、南東部ウィスコンシン州のバイオメディカルテクノロジーアライアンス、Milwauke、WI、アメリカ合衆国、バイロンリーシュ麻痺財団、ミルウォーキー、ウィスコンシン州、米国、イーグルスの友愛会、中西部章USPHS助成NS38500、神経疾患や脳卒中の国立研究所、NIH、米国後援： 、米国、国立脊髄損傷協会、ミルウォーキー章、米国、北東イングランド幹細胞研究所、ニューカッスル大学、英国。

成体マウスのウィスカの卵胞からバルジの解剖<ol><li>我々は、10週間〜6ヵ月齢のマウスを使用してください。若いマウスでは、しばしば複数のセルをもたらす。 betadine（ヨウ素溶液）と約3分間過酸化水素（薬局から入手可能）の1:1混合物中にマウスと水没全体の動物を安楽死させる。 </li><li>潮吹き全体、特にマウス、顔領域、75％エタノールで、そして層流フードで解剖顕微鏡にマウスを運ぶ。 </li><li>慎重に毛の球根にカットし、HBSSでプールを避け、ウィスカーパッドを解剖。 </li><li>室温でのHBSSでウィスカの卵胞とプールを細かく分析。ピンセットで毛包に次のスキンを保持し、ストレートはさみで卵胞の周りにカットすることでこれを行います。毛の球根を負傷を避けるために深くカット。 </li><li>ウィスカーパッドのウィスカの卵胞を持ち上げて、新鮮なHBSSで新しい皿に入れ。 </li><li>ウィスカの卵胞がきれいになるまで、バッファのホヤとゆるいadipousと皮膚組織をフラッシュします。必要に応じて、ウィスカの卵胞に神経を切断し、こするとHBSSを繰り返しフラッシュすることによって付着組織を除去する。 </li><li>卵胞の隣にある皮膚の部分に保持するためのmicroforcepsを使用して、シャープにタングステン針を使用してSylgardコーティングされたガラスペトリ皿にきれいな毛のfollilceを固定。 </li><li>マイクロブレードでlongitudicallyウィスカの卵胞をカット。この膨らみを傷つけるので、あまり深く切断は避けてください。なくなってまで、HBSSの繰り返しホヤで血を取り外します。血液の外観は、カットが十分な深さだったことを示しています。 longitudialカットが十分に長いではない場合、それが曲がっmicroscissorsと長くなるかもしれない。 </li><li>海綿静脈洞のレベルより上の横方向のカットとリングの洞内のレベルで続いて第二横カットを行い、肌に近い。 </li><li>これで、カプセル内部の出っ張りが表示されます。 </li><li>ピンセットでのコラーゲンのカプセルの端をつかんで、曲がったタングステン針で膨らみをロールアウト。これで、空のカプセルと分離された膨らみが表示されます。 </li><li>プールは室温でHBSSで別々の培養プレートにふくらみを分離。他の組織がプールされたふくらみを汚染されていないことを確認します。 </li></ol>バルジ植の文化<ol><li> 50μlのコラーゲンと隣同士に10μlの滅菌六パーセントのNaClを配置することによって、コラーゲンでコーティング35mmの培養プレートを。ダブル曲がったPasteyrのピペットでよく混和して、培養プレートの端に向かって押し込みます。クリーンデシケーター内で一晩インキュベートするが、乾燥させてください。使用前に、生理食塩水でプレートをすすぎます。 </li><li>プレインキュベーションコラーゲンコート培養培地で約3時間とすすぎ培養プレート。培地は85％のα-改変MEM、10％ウシ胎児血清、5％の11日目ニワトリ胚抽出物で構成されています。 </li><li>再インキュベーション後、プレートから培地を除去し、できるだけ少ない培地でいくつかのふくらみを追加。過剰培地を削除します。 </li><li> 10％酸素、5％CO 2の加湿雰囲気で細胞インキュベーターで、1時間インキュベートする、ではなく、より長い。 </li><li> 1時間後、静かに培養液の1.5 mlを加える。バルジの外植片は、コラーゲン基質に従ってください。毎日培養液の50％を交換してください。 </li><li> 3内に - 4日間、高度回遊性細胞は、バルジの外植片から移住予定。それらの星状の形態、運動性、および細胞間接触の支配的な欠如を注意してください。今後数日にわたって、より多くの細胞が移住し、細胞が急速に増殖するでしょう移住予定。 </li><li>シャープタングステン針とEPI - NCSCの移民の発症後3日間 - 膨らみ2を取り外します。バルジが長く残っている場合、それらは平坦化し、それが不可能な純粋なEPI - NCSCの文化を得るためにする傾向がある。 </li><li> 重要 ：EPI - NCSCより数日後、時々明らかになる、と板状形態を持つ希少な細胞では、EPI - NCSC しない場合に少ない運動ですが、推定上の表皮幹細胞/前駆細胞。 EPI - NCSCと推定表皮幹細胞/前駆細胞を含むこれらの細胞で構成される文化、または混合培養では、破棄する必要があります。 </li><li>ヒント：彼らが推定されるオートクリン/パラクリン増殖因子のシグナル伝達のために、高い細胞密度で急速に差別化する傾向があるので、長すぎる一次外植培地中でのために細胞を保持しない。 </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>

それらの回遊能力のおかげで、EPI - NCSCは、in vitroで拡張できる幹細胞の高度に純粋な人口として単離することができる。大人の場所にある胚の残党として、EPI - NCSCは、将来の細胞置換療法、生物医学工学および/または再生医療のための潜在的に魅力的な候補です。我々は、彼らが望ましい形質を示す脊髄損傷のマウスモデルでEPI - NCSCをテストしている。我々が期待どおりに、ことを示したLongSAGE（www.ncbi.nl...

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

EPI - NCSCは大人の場所、毛包のバルジの胚神経堤の名残です。彼らは神経細胞、神経支持細胞、平滑筋細胞、骨/軟骨細胞とメラノサイトを含む分化した細胞型の広い配列を生成するために生理学的性質を持つ多能性幹細胞である。 EPI - NCSCは、有毛皮膚に容易にアクセス可能であり、幹細胞の純度の高い人口として単離することができる。このビデオでは、ウィスカの卵胞からマウスEPI - NCSC培養を調製するための詳細なプロトコルを提供します。成体マウスのウィスカーパッドが削除され、ウィスカの卵胞は、解剖される。卵胞は、バルジ領域の上下に縦方向にし、続いて横方向にカットされています。膨らみは、コラーゲンのカプセルから削除され、培養プレートに配置されます。 EPI - NCSCは3〜4日後にバルジの外植片からの移住を開始します。

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

ここでは、マウス表皮神経堤幹細胞（EPI - NCSC）を分離する手法を示す。テクニックは、膨らみを分離し、組織培養にそれをplaceing、ミクロ解剖ウィスカの卵胞を伴います。 4日間 - EPI - NCSCは、3つの内の基層の上に膨らみの外植片からの移住を開始します。

マウス表皮神経堤幹細胞（EPI - NCSC）文化

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 ビュー.  Newcastle University. ここでは、マウス表皮神経堤幹細胞（EPI - NCSC）を分離する手法を示す。テクニックは、膨らみを分離し、組織培養にそれをplaceing、ミクロ解剖ウィスカの卵胞を伴います。 4日間 - EPI - NCSCは、3つの内の基層の上に膨らみの外植片からの移住を開始します。

ビデオ: 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細胞由来の神経上皮細胞の培養

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 ...

生物学

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.

マウス神経幹細胞の前駆体の文化

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]).

6.5 DPCのマウス胚の解剖

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.

ヒト神経幹細胞を数える

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.

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

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.

マウス神経組織から単細胞懸濁液の生成

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.

動物の無血清ヒト幹細胞培養のための効率的なサプリメントとしてプールしたヒト血小板可溶化物（pHPL）の調製

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.

マウスES胚細胞の単離と導出

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.

細胞運動やゼブラフィッシュ胚における個々の神経細胞と神経堤細胞のアクチン細胞骨格のライブイメージング

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.

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

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

マウス表皮神経堤幹細胞（EPI - NCSC）文化

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ES細胞由来の神経上皮細胞の培養

マウス神経幹細胞の前駆体の文化

6.5 DPCのマウス胚の解剖

ヒト神経幹細胞を数える

アガロースゲルベースのマイクロ流体デバイスを用いた神経幹細胞運動の評価

マウス神経組織から単細胞懸濁液の生成

動物の無血清ヒト幹細胞培養のための効率的なサプリメントとしてプールしたヒト血小板可溶化物（pHPL）の調製

マウスES胚細胞の単離と導出

細胞運動やゼブラフィッシュ胚における個々の神経細胞と神経堤細胞のアクチン細胞骨格のライブイメージング

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

マウス表皮神経堤幹細胞（EPI - NCSC）文化

要約

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ES細胞由来の神経上皮細胞の培養

マウス神経幹細胞の前駆体の文化

6.5 DPCのマウス胚の解剖

ヒト神経幹細胞を数える

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

マウス神経組織から単細胞懸濁液の生成

動物の無血清ヒト幹細胞培養のための効率的なサプリメントとしてプールしたヒト血小板可溶化物（pHPL）の調製

マウスES胚細胞の単離と導出

細胞運動やゼブラフィッシュ胚における個々の神経細胞と神経堤細胞のアクチン細胞骨格のライブイメージング

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

アガロースゲルベースのマイクロ流体デバイスを用いた神経幹細胞運動の評価