Güneydoğu Wisconsin Biyomedikal Teknoloji Alliance Milwauke, WI, ABD; Bryon Riesch Felç Vakfı&#39;nın, Milwaukee, WI, ABD; Eagles Kardeş Sipariş, Midwest Bölüm USPHS hibe NS38500, Ulusal Nörolojik Hastalıklar Enstitüsü ve İnme, NIH, ABD tarafından desteklenen: ABD Ulusal Spinal Kord Yaralanma Birliği, Milwaukee Bölüm, ABD, Kuzey Doğu İngiltere Kök Hücre Enstitüsü, Newcastle Üniversitesi, Birleşik Krallık.

Yetişkin Fare Whisker foliküllerin Bulge diseksiyonu <ol><li> Biz 10 hafta ile 6 ay eski fareler kullanın. Genç farelerin, genellikle daha fazla hücre verim. Fare ve betadin (iyot çözeltisi) ve yaklaşık 3 dakika boyunca hidrojen peroksit (eczane) 1:1 karışımın içine daldırın tüm hayvan Euthanize. </li><li> Squirt tüm fare,% 75 etanol ile özellikle yüz bölgesi, ve laminer akış kaput diseksiyon mikroskobu fare taşımak. </li><li> Saç soğanı içine kesme, HBSS havuz ve dikkatle kaçınarak, bıyık pedleri parçalara ayır. </li><li> Oda sıcaklığında HBSS bıyık kıl follikülleri ve havuz teşrih. Bir forseps ile saç folikülü cilt tutan ve daha sonra düz makas ile folikül etrafında kesme bunu. Saç soğanı yaralamaktan kaçınmak için derin kesin. </li><li> Bıyık yastık bıyık folikül kaldırın ve taze HBSS ile yeni bir levha koymak. </li><li> Bıyık folikülleri temiz olana kadar tampon squirts gevşek adipous ve dermal doku yıkayın. Gerekirse, bıyık folikül sinir kesme ve kazıma ve HBSS tekrarlanan basması yapışık doku kaldırmak. </li><li> Deri parçası üzerine folikül yanında tutmak için microforceps kullanarak, bilenmiş tungsten iğneler kullanarak Sylgard kaplı cam Petri kabı üzerine temiz saç follilce Pin. </li><li> Kes bıyık MicroBlade longitudically folikül. Bu çıkıntı zarar gibi, çok derin bir kesme kaçının. Gitmiş kadar HBSS tekrarlanan squirts kan çıkarın. Kan Görünüm kesim yeterince derin olduğu için iyi bir göstergedir. Longitudial kesim yeterince uzun değilse, bükülmüş microscissors uzatılabilir. </li><li> Kavernöz sinüs seviyesinden enine kesilmiş ve daha sonra ikinci bir halka sinüs içinde düzeyinde enine kesilmiş, cilde yakın. </li><li> Artık kapsül içindeki çıkıntı göreceksiniz. </li><li> Forseps ile kollajen kapsül sona tut ve şişkinliği bükük bir tungsten iğne ile dışarı rulo. Artık boş kapsül ve izole bir çıkıntı göreceksiniz. </li><li> Havuzu, oda sıcaklığında HBSS bir ayrı bir kültür plakasına şişkinlikler izole edilmiştir. Başka hiçbir doku toplanmış şişkinlikler kirlenmesine neden olduğundan emin olun. </li></ol> Çıkıntı eksplantlar Kültür <ol><li> 50 ul kolajen ve birbirlerine önümüzdeki 10 ul steril% 6 NaCl koyarak Coat kollajen ile 35 mm kültür plakaları. Bir çift bükülmüş Pasteyr pipetlemeyin ile iyice karıştırınız ve kültür plaka kenarına doğru itin. Temiz bir desikatöre gece inkübe, ancak kuru izin vermeyin. Kullanmadan önce, tabak tuzlu su ile durulayın. </li><li> Kültür ortamı ile yaklaşık 3 saat önceden inkübe kollajen kaplı ve durulanır kültür plakaları. Kültür orta,% 85,% 10 fetal sığır serumu ve% 5 gün 11 civciv embriyo ekstresi Alpha-modifiye MEM oluşur. </li><li> Re-inkübasyondan sonra, tabak kültür ortamı kaldırmak ve mümkün olduğunca az orta birkaç şişkinlikler ekleyin. Aşırı kültür ortamı çıkarın. </li><li> Hücre kuvöz, 1 saat% 10 oksijen ve% 5 CO 2 ile nemlendirilmiş bir atmosferde inkübe edin, ama uzun. </li><li> 1 saat sonra 1,5 ml kültür ortamı yavaşça ekleyin. Bulge eksplantlar kollajen altyapısını uymalıdır. % 50 günlük kültür ortamı değiştirin. </li><li> Içinde 3 - 4 gün, yüksek göç hücrelerinin şişkinliği eksplantlar göç edecektir. Stellat morfoloji, motilite ve hücre-hücre kişilerin hakim olmaması dikkat edin. Önümüzdeki birkaç gün içinde, daha fazla hücre göç ve hücreler hızla çoğalacak göç etti. </li><li> EPI-NCSC göç başlangıcı, ucu sivriltilmiş bir tungsten iğne ile şişkinliği 2 - 3 gün sonra çıkarın. Şişkinlikler daha uzun süre kalır, onlar düzleştirmek ve EPI-NCSC saf kültürleri elde etmek imkansız hale eğilimindedir. </li><li> Önemli: Nadir hücreler bazen birkaç gün sonra EPI-NCSC daha belirgin hale gelir ve basık morfolojisi, EPI-NCSC daha az hareketli, ancak varsayılan epidermal kök hücre / atalarıdır. EPI-NCSC ve varsayılan epidermal kök hücre / atalarıdır içeren bu hücrelerin oluşan Kültürler, ya da karma kültürlerin atılır gerekir. </li><li> İpucu: varsayılan parakrin / otokrin büyüme faktörü sinyalizasyon nedeniyle, yüksek hücre yoğunluğu hızla ayırt etmek eğilimi olarak hücreler, primer eksplant orta çok uzun süre tutmak. </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>

Onların göç yeteneği sayesinde, EPI-NCSC, kök hücreler, in vitro açılabilir son derece saf bir nüfus olarak izole edilebilir. Yetişkin bir yerde embriyonik kalıntıları olarak, EPI-NCSC gelecek hücre replasman tedavileri, biyomedikal mühendislik ve / veya rejeneratif tıp için çekici potansiyel adaylardır. Arzu özellikleri göstermektedir Biz, spinal kord yaralanması bir fare modelinde EPI-NCSC test ettik. LongSAGE (www.ncbi.nlm.nih.gov / coğrafi, seri numarası GSE4680) gen ekspresyon profili sayesinde gösterdi ki, bek...

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

EPI-NCSC yetişkin bir konuma, saç köklerinin şişkinliği embriyonik nöral krest kalıntıları vardır. Onlar multipotent kök hücreleri nöronların, sinir destekleyen hücreler, düz kas hücreleri, kemik / kıkırdak hücreleri ve melanositler de dahil olmak üzere geniş bir yelpazede, farklılaşmış hücre türleri oluşturmak için fizyolojik özelliği vardır. EPI-NCSC saçlı deride kolaylıkla erişilebilir ve kök hücreler, son derece saf bir nüfus olarak izole edilebilir. Bu video bıyık kıl follikülleri fare EPI-NCSC kültürlerin hazırlanması için ayrıntılı bir protokol sağlar. Yetişkin bir farenin bıyık ped kaldırılır ve bıyık kıl follikülleri diseke. Folikülleri sonra şişkinliği bölgenin altında ve üstünde boydan boya ve daha sonra enine kesilir. Çıkıntı kollajen kapsül kaldırılır ve bir kültür plaka yerleştirilir. Epi-NCSC şişkinliği eksplantlar 3 ila 4 gün sonra göç etmeye başlar.

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

Burada fare epidermal nöral krest kök hücreleri (EPI-NCSC) izole etmek için yöntem gösteriyor. Tekniği içerir mikro diseksiyon, bıyık kıl follikülleri, şişlik izole ve doku kültürü içine placeing. 4 gün - 3 gün içinde EPI-NCSC alt tabaka üzerine çıkıntı eksplantlar göç etmeye başlar.

Fare Epidermal Sinir Crest Kök Hücre (EPI-NCSC) Kültürler

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

11.5K Görüntüleme.  Newcastle University. Burada fare epidermal nöral krest kök hücreleri (EPI-NCSC) izole etmek için yöntem gösteriyor. Tekniği içerir mikro diseksiyon, bıyık kıl follikülleri, şişlik izole ve doku kültürü içine placeing. 4 gün - 3 gün içinde EPI-NCSC alt tabaka üzerine çıkıntı eksplantlar göç etmeye başlar.

Video: 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.

Fare Nöral Kök Hücre Öncüleri Kültür

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

Biyoloji

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]).

6.5 DPC Fare Embriyolar diseksiyonu

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.

İnsan Nöral Kök Hücreler Sayma

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.

İmmünositokimya: İnsan Sinir Kök Hücreler

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.

Nöral Kök Hücre Motilite değerlendirilmesi Agaroz Jel tabanlı mikroakışkan Cihaz Kullanımı

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.

Fare Sinir Doku Hücre Tek Cezalar Üretimi

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.

Hayvan Serum Ücretsiz İnsan Kök Hücre Kültürleri Verimli Ek olarak Pooled İnsan Trombosit Lizat (pHPL) hazırlanması

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.

Zebra balığı Embriyolar Bireysel Nöronlar ve Sinir Crest Hücreler Aktin Sitoiskelet Hücre Motilite ve Canlı Görüntüleme

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.

Yüksek Yoğunluklu İnsan Embriyonik Kök Hücre Kültürleri gelen Rapid Fibroblast Kaldırma

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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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