los rigores de aislamiento de células individuales, esto sólo debe llevarse a cabo una vez que las colonias se puede ampliar a granel células CD133 + aisladas. Una descripción más detallada de las condiciones de cultivo diferentes y proteínas de andamiaje que pueden utilizarse para detener la cultura del hígado y las células progenitoras se ha caracterizado bien por Lola Reid. Este trabajo ofrece condiciones alternativas y modificaciones, que los investigadores pueden incorporar a su programa de investigación una vez que las técnicas básicas de aislamiento se dominan. 27,28 Dr. El trabajo de Reid también proporciona un análisis más detallado de la biología y la maduración de linaje entre las células madre hepáticas y progenitores comprometidos. En términos de análisis de tumores in vivo, que han tenido éxito utilizando células recién aisladas y células clonal ampliado CD133 + células in vitro, utilizando principalmente 1x10 6 células. Nos hemos centrado ontwo cepas genéticas de la lesión crónica del hígado, el MAT1a - / - y PTEN específica del hígado - / - ratones, y se han utilizado tanto en ratones nude y ratones de tipo salvaje como anfitriones para el crecimiento del tumor. En nuestra experiencia, CD133 + células madre del hígado sólo se forman tumores cuando están aisladas de importantes modelos de lesión hepática que se pre-malignas. Tenga en cuenta que los tumores formados de células CD133 + en general tienen tanto el carcinoma hepatocelular y colangiocarcinoma características, lo que sugiere que una célula madre o el origen de células progenitoras de los tumores. 2-4,29 El trabajo de seguimiento después de los tumores están documentadas incluye el análisis estándar patológico del tejido tumoral (tinción H &amp; E) y la tinción inmunohistoquímica. Además, los tumores pueden ser picada y digerido para el análisis de FACS o volver a la cultura. 2-4,30 En conclusión, hemos detallado un procedimiento para el aislamiento, la expansión, y la caracterización básica de las células madre CD133 + CD133 + hígado y las células madre del cáncer. Solución de problemas: CD45 contaminación: Para el paso 3, si hay contaminación de las células CD45 +, que puede ser evaluado mediante la adición de un Ab CD45-FITC antes del análisis FACS y el aislamiento, asegúrese de que el anticuerpo microesferas CD45 no ha caducado y que el filtrado se recogió sólo mientras el filtro fue en el soporte magnético. Cualquier filtrado colectado mientras que el filtro no está en el soporte magnético contendrá las células CD45 +. Bajo número de células: Para el hígado lesionado, el número total de CD133 + no parenquimatosas aisladas puede ser inferior a 10.000. Estas células son poco frecuentes en el hígado en reposo. Para un modelo de lesión crónica, como la dieta DDC 0,1%, el número se incrementará en gran medida a 100.000 células. Si el número total de células es muy por debajo de estos números, una cuestión a considerar es la tinción FACS Ab. Compruebe que el Ab CD133 no ha expirado, como la tinción de los pobres se traducirá en un rendimiento pobre. Además, se recomienda realizar un análisis de FACS de hígado de células no parenquimatosas para determinar la población relativa antes de intentar el aislamiento FACS. La viabilidad celular de baja después de FACS: Uno de los problemas de viabilidad puede estar relacionado con cómo las células se procesan y durante cuánto tiempo. Idealmente, el procedimiento de celda de aislamiento total, los pasos 1-4, debe llevarse a cabo sin ningún tipo de retrasos entre los pasos y se terminó en el mismo día. Cualquier demora significativa entre los pasos 1-4 reducirá en gran medida la viabilidad de las células. Una segunda cuestión relacionada con la viabilidad celular puede estar relacionado con la presión de funda para el aislamiento FACS. Se recomienda utilizar una presión de funda inferior. Por último, una vez que las células son aisladas, deben ser inmediatamente plateado, como cualquier sistema de almacenamiento significativo en el hielo después de la clasificación también se reducirá la viabilidad. CD133 + no parenquimatosas heterogeneidad: Informes recientes indican que las células estrelladas hepáticas también pueden tener CD133 expresión y tienen cierta plasticidad. 25 Por lo tanto, además de verificar la potencia bi-genes con albúmina y Krt19, de verificación adicionales pueden incluir genes asociados con las células estrelladas, como la proteína glial fibrilar ácida de las células pasivas, estrelladas y alfa-actina de músculo liso y desmina en las células estrelladas activadas. 3,4,26 Además, la población CD133 + representa una población de progenitores en general. La adición de un segundo marcador, como EpCAM, puede ayudar a perfeccionar aún más la población, y la heterogeneidad límite.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> DMEM: F12 </td><td> Invitrogen </td><td> 10565-018 </td><td> Con rojo fenol </td></tr><tr><td> CD45 microbolas </td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td> Factor de crecimiento de hepatocitos </td><td> Sigma </td><td> H1404 </td><td></td></tr><tr><td> Factor de Crecimiento Epidérmico </td><td> Sigma </td><td> E4127 </td><td></td></tr><tr><td> DNasa </td><td> Sigma </td><td> DN25 </td><td> 1 gramo </td></tr><tr><td> Colagenasa D </td><td> Roche </td><td> 1088874 </td><td></td></tr><tr><td> Pronasa </td><td> Roche </td><td> 0165921 </td><td></td></tr><tr><td> 70 micras colador </td><td> Pescador </td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50 reacciones </td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi LD columna </td><td> Miltenyi </td><td> 130-042-901 </td><td></td></tr><tr><td> CD133-PE FACS Ab </td><td> eBioscience </td><td> 12-1331-82 </td><td></td></tr><tr><td> Laminina placas revestidas </td><td> BD </td><td> 354410 </td><td> 96 y </td></tr><tr><td> Tripsina EDTA 0,05% </td><td> Invitrogen </td><td> 25300-354 </td><td> 100 ml </td></tr><tr><td> RNeasy Micro Kit </td><td> Quaigen </td><td> 74004 </td><td> 50 columnas de 5x10 5 células o menos </td></tr><tr><td> Pharm Lyse </td><td> BD </td><td> 555899 </td><td> Concentración 10 veces </td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

Células madre del hígado, o células ovales, proliferan durante la lesión crónica del hígado, y se proponen de diferenciarse en hepatocitos y cholangiocytes. Además, las células madre del hígado son la hipótesis de que los precursores de un subgrupo de cáncer de hígado, carcinoma hepatocelular. Uno de los principales desafíos para frenar el trabajo de células de cualquier órgano sólido como el hígado es el aislamiento de una población poco común de células para su análisis detallado. Por ejemplo, la gran mayoría de las células en el hígado son los hepatocitos (fracción de parénquima), que son significativamente mayores que las células no parenquimatosas. Enriqueciendo los compartimentos celulares específicos del hígado (es decir, del parénquima y las fracciones no parenquimatosas), y la selección de células CD45 negativas, que son capaces de enriquecer a la población a partir de células madre de más de 600 fold.The proceduresdetailed en este informe permiten una población relativamente raro de células de un órgano sólido que se ordenan de manera eficiente. Este proceso puede ser utilizado para las células madre del hígado isolateliver murino normal, así como la lesión hepática crónica modelos, que demuestran los tallos aumentó la proliferación de células del hígado. Este método tiene claras ventajas sobre técnicas de inmunohistoquímica estándar de hígado congelado o fijado en formol como los estudios funcionales utilizando células vivas se puede realizar después de la primera co-localización de los experimentos. Para llevar a cabo el procedimiento descrito en este informe, una relación de trabajo con una investigación basada en la citometría de flujo principal se recomienda que los detalles de aislamiento FACS son altamente dependientes de instrumentación especializada y un gran conocimiento práctico de la citometría de flujo básico procedimientos. El objetivo específico de este proceso consiste en aislar a una población de células madre del hígado que puede ser ampliado clonal in vitro.

Watch this Scientific Journal Video about Isolation of CD133+ Liver Stem Cells for Clonal Expansion at JoVE.com

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

A continuación se describe el aislamiento de CD133 que expresan las células madre del hígado y las células madre del cáncer de hígado en su totalidad murino, un proceso que requiere de digestión de los tejidos, el enriquecimiento celular y citometría de flujo aislamiento. Se incluyen métodos para el aislamiento avanzado de células individuales y la expansión clonal.

El aislamiento de células madre CD133 + hígado para la expansión clonal

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

isolation-of-cd133-liver-stem-cells-for-clonal-expansion

Research

JoVE Journal

Biology

22.2K vistas.  Pennsylvania State College of Medicine. A continuación se describe el aislamiento de CD133 que expresan las células madre del hígado y las células madre del cáncer de hígado en su totalidad murino, un proceso que requiere de digestión de los tejidos, el enriquecimiento celular y citometría de flujo aislamiento. Se incluyen métodos para el aislamiento avanzado de células individuales y la expansión clonal.

Video: Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Aislamiento y trasplante de células madre hematopoyéticas (HSC)

Investigación

Biología

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 ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Aislamiento de células de la glándula salival del ratón madre

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 ...

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

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

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

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and 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 ...

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 ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Aislamiento, cultivo y trasplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocolo para el aislamiento de hepatocitos primarios humanos y la correspondiente principales poblaciones de células hepáticas no parenquimatosas

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...