Este trabajo fue financiado por una Nueva Facultad II Premio del Instituto de Medicina Regenerativa de California (RN2-00921-1), y un premio financiado por los NIH de Investigación Nacional (F32-HL104924)

1. Preparación de los Alimentadores de embriones de ratón <ol><li> El MEFs utilizado en el co-cultivo de hESCs debe ser previamente tratada por irradiación o mitomicina C para inhibir las células de sufriendo división. </li><li> Dos horas antes de la siembra MEF, bata de cada plasma de 60 mm placa de cultivo tratado con 2 ml de gelatina al 0,05%. </li><li> Descongelar un vial de MEFs tratada y placas a ~ 20.000 células / cm 2. </li><li> Permitir que las células se adhieran durante la noche. </li></ol> 2. Siembra Co-hESCs en MEFs <ol><li> Las nuevas colonias se puede iniciar a partir de cultivos crioconservados o pases culturas actuales sobre nuevos MEF platos recubiertos, como a continuación. </li><li> Lavar la placa que contiene las colonias hESC grandes para ser pasadas 1 hora con 3 ml de PBS 1x calentado. </li><li> Añadir 3 ml de colagenasa IV / solución de PBS (1 mg / ml), y se incuba durante 5-10 minutos a 37 ° C. (Nota: Las células no se harán distintos o balón como se ha visto con tripsina). </li><li> Mientras que las células están todavía en lacolagenasa, utilizar un borde de una punta de pipeta de 5 ml, crear un patrón de rejilla en las colonias, romper en pequeños grupos de células. </li><li> Con la punta de la punta de la pipeta (realizado de una manera perpendicular a la placa de cultivo) para raspar el fondo de la placa de cultivo, desplazando todas las células de la superficie. </li><li> Recoger las células en un tubo Falcon y giran a (1000 rpm) durante 5 min. </li><li> Después de la centrifugación, se aspira el medio, dejando el sedimento de células en el tubo Falcon. </li><li> Aspirar el medio desde el MEF MEFs chapado en el día antes. </li><li> Re-placa las células a una proporción de 1:3 de la placa original, y se alimentan con 3 ml de medio de hESC </li></ol> 3. Agotan MEFs de la Co-cultura Nota: Humanos culturas ESC debe ser a alta densidad, por lo general entre 10 y 14 días de cultivo. <ol><li> Colocar la punta de una pipeta de aspiración en el borde de la placa y permitir la succión para tirar suavemente hasta el borde de la hoja de MEF. </li><li> ReMove los medios de comunicación de la antena, y permita que la punta para coger la capa celular y mueva lentamente la punta de forma arqueada por encima de la placa. Nota: La hoja puede atascar en la punta de la pipeta, pero esto no es problemático ya que no debe impedir que el usuario manualmente tirando de la hoja de la superficie de la placa de cultivo. Si las células están pegados a la final del aspirador, puede usar la parte superior de la placa de cultivo para romper la lámina de células para la aspiración completa. </li><li> Reemplace inmediatamente hESC medio, y poner la placa en la incubadora. </li></ol> 4. Los resultados representativos El resultado final del proceso de eliminación MEF produce pequeñas colonias hESC inalteradas (Figura 1D) capaces de someterse a la expansión significativa en colonias muy grandes (Figura 2) mientras se mantiene la expresión de los fabricantes de pluripotencia SSEA-4, octubre ¾, y TRA 1-81 (figura 3). Figura 1. ESC morfología de las colonias antes y después de la extracción MEF. Humanos colonias de células madre embrionarias en un día) 1 y B) el día 2 después de la siembra en placas de cultivo. C) de alta densidad Humanos colonias de células madre embrionarias (H7), rodeada de MEFs. Las dos colonias hESC se describen en rayas rojas. D) Después de la eliminación MEF mediante la técnica de aspiración descrito, nos quedamos con una colonia de células madre aisladas con muy pocos MEFs que rodea la colonia intacta. <img alt="Figura 2" src="/files/ftp_upload/3951/3951fig2.jpg" /> Figura 2. Expansión ESC Colonia 10 días después de la extracción MEF. HESC La colonia original inmediatamente después de agotamiento MEF (izquierda) se le permite expandirse en una colonia muy grande (derecha) aproximadamente 800 micras, 10x. Tenga en cuenta que la colonia original se forma la imagen con el mismo aumento (10x) como la colonia compuesto. <img alt="Figura 3" src="/files/ftp_upload/3951/3951fig3.jpg" /> Figura 3. La inmunotinción de las colonias hESC MEF-agotado. Identificación por inmunofluorescencia de las colonias de hasta 10 días después del agotamiento MEF demuestra que las colonias de mantener su expresión de marcadores de pluripotencia como evidente por octubre ¾, SSEA-4, y la tinción de Tra-1-81. Además, estas colonias de células madre no muestran diferenciación hacia un destino mesodérmico, indicado por la ausencia de Flk-1. A, E, I, y M) son imágenes de transmisión de luz de las colonias immunoflouscently manchadas, 20, 10, 10 y 2x, . respectivamente B, F, J y N) mostrar los núcleos -. células teñidas con DAPI de las colonias immunofluorescently manchadas, 20, 10, 10 y 2x, respectivamente CD) muestran la expresión demarcador de células madre humanas SSEA-4 solamente, y la imagen compuesta, 20x. GH) muestran la expresión de células madre humano octubre marcador ¾ sólo, y la imagen compuesta, KL 10x.) muestran la expresión de marcadores de células madre humanas Tra 1-81 sólo y la imagen compuesta, 10x. MP) La última fila si las imágenes muestran una colonia retratar las fronteras típicas liso visto con este aumento, 2x, y estas colonias no expreso O) Flk-1, un marcador temprano de la diferenciación del mesodermo, ni que P) contiene fibroblastos como se muestra por la ausencia de tinción de DDR2. Vídeo Suplementario. Extracción de fibroblastos por aspiración de planos. <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">Haga clic aquí para ver la película</a> .

<ol>
	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <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+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. 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+feeder+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

No hay conflictos de interés declarado.

El método presentado en este manuscrito ofrece una alternativa rápida y menos costosa para la eliminación de células alimentadoras de fibroblastos humanos de cultivos de células embrionarias. La eliminación exitosa de los fibroblastos es dependiente de la existencia de una monocapa confluente herméticamente de estas células que se desarrolla en más cultivos de células madre. Después de 7-10 días, las colonias que crecen hESC empujará los fibroblastos de conexión en una dirección hacia fuera, la generació...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Fibroblastos Marker </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> Núcleo Celular Marker </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> Human ESC Marker </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> Human ESC Marker </td></tr><tr><td> Flk-1 </td><td> BD Pharma </td><td> 555307 </td><td> Marcador de diferenciación temprana </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> Human ESC Marker </td></tr></tbody></table> Tabla 1. Tabla de razones específicascaballeros utilizada para la inmunotinción hESC.

rapid fibroblast removal from high density human embryonic stem cell cultures

Fibroblastos de embriones de ratón (MEFs) se utilizaron para establecer células madre embrionarias humanas (hESCs) los cultivos tras el aislamiento de blastocisto 1. Este sistema de alimentación mantiene hESCs de someterse a la diferenciación espontánea durante la expansión celular. Sin embargo, este método de co-cultivo es laborioso, requiere de personal altamente capacitado y pureza rendimientos hESC bajo 4. Muchos laboratorios han intentado reducir al mínimo el número de células de alimentación en cultivos de células madre (es decir, la incorporación de la matriz platos recubiertos o otros tipos de células alimentadoras 5-8). Estos sistemas de cultivos modificados han mostrado cierta promesa, pero no han suplantado el método estándar para el cultivo de hESCs con mitomicina C tratados con fibroblastos de ratón embrionaria, con el fin de retardar la diferenciación espontánea no deseada de los cultivos de células madre. Por lo tanto, las células de alimentación utilizados en la expansión de células madre debe ser removido durante experimentos de diferenciación. Aunque se dispone de varias técnicas para purificar el co hESClonies (FACS, Mac, o el uso de vectores resistentes a los fármacos) de los alimentadores, estas técnicas son mano de obra intensiva, costosa y / o contrarios a la hESC. El objetivo de este proyecto era inventar un método de purificación que permite la recolección de una población pura de hESCs. Hemos observado que en un cultivo confluente hESC, la población MEF se puede eliminar mediante una aspiración simple y rápido de la hoja de MEF. Esta eliminación es dependiente de varios factores, incluyendo lateral de célula a célula de unión de MEFs que tienen una menor afinidad de unión a la placa de cultivo de estireno, y la capacidad de las colonias de células madre para empujar los fibroblastos hacia el exterior durante la generación de su propio &quot; nicho &quot;. El hESC fueron examinados por SSEA-4, Oct3 / 4 y expresión Tra 1-81 hasta 10 días después de la eliminación del MEF para asegurar el mantenimiento de la pluripotencia. Por otra parte, las colonias de células madre fueron capaces de continuar creciendo en grandes formaciones de después de la eliminación MEF, que proporciona un nivel adicional de expansión hESC.

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

A pesar de los esfuerzos en curso a las culturas de transición a la alimentación libre de condiciones, la derivación y la cultura de células madre embrionarias humanas (hESC) siguen siendo en gran parte dependiente en co-cultivos con alimentadores de embriones de ratón (MEFs). A continuación, se muestra una nueva metodología para la rápida eliminación de los alimentadores a partir de cultivos celulares de grado terapéutico antes de la experimentación.

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 ...

rapid-fibroblast-removal-from-high-density-human-embryonic-stem-cell

Research

JoVE Journal

Biology

11.7K vistas.  University of California, Merced. A pesar de los esfuerzos en curso a las culturas de transición a la alimentación libre de condiciones, la derivación y la cultura de células madre embrionarias humanas (hESC) siguen siendo en gran parte dependiente en co-cultivos con alimentadores de embriones de ratón (MEFs). A continuación, se muestra una nueva metodología para la rápida eliminación de los alimentadores a partir de cultivos celulares de grado terapéutico antes de la experimentación.

Video: Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES pases madre de embriones humanos líneas celulares con tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Investigación

Biología

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Ratón epidérmico de la cresta neural de células madre (EPI-NCSC) Culturas

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparación de combinados lisado plaquetario humano (pHPL) como un suplemento eficaz de los animales libres de suero humano culturas de células madre

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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

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