Supportato da: USPHS concedere NS38500, Istituto nazionale dei disordini neurologici e colpo, NIH, USA; Alleanza Tecnologie Biomediche del Sudest Wisconsin, Milwauke, WI, USA; Bryon Riesch Paralisi Fondazione, Milwaukee, WI, USA; confraternita delle aquile, Midwest capitolo , USA; Nazionale Spinal Cord Injury Association, Milwaukee capitolo, Stati Uniti, North East England Stem Cell Institute di Newcastle University, UK.

Dissezione del Bulge dai follicoli adulti Whisker mouse <ol><li> Noi utilizzare 10 settimane a 6 mesi topi vecchi. Giovani topi spesso produrre più cellule. Euthanize mouse e animali sommergere tutto in miscela 1:1 di betadine (soluzione di iodio) e perossido di idrogeno (disponibile da farmacia) per circa 3 minuti. </li><li> Bagnate topo intero, in particolare la regione del viso, con il 75% di etanolo, e portare il mouse microscopio a dissezione in cappa a flusso laminare. </li><li> Sezionare portabaffi, evitando accuratamente di taglio in bulbi dei capelli e piscina in HBSS. </li><li> Sezionare i follicoli baffi e piscina in HBSS a temperatura ambiente. Farlo tenendo la pelle accanto al follicolo pilifero con una pinza, e poi il taglio di tutto il follicolo con le forbici diritte. Tagliato profondamente per evitare di ferire bulbi piliferi. </li><li> Sollevare follicolo soffio fuori pad baffi e mettere in nuova piastra con HBSS fresca. </li><li> Flush adipous sciolti e tessuto cutaneo con schizzi di buffer, fino a quando i follicoli baffi sono pulite. Se necessario, tagliare coraggio di follicolo baffi e rimuovere il tessuto aderente raschiando e vampate di ripetute HBSS. </li><li> Pin follilce pulire i capelli sul piatto di vetro rivestite Sylgard Petri con aghi affilati tungsteno, utilizzando microforceps per tenere sulla parte della pelle accanto al follicolo. </li><li> Baffi tagliati follicolo longitudically con un MicroBlade. Evitare di tagliare troppo profondamente, in modo da ferire il rigonfiamento. Rimuovere il sangue con schizzi ripetuti fino HBSS andato. Aspetto di sangue è una buona indicazione che il taglio era abbastanza profonda. Se il taglio longitudial non è sufficientemente lunga, può essere allungato con microscissors piegato. </li><li> Fare un taglio trasversale sopra il livello del seno cavernoso e successivamente un secondo taglio trasversale a livello all&#39;interno del seno anello, vicino alla pelle. </li><li> Vedrete ora il rigonfiamento all&#39;interno della capsula. </li><li> Afferra la fine di capsula di collagene con le pinze e stendere il rigonfiamento con un ago di tungsteno piegato. Vedrete ora la capsula vuota e il rigonfiamento isolato. </li><li> Piscina isolato rigonfiamenti in una piastra di coltura separata HBSS a temperatura ambiente. Assicurarsi che nessun altro tessuto sta contaminando i rigonfiamenti pool. </li></ol> Cultura della Espianti rigonfiamento <ol><li> Cappotto 35 piatti millimetri cultura con il collagene mettendo 50 microlitri di collagene e 10 microlitri sterile di NaCl al 6% l&#39;uno accanto all&#39;altro. Mescolare bene con un doppio piegato pipetta Pasteyr e spingere verso il bordo della piastra di coltura. Incubare per una notte in un essiccatore pulito, ma non lasciate asciugare. Prima dell&#39;uso, lavare i piatti con soluzione salina. </li><li> Pre-incubazione piastre di coltura collagene rivestito e risciacquati per circa 3 ore con il terreno di coltura. Terreno di coltura è costituito da 85% alfa-MEM modificato, il 10% di siero fetale bovino e il 5% giorno 11 estratto di embrione di pollo. </li><li> Dopo aver ri-incubazione, togliere terreno di coltura da piatti e aggiungere con rigonfiamenti vari media il meno possibile. Rimuovere l&#39;eccesso di media cultura. </li><li> Incubare per 1 ora, ma non di più, nella cella incubatore in atmosfera umidificata con 10% di ossigeno e 5% di CO 2. </li><li> Dopo 1 ora, delicatamente aggiungere 1,5 ml di terreno di coltura. Espianti rigonfiamento dovrebbero aderire al substrato di collagene. Sostituire il 50% del terreno di coltura quotidiano. </li><li> Entro 3 - 4 giorni, le cellule altamente migratorie emigreranno dal espianti rigonfiamento. Nota la loro morfologia stellate, la motilità, e l&#39;assenza predominante del cellula-cellula contatti. Nel corso dei prossimi giorni, più celle emigreranno, ed emigrò cellule si moltiplicano rapidamente. </li><li> Rimuovere rigonfiamento 2 - 3 giorni dopo l&#39;inizio dei EPI-NCSC emigrazione con un ago di tungsteno affilata. Se i rigonfiamenti sono rimasti più a lungo, tendono ad appiattire e rendere l&#39;impossibilità di ottenere puro EPI-NCSC culture. </li><li> Importante: le cellule rare con morfologia appiattita, che diventano a volte evidenti alcuni giorni più tardi EPI-NCSC, e che sono meno mobili NON sono EPI-NCSC, ma putativo cellule staminali epidermiche / progenitori. Culture composto di queste cellule, o colture miste contenenti EPI-NCSC e putativo cellule staminali epidermiche / progenitori devono essere scartati. </li><li> Suggerimento: NON mantenere le cellule troppo a lungo in media espianto primario, in quanto tendono a differenziare rapidamente ad alta densità cellulare, presuntamente a causa di autocrino / paracrino di segnalazione del fattore di crescita. </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>

In virtù della loro capacità migratorie, l&#39;EPI-NCSC può essere isolata come una popolazione altamente pura di cellule staminali, che possono essere espanse in vitro. Come resti embrionale in una posizione adulta, l&#39;EPI-NCSC sono candidati potenzialmente interessanti per future terapie di sostituzione cellulare, ingegneria biomedica e / o medicina rigenerativa. Abbiamo testato EPI-NCSC in un modello murino di lesioni del midollo spinale, dove si mostrano i tratti desiderabili. Attraverso profili di espressione genica LongSAGE (www...

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

EPI-NCSC sono i resti della cresta neurale embrionale in una posizione adulta, il rigonfiamento dei follicoli piliferi. Si tratta di cellule staminali multipotenti che hanno la proprietà fisiologiche di generare una vasta gamma di tipi di cellule differenziate, tra i neuroni, cellule nervose di supporto, le cellule muscolari lisce, osso / le cellule della cartilagine e melanociti. EPI-NCSC sono facilmente accessibili nella pelle pelosa e può essere isolato come una popolazione altamente pura di cellule staminali. Questo video fornisce un protocollo dettagliato per la preparazione del mouse EPI-NCSC culture dai follicoli dei baffi. Il pad baffi di un topo adulto viene rimosso, e follicoli baffi sezionato. I follicoli vengono quindi tagliati longitudinalmente e poi trasversalmente sopra e sotto la regione di rigonfiamento. Il rigonfiamento viene rimosso dalla capsula collagene e posto in una piastra di coltura. EPI-NCSC cominciano a emigrare dal espianti rigonfiamento 3 o 4 giorni dopo.

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

Qui mostriamo il nostro metodo per isolare il mouse epidermiche cresta neurale delle cellule staminali (EPI-NCSC). Tecnica prevede micro-dissezione follicoli baffi, isolando il rigonfiamento e placeing in colture di tessuti. EPI-NCSC iniziano a emigrare da espianti rigonfiamento sul substrato entro 3 - 4 giorni.

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

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 Views.  Newcastle University. Qui mostriamo il nostro metodo per isolare il mouse epidermiche cresta neurale delle cellule staminali (EPI-NCSC). Tecnica prevede micro-dissezione follicoli baffi, isolando il rigonfiamento e placeing in colture di tessuti. EPI-NCSC iniziano a emigrare da espianti rigonfiamento sul substrato entro 3 - 4 giorni.

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

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES Cell-derivati ​​colture cellulari neuroepiteliale

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Ricerca

Biologia

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 di cellule precursori staminali neurali del mouse

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

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]).

Dissezione di 6,5 embrioni di topo 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.

Conteggio cellule staminali neuronali

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 ...

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.

Valutare motilità delle cellule staminali neurali L&#39;utilizzo di un gel a base di Agarose dispositivo a microfluidi

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.

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

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.

Preparazione del pool lisato piastrinico umani (pHPL) come supplemento efficiente per animali siero-Free Culture sulle cellule staminali umane

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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.

Immagini dal vivo della motilità cellulare e citoscheletro dei singoli neuroni e cellule della cresta neurale in embrioni di 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.

Fibroblast rimozione veloce da alta densità Culture staminali embrionali umane

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