Este trabalho foi financiado por uma Nova Faculdade Prêmio II do California Institute of Regenerative Medicine (RN2-00921-1), e um NIH-financiado Prêmio Nacional de Pesquisa (F32-HL104924)

1. Preparação de Alimentadores embrionárias de rato <ol><li> O MEFs utilizado na co-cultura de hESCs devem ser previamente tratadas por irradiação ou mitomicina C para inibir as células de sofrer divisão. </li><li> Duas horas antes da semeadura MEF, revestimento de plasma 60 milímetros cada prato de cultura tratada com 2 ml de 0,05% de gelatina. </li><li> Descongelar um frasco de MEFs tratada e da placa de ~ 20.000 células / cm 2. </li><li> Permitir que as células a aderir durante a noite. </li></ol> 2. Co-semeadura hESCs em MEFs <ol><li> Novas colônias pode ser iniciada a partir de culturas criopreservados ou passagens em culturas atuais para novos MEF-revestidos pratos, como abaixo. </li><li> Lave a placa contendo as colónias grandes CTEh a ser passadas uma vez com 3 mL de PBS 1x aquecido. </li><li> Adicionar 3 ml de colagenase IV / solução de PBS (1 mg / ml) e incubar durante 5-10 min a 37 ° C. (Nota: As células não ficará visível ou bola para cima, como visto com tripsina). </li><li> Enquanto que as células estão ainda nacolagenase, utilizar uma ponta de uma pipeta de 5 ml, crie um padrão de grade sobre as colónias, quebrando-as em pedaços de pequenas células. </li><li> Usar o fim da ponta de pipeta (realizada de uma forma perpendicular à placa de cultura) para raspar o fundo da placa de cultura, deslocando todas as células da superfície. </li><li> Recolher as células para um tubo Falcon e em rotação (1000 rpm) durante 5 min. </li><li> Após a centrifugação, aspirar o meio, deixando o sedimento de células no tubo de Falcon. </li><li> Aspirar o meio do dia MEF MEFs do folheado antes. </li><li> Re-placa das células a uma razão de 1:3 a partir da placa original, e alimentos com 3 ml de meio de hESC </li></ol> 3. Esgotando MEFs da co-cultura Nota: As culturas humanas CES devem estar em alta densidade, normalmente entre 10 e 14 dias de cultura. <ol><li> Colocar a ponta de uma pipeta de aspiração na extremidade da placa e permitir que a sucção puxe para cima da borda da folha de MEF. </li><li> Remove a mídia do prato, e permita que a ponta para pegar a folha de celular e mova lentamente a ponta de forma arqueada acima da placa. Nota: A folha pode entupir a ponta da pipeta, mas isto não é problemática, uma vez que não deve impedir que o utilizador manualmente puxando a folha a partir da superfície da placa de cultura. Se as células são presos à extremidade do aspirador, é possível utilizar a parte superior do prato de cultura para quebrar a camada de células por aspiração completa. </li><li> Substitua meio hESC imediatamente, e colocar a placa de volta para a incubadora. </li></ol> 4. Resultados representativos O resultado final do processo de remoção MEF produz pequenas colónias CTEh não perturbadas (Figura 1D), capazes de sofrer uma expansão significativa em colónias grandes (Figura 2), mantendo a expressão de fabricantes de pluripotência SSEA-4, Out ¾ e Tra 1-81 (Figura 3). Figura 1. CES morfologia da colônia antes e após remoção MEF. Humanos Colônias células-tronco embrionárias em um dia) 1 e B) dias após a semeadura em duas placas de cultura. C) de Alta Densidade Humanos Colônias células-tronco embrionárias (H7) cercado por MEFs. As duas colônias CTEh estão descritos no traços vermelhos. D) Após a remoção MEF usando a técnica de aspiração descrito, ficamos com uma colônia isolada com hESC MEFs muito poucos em torno da colônia intacta. <img alt="Figura 2" src="/files/ftp_upload/3951/3951fig2.jpg" /> Figura 2. CES Expansão Colônia 10 dias após a remoção MEF. HESC A colônia original imediatamente após a depleção MEF (à esquerda) é permitido a expansão em uma colônia muito grande (direita) adeximately 800 pm, 10x. Note-se que a colónia original, é criada uma imagem com a mesma ampliação (10x) quanto à colónia composto. <img alt="Figura 3" src="/files/ftp_upload/3951/3951fig3.jpg" /> Figura 3. A imunocoloração de colónias MEF com depleção CTEh. Imunofluorescente identificação de colónias de até 10 dias após a depleção MEF mostra que as colónias manter a sua expressão de marcadores de pluripotência, como é evidente pelos outubro ¾, SSEA-4, e-Tra 1-81 coloração. Além disso, estas colónias CTEh não apresentam diferenciação para um destino mesodérmica, indicado pela ausência de Flk-1. A, E, I e M) são imagens de transmissão de luz das colónias coradas immunoflouscently, 20, 10, 10 e 2x, . respectivamente B, F, J e N) mostram os núcleos -. células DAPI corados das colónias coradas imunofluorescência, 20, 10, 10 e 2x, respectivamente CD) mostram a expressão demarcador de células estaminais humanas SSEA-4 apenas, e imagem composta, 20x. GH) mostram a expressão de células estaminais humanas marcador outubro ¾ só, e imagem composta, KL 10x.) mostram a expressão do marcador de células estaminais humanas Tra 1-81 só e imagem composta, 10x. MP) A última linha se imagens retratam uma colônia retratar as fronteiras típicas suaves visto com este aumento, 2x, e essas colônias não expressa O) Flk-1, um marcador precoce de diferenciação mesoderme, nem eles P) contém fibroblastos como mostrado pela ausência de coloração DDR2. Vídeo suplementar. Remoção fibroblastos por aspiração Folha. <a href="http://www.jove.com/files/ftp_upload/3951/3951movie1.mp4" target="_blank">Clique aqui para ver filme</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>

Não há conflitos de interesse declarados.

O método apresentado neste manuscrito oferece uma alternativa rápida e menos onerosa para eliminar células alimentadoras de fibroblastos humanos de culturas de células embrionárias. A eliminação bem sucedida de fibroblastos é dependente da existência de uma monocamada confluente de firmemente estas células que se desenvolvem em culturas de células estaminais mais longos. Depois de 7-10 dias, as colónias que crescem CTEh vai empurrar os fibroblastos de alimentação numa direcção para fora, gerando uma mono...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Marcador de fibroblastos </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> Marcador Núcleo Celular </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> Humana ESC Marcador </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> Humana ESC Marcador </td></tr><tr><td> Flk-1 </td><td> BD Pharma </td><td> 555307 </td><td> Marcador de diferenciação precoce </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> Humana ESC Marcador </td></tr></tbody></table> Tabela 1. Tabela de rea específicagents utilizado para a imunocoloração o hESC.

rapid fibroblast removal from high density human embryonic stem cell cultures

Fibroblastos de rato embrionárias (MEFs) foram utilizados para estabelecer células-tronco embrionárias (hESCs) culturas após blastocisto isolamento 1. Este sistema de alimentação mantém hESCs de sofrer diferenciação espontânea durante a expansão de células. No entanto, este método de co-cultura é um trabalho intensivo, requer pessoal altamente treinado, e os rendimentos a pureza hESC baixo 4. Muitos laboratórios têm tentado minimizar o número de células de alimentação em culturas CTEh (isto é, a incorporação da matriz revestidos pratos ou outros tipos de células alimentadoras 5-8). Estes sistemas de cultura modificados têm mostrado alguma promessa, mas não suplantou o método padrão para hESCs cultura com mitomicina C tratados com fibroblastos de rato embyronic a fim de retardar a diferenciação espontânea indesejada das culturas CTEh. Portanto, as células de alimentação utilizados na expansão hESC deve ser removido durante as experiências de diferenciação. Embora várias técnicas disponíveis para a purificação do co hESClonies (FACS, Macs, ou o uso de vetores resistentes aos medicamentos) de alimentadores, estas técnicas são de trabalho intensivo, caro e / ou destruidores para o hESC. O objetivo deste projeto foi o de inventar um método de purificação que permite a colheita de uma população mais pura de hESCs. Temos observado que em uma cultura confluente hESC, a população MEF pode ser removido usando uma aspiração simples e rápida da folha de MEF. Esta remoção é dependente de vários factores, incluindo a ligação de célula-a-célula lateral MEFs que tem uma menor afinidade de ligação para o prato de cultura de estireno, e a capacidade de as colónias de células-tronco para empurrar para fora os fibroblastos durante a geração do seu próprio &quot; nicho &quot;. O hESC foram então examinadas para SSEA-4, Oct3 / 4 e expressão Tra 1-81 até 10 dias após a remoção do MEF para assegurar a manutenção da pluripotência. Além disso, as colônias CTEh foram capazes de continuar crescendo de em formações maiores após a remoção do MEF, proporcionando um nível adicional de expansão 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

Apesar dos esforços em curso para as culturas de transição para a alimentação livres de condições, a derivação ea cultura de células estaminais embrionárias humanas (CTEh) permanecem em grande parte dependente de co-culturas com alimentadores embrionárias (MEFs). Aqui, mostramos uma nova metodologia para a rápida remoção de alimentadores de culturas CTEh antes da experimentação.

A remoção rápida de fibroblastos de Alta Densidade Humanos culturas de células estaminais embrionárias

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 visualizações.  University of California, Merced. Apesar dos esforços em curso para as culturas de transição para a alimentação livres de condições, a derivação ea cultura de células estaminais embrionárias humanas (CTEh) permanecem em grande parte dependente de co-culturas com alimentadores embrionárias (MEFs). Aqui, mostramos uma nova metodologia para a rápida remoção de alimentadores de culturas CTEh antes da experimentação.

Vídeo: 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.

MATIZ Passaging-tronco embrionárias humanas Cell-linhas com 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 ...

Biologia

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.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

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.

Derivação de células estaminais embrionárias 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.

Rato Epidérmico Neural Stem Cell Crest (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.

Cromatina imunoprecipitação de células estaminais embrionárias 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.

Preparação de pool de plaquetas Lysate Humanos (pHPL) como um suplemento eficiente para animais de soro livre de culturas de células estaminais humanas

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 Análise de Crescimento Humano embrionárias Colony Stem Cell

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.

Diferenciação de células-tronco embrionárias em precursores oligodendroglial

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.

Estabelecer culturas de fibroblastos adultos primária de 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.