Apoiado por: USPHS conceder NS38500, Instituto Nacional de Distúrbios Neurológicos e Derrame, NIH, EUA; Alliance Tecnologia Biomédica da Southeastern Wisconsin, Milwauke, WI, EUA; Bryon Paralysis Foundation Riesch, Milwaukee, WI, EUA; Ordem Fraternal de Eagles, Centro-Oeste Capítulo , EUA; National Spinal Cord Injury Association, Capítulo Milwaukee, EUA; North East England Stem Cell Institute, da Universidade de Newcastle, no Reino Unido.

Dissecção do Bulge de Folículos Whisker Adulto rato <ol><li> Usamos 10 semanas a 6 meses ratos velhos. Ratos mais jovens, muitas vezes produzir mais células. Euthanize rato e animal submerge inteiro em 1:01 mistura de betadine (solução de iodo) e peróxido de hidrogênio (disponível em farmácias) para cerca de 3 minutos. </li><li> Squirt do mouse todo, especialmente a região facial, com etanol 75%, e levar o mouse para microscópio de dissecção em capela de fluxo laminar. </li><li> Dissecar almofadas bigode, evitando cuidadosamente corte em bulbos capilares e piscina em HBSS. </li><li> Dissecar folículos bigode e piscina em HBSS à temperatura ambiente. Fazer isso, mantendo a pele junto ao folículo piloso com uma pinça, e depois corte em torno do folículo com a tesoura reta. Cortar profundamente para evitar ferir os bulbos de cabelo. </li><li> Levante folículo whisker fora do bloco de bigode e colocado em placa de novo com HBSS fresco. </li><li> Lave adiposo frouxo e tecido dérmico com esguichos de tampão, até que os folículos whisker estão limpos. Se necessário, corte coragem de folículo bigode e remover o tecido aderente por raspagem e flushes repetida de HBSS. </li><li> Pin follilce cabelo limpo para Sylgard revestido prato de vidro Petri utilizando agulhas de tungstênio afiadas, usando micropinças para se agarrar a parte da pele ao lado do folículo. </li><li> Whisker corte folículo longitudically com um MicroBlade. Evitar o corte muito fundo, pois isso irá prejudicar a protuberância. Remoção de sangue com jatos repetida de HBSS até ido. Aparecimento de sangue é uma boa indicação de que o corte foi profundo o suficiente. Se o corte longitudial não é suficientemente longo, pode ser alongado com microtesoura dobrados. </li><li> Faça um corte transversal acima do nível do seio cavernoso e, posteriormente, um segundo corte transversal ao nível do anel no interior do seio, perto da pele. </li><li> Você vai ver agora a protuberância dentro da cápsula. </li><li> Pegar um final de à cápsula de colágeno com a pinça e roll out do bojo com uma agulha de tungstênio dobrados. Você vai ver agora a cápsula vazia e isolada do bojo. </li><li> Piscina isolada protuberâncias em uma placa de cultura separados em HBSS à temperatura ambiente. Certifique-se de qualquer outro tecido está contaminando as protuberâncias pool. </li></ol> Cultura de explantes bojo <ol><li> 35 placas de revestimento milímetros cultura com colágeno colocando 50 mL de colágeno e 10 ml estéril NaCl 6% ao lado do outro. Misture bem com uma pipeta Pasteyr duplo dobrado e empurrar para a borda da placa de cultura. Incubar durante a noite em um dessecador limpo, mas não deixe secar. Antes do uso, lavar as placas com solução salina. </li><li> Pré-incubar colágeno revestido e lavadas placas de cultura por aproximadamente 3 horas com meio de cultura. Meio de cultura é composto por 85% de alfa-MEM modificado, 10% de soro fetal bovino e 5% dia 11 extrato de embrião de galinha. </li><li> Depois de re-incubação, remova o meio de cultura a partir de chapas e adicionar várias protuberâncias com o meio o menos possível. Remova o meio de cultura em excesso. </li><li> Incubar por 1 hora, mas não mais, na célula incubadora em uma atmosfera com oxigênio umidificado 10% e 5% CO 2. </li><li> Após 1 hora, gentilmente adicionar 1,5 ml de meio de cultura. Explantes bojo devem aderir ao substrato de colágeno. Substituir 50% do meio de cultura diária. </li><li> Dentro de 3-4 dias, as células altamente migratórias vai emigrar de explantes bojo. Nota sua morfologia estreladas, motilidade e ausência predominante de célula-contatos. Ao longo dos próximos dias, mais células vão emigrar, e emigrou células irão proliferar com rapidez. </li><li> Remover bojo 2-3 dias após o início da EPI-NCSC emigração com uma agulha de tungstênio afiadas. Se as protuberâncias ficam mais tempo, eles tendem a achatar e tornar impossível a obtenção de pura EPI-NCSC culturas. </li><li> Importante: Rare células com morfologia achatada, que se tornam, por vezes, aparente vários dias mais tarde do que EPI-NCSC, e quais são menos móveis NÃO são EPI-NCSC, mas putativo de células-tronco da epiderme / progenitores. Culturas consistindo destas células, ou culturas mistas contendo EPI-NCSC e putativo de células-tronco da epiderme / progenitores precisam ser descartados. </li><li> Dica: Não manter as células por muito tempo em meio explante primário, como eles tendem a diferenciar rapidamente em alta densidade celular, supostamente devido a autócrina / parácrina de sinalização do fator de crescimento. </li></ol>

<ol>
	<li>Sieber-Blum, M., Grim, M., YF, H. u., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Sieber-Blum, M., Grim, M., Tuan, R., Grim, 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+adult+hair+follicle+-+cradle+for+pluripotent+neural+crest+stem+cells.">The adult hair follicle - cradle for pluripotent neural crest stem cells.</a> Birth Defects Research (Part C). 72, 162-172 (2004).</li><li>Sieber-Blum, M., Schnell, L., Grim, M., Schneider, R., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+epidermal+neural+crest+stem+cell+(EPI-NCSC)+grafts+in+the+lesioned+spinal+cord.">Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.</a> Pub. 32, 67-81 (2006).</li><li>Hu, Y. F., Zhang, Z. -. J., Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidermal+neural+crest+stem+cell+(EPI-NCSC)+molecular+signature.">An epidermal neural crest stem cell (EPI-NCSC) molecular signature.</a> Stem Cells. 24, 2692-2702 (2006).</li></ol>

Em virtude da sua capacidade migratória, EPI-NCSC pode ser isolado como uma população altamente puro de células-tronco, que podem ser expandidos in vitro. Como restos embrionários em um local adulto, EPI-NCSC são candidatos potencialmente atraente para futuras terapias com células de substituição, engenharia biomédica e / ou medicina regenerativa. Nós testamos EPI-NCSC em um modelo do rato de lesão da medula espinhal, onde eles mostram características desejáveis. Através do perfil de expressão gênica por LongSAGE (www.ncbi....

mouse epidermal neural crest stem cell (epi-ncsc) cultures

EPI-NCSC são remanescentes da crista neural embrionária em um local adulto, o bojo dos folículos pilosos. Eles são células-tronco multipotentes que têm a propriedade fisiológica de gerar uma grande variedade de tipos de células diferenciadas, inclusive neurônios, células nervosas de apoio, células musculares lisas, osso / células de cartilagem e melanócitos. EPI-NCSC são facilmente acessíveis na pele aveludada e podem ser isoladas como uma população altamente puro de células-tronco. Este vídeo fornece um protocolo detalhado para a preparação do mouse EPI-NCSC culturas de folículos bigode. A almofada de bigode de um rato adulto é removido, e os folículos whisker dissecados. Os folículos são então cortadas longitudinalmente e, posteriormente, transversalmente acima e abaixo da região bojo. A protuberância é removida a cápsula de colágeno e colocados em uma placa de cultura. EPI-NCSC começam a emigrar de explantes bojo 3-4 dias depois.

Watch this Scientific Journal Video about Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures at JoVE.com

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Aqui nós mostramos o nosso método para isolar do mouse epidérmicas células da crista neural haste (EPI-NCSC). Técnica envolve a micro-dissecando folículos bigode, isolando o bojo e placeing-lo em cultura de tecidos. EPI-NCSC começam a emigrar a partir de explantes saliência para o substrato dentro de 3-4 dias.

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 ...

mouse-epidermal-neural-crest-stem-cell-epi-ncsc-cultures

Research

JoVE Journal

Biology

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

11.5K visualizações.  Newcastle University. Aqui nós mostramos o nosso método para isolar do mouse epidérmicas células da crista neural haste (EPI-NCSC). Técnica envolve a micro-dissecando folículos bigode, isolando o bojo e placeing-lo em cultura de tecidos. EPI-NCSC começam a emigrar a partir de explantes saliência para o substrato dentro de 3-4 dias.

Vídeo: Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura de rato precursores das células-tronco neurais

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Biologia

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissecção de 6,5 embriões de camundongos dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Contando células-tronco neurais

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passaging células-tronco neurais

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Imunocitoquímica: células-tronco neurais

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Geração de Single-Cell Tissue Suspensões de rato Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

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 ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imagens ao vivo da motilidade celular e citoesqueleto de actina de neurônios individuais e células da crista neural em embriões Zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

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

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

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 ...