Unterstützt durch: USPHS gewähren NS38500, National Institute of Neurological Disorders and Stroke, NIH, USA; Biomedical Technology Alliance von Southeastern Wisconsin, Milwauke, WI, USA; Bryon Riesch Paralysis Foundation, Milwaukee, WI, USA; Fraternal Order of Eagles, Midwest Kapitel , USA, National Spinal Cord Injury Association, Milwaukee Kapitel, USA, North East England Stem Cell Institute, Universität Newcastle, UK.

Dissection of the Bulge von erwachsenen Maus Whisker Follikel <ol><li> Wir verwenden 10 Wochen bis 6 Monate alten Mäusen. Jüngere Mäusen liefern oft mehr Zellen. Euthanize Maus und tauchen ganze Tier in 1:1-Mischung von betadine (Iodlösung) und Wasserstoffperoxid (erhältlich von der Apotheke) für ca. 3 Minuten. </li><li> Squirt gesamte Maus, vor allem das Gesicht, mit 75% Ethanol, und tragen Maus Dissektionsmikroskop in Laminarströmungshaube. </li><li> Dissect Schnurrhaarkissen, sorgfältig zu vermeiden Einschneiden Haarzwiebeln, und Pool in HBSS. </li><li> Dissect Whisker Follikel und Pool in HBSS bei Raumtemperatur. Tun Sie dies, indem Haut neben Haarfollikel mit einer Pinzette und dann Schneiden um den Follikel mit der geraden Schere. Cut tief zu verletzen Haarwurzeln. </li><li> Heben Sie Whisker Follikel von Whisker-Pad und in neue Platte mit frischem HBSS. </li><li> Flush verlieren adipous und Hautgewebe mit spritzt Puffer, bis Whisker Follikel sauber sind. Wenn nötig, schneiden Nerv Whisker Follikel und entfernen anhaftendem Gewebe durch Schaben und wiederholte Spülungen der HBSS. </li><li> Pin sauberes Haar follilce auf Sylgard-beschichtetes Glas-Petrischale mit geschärften Wolfram Nadeln, mit microforceps zur Aufnahme auf die Haut Teil neben dem Follikel. </li><li> Cut Whisker Follikel longitudically mit einem MicroBlade. Vermeiden Sie zu tief, da dies die Ausbuchtung verletzen wird. Entfernen Sie Blut spritzt wiederholt HBSS bis gegangen. Auftreten von Blut ist ein gutes Indiz dafür, dass der Schnitt tief genug war. Wenn der Längsschlitten Schnitt ist nicht lang genug, kann es mit gebogenen Mikroschere verlängert werden. </li><li> Machen Sie einen Schnitt quer über dem Niveau des Sinus cavernosus und anschließend eine zweite Schnitt quer auf der Ebene innerhalb des Rings Sinus, um die Haut zu schließen. </li><li> Sie sehen nun die Ausbuchtung im Inneren der Kapsel. </li><li> Schnappen Sie ein Ende zu Kollagenkapsel mit der Zange und Roll-out der Ausbuchtung mit einem gebogenen Wolframnadel. Sie sehen nun die leere Kapsel und das isolierte Ausbuchtung. </li><li> Pool isoliert Ausbuchtungen in einem separaten Kulturplatte in HBSS bei Raumtemperatur. Achten Sie darauf, keine andere Gewebe ist eine Verunreinigung des gepoolten Ausbuchtungen. </li></ol> Culture of Bulge Explantate <ol><li> Coat 35 mm Kulturschalen mit Kollagen, indem 50 pl Kollagen und 10 ul sterilem 6% NaCl nebeneinander. Gut mischen mit einem Doppel-gebogen Pasteyr Pipette und drücken Sie auf den Rand der Kulturschale. Inkubieren über Nacht in einem sauberen Exsikkator, aber nicht trocknen lassen. Vor dem Gebrauch gründlich die Platten mit Kochsalzlösung. </li><li> Pre-Inkubation Kollagen-beschichteten und gespült Kultur-Platten für ca. 3 Stunden mit Kulturmedium. Kulturmedium besteht aus 85% Alpha-modifizierten MEM, 10% fötalem Rinderserum und 5% Tag 11 Hühnerembryo-Extrakt. </li><li> Nach erneuter Inkubation entfernen Kulturmedium aus Platten und fügen mehrere Ausbuchtungen mit möglichst wenig Medium wie möglich. Entfernen Sie das überschüssige Kulturmedium. </li><li> Inkubation für 1 Stunde, aber nicht mehr, in Zellinkubator in einer befeuchteten Atmosphäre mit 10% Sauerstoff und 5% CO 2. </li><li> Nach 1 Stunde sanft 1,5 ml Kulturmedium. Bulge Explantate sollten, um das Kollagen Substrat haften. Ersetzen Sie 50% des Kulturmediums täglich. </li><li> Innerhalb von 3 bis 4 Tagen werden weit wandernden Zellen aus dem Wulst Explantate auszuwandern. Beachten Sie ihre sternförmige Morphologie, Motilität und vorherrschenden Abwesenheit von Zell-Zell-Kontakten. In den nächsten Tagen werden mehrere Zellen auswandern, und emigrierte Zellen rasch vermehren. </li><li> Entfernen Ausbuchtung 2 bis 3 Tage nach Beginn der EPI-NCSC Auswanderung mit einem geschärften Wolframnadel. Wenn die Ausbuchtungen länger bleiben, neigen sie zu glätten und machen es unmöglich, reine EPI-NCSC Kulturen zu erhalten. </li><li> Wichtig: Rare Zellen mit abgeflachten Morphologie, die manchmal sichtbar werden einige Tage später als EPI-NCSC, und welche weniger beweglich sind NICHT EPI-NCSC, aber putative epidermalen Stammzellen / Vorläuferzellen. Kulturen aus diesen Zellen, oder Mischkulturen mit EPI-NCSC und vermeintlichen epidermalen Stammzellen / Vorläuferzellen müssen entsorgt werden. </li><li> Tipp: Bewahren Sie keine der Zellen zu lange in primäre Explantat Medium, da sie schnell zu differenzieren bei hoher Zelldichte, vermeintlich durch autokrine / parakrine growth factor Signalisierung neigen. </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>

Aufgrund ihrer Fähigkeit, wandernde, kann EPI-NCSC als hochreines Population von Stammzellen, die in vitro erweitert werden kann isoliert werden. Da embryonale Reste eines erwachsenen Lage sind EPI-NCSC potenziell attraktive Kandidaten für die zukünftige Zellersatztherapien, Biomedizintechnik und / oder der regenerativen Medizin. Wir haben EPI-NCSC in einem Mausmodell für Rückenmarksverletzungen getestet, wo sie wünschenswerte Merkmale zeigen. Durch Genexpressionsanalysen von LongSAGE (www.ncbi.nlm.nih.gov / geo, Seriennummer GSE4680)...

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

EPI-NCSC sind Reste der embryonalen Neuralleiste bei einem Erwachsenen Lage, die Wölbung der Haarfollikel. Sie sind multipotente Stammzellen, die die physiologische Eigenschaft auf eine breite Palette von differenzierten Zelltypen, einschließlich Neuronen, Nerven Stützzellen, glatte Muskelzellen, Knochen / Knorpel-Zellen und Melanozyten zu generieren. EPI-NCSC sind leicht zugänglich in der behaarten Haut und kann als hoch reinen Population von Stammzellen isoliert werden. Dieses Video gibt einen detaillierten Protokoll für die Vorbereitung Maus EPI-NCSC Kulturen aus Whisker Follikel. Die Whisker pad einer erwachsenen Maus entfernt wird, und Whisker Follikel zerlegt. Die Follikel sind dann in Längsrichtung und anschließend quer geschnitten oberhalb und unterhalb der Ausbuchtung Region. Die Ausbuchtung wird aus dem Kollagen Kapsel entfernt und in eine Kulturschale. EPI-NCSC beginnen mit der Ausbuchtung Explantate 3 bis 4 Tage später emigrieren.

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

Hier zeigen wir unsere Methode zur Maus epidermalen Neuralleiste Stammzellen (EPI-NCSC) zu isolieren. Technik beinhaltet Mikro-Sezieren Whisker Follikel, die Isolierung des Bulge und Silber reflektiert sie in Gewebekultur. EPI-NCSC beginnen mit Ausbuchtung Explantate auf das Substrat innerhalb von 3 auswandern - 4 Tage.

Maus Epidermal Neural Crest Stem Cell (EPI-NCSC) Kulturen

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 ...

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Research

JoVE Journal

Biology

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

11.4K Aufrufe.  Newcastle University. Hier zeigen wir unsere Methode zur Maus epidermalen Neuralleiste Stammzellen (EPI-NCSC) zu isolieren. Technik beinhaltet Mikro-Sezieren Whisker Follikel, die Isolierung des Bulge und Silber reflektiert sie in Gewebekultur. EPI-NCSC beginnen mit Ausbuchtung Explantate auf das Substrat innerhalb von 3 auswandern - 4 Tage.

Serie: 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.

Kultur der Maus 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 ...

Forschung

Biologie

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]).

Dissection von 6,5 dpc muriner Embryonen

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.

Counting Nervenstammzellen

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 ...

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.

Immunzytochemie: Human Neural Stem Cells

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.

Die Beurteilung Neural Stem Cell Motilität Mit einem Agarosegel-basierte Mikrofluidikvorrichtung

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.

Generierung von Einzel-Zellsuspensionen aus Maus Nervengewebe

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.

Vorbereitung der gepoolten menschlichen Thrombozyten-Lysat (PHPL) als effiziente Ergänzung zum tierischen Serum-freien menschlichen Stammzell-Kulturen

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.

Live-Imaging von Zellbewegung und Aktin-Zytoskelett von einzelnen Neuronen und Zellen der Neuralleiste in Zebrafisch-Embryos

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.

Schnelle Fibroblast Entfernung aus High Density humanen embryonalen Stammzellen Kulturen

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...