一般我々は、容易な取り扱いを確保するために与えられた一度に1つ以上のサンプルを解凍することをお勧めします。ハンドラは、長時間室温と低CO2で培養を残さないよう注意する必要があります。生細胞の全ての遠心は室温で5分間、500〜600 × gで行われます。 MEFを（マウス胚性フィーダー細胞） <ol><li> 37〜プレ暖かいMEFのメディア℃の-80から直ちに° Cおよび37 ° Cの水浴中に浸し、チューブの下半分をMEFバイアルを取り外します。細胞が80％（左の小さな冷凍部分）に解凍する前に、これは30〜45秒ほどかかります。 </li><li>迅速に、層流フードへのチューブをもたらす70％アルコールでダウンスプレー、と予め温めておいた培地に細胞を移す。 </li><li>前の準備プレートからゼラチン溶液を吸引除去する。 </li><li> sixのウェルの各々に滴下方法でMEFの溶液のアリコート2ミリリットル。よく約均等にMEFのを配布してください。 MEFのは、プレートにアタッチできるように一晩MEFプレート、および37℃のインキュベータ内の場所を日付。 </li><li> 6時間後のMEFは、プレートに添付されます。 24〜48時間、めっき後MEFプレートを使用するのが最良です。 4日以上経過であるMEFのプレートを使用しないでください。 </li><li> 37にメディア及び0.05％トリプシン/ EDTA ° Cボンネット、prelabel下の1滅菌50 mlコニカルチューブプリ暖かい色合い。 </li><li>慎重に分割する文化から色相の培地を吸引除去する。優しく1Xリン酸緩衝液（PBS）ウェルあたりの2mLのでウェルを洗浄。 PBSを吸引除去する。ウェルに0.75ミリリットル0.05％トリプシン/ EDTAを追加。 </li><li>蓋を交換し、4倍で細胞を観察します。色相のコロニーを周囲のMEFは撤回を開始する必要があります。 MEFのは十分に丸められると色相のコロニーの境界線が荒いときは、フードにプレートを返します。このプロセスは、まったくの10分より長くしたり、細胞がしない再成長、約5分かかりますありません。 </li><li> MEFの単分子層が完全に（単層が固定して、1つの部品のままとなる場合がある）切り離されるまで、静かに、井戸の底を洗い、上下にピペッティングし。 </li><li>追加の色合いのメディアを含む50 mlコニカルチューブに細胞懸濁液を移す。メディアをオフ吸引、5分間500xg細胞のチューブをスピン、細胞ペレットを乱さないように注意してください。 </li><li> MEFの新しいプレート上にプレートに、追加の色合いのメディアへの細胞の適切に定める量を追加し、自動化されたピペットでソリューション5〜7倍のピペット。 </li><li> MEFの新鮮なプレートからMEFメディアを取り出します。 </li><li>分注し、各ウェルに賢明な色合いのソリューションのドロップを、よくに関して均等にドロップを配布することを確認すること。プレートを振とうすることなく、慎重な色合いのコロニーのシードように一晩37℃のインキュベーターにセルを返します。 </li><li>スプリットから出てくる色合いの細胞は、雪解けから出てくるものと同様に動作する必要があります。 </li></ol>

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

このビデオでは、我々の研究室でどのように日常的に通過の色合いヒト胚性幹細胞株を示した。効率的かつ定期的に分割し、継代は、健康なヒト胚性幹細胞株を維持するために必須です。トリプシンを介した通路は、色相の細胞株の大きな利点である、しかし、それは上の文化をトリプシンしたり、再platting中の単一細胞の大部分を持たないことが重要です。

色合いメディア651.5ミリリットルKO - DMEM 500ミリリットルPenStrep 6.5ミリリットルGlutaMAXTM 6.5ミリリットルNEAA 6.5ミリリットル2 - メルカプトエタノール0.5ul KO血清の交換65ミリリットルPlasmanate 65ミリリットルbFGF2（10 ng / mLの最終的な）0.65ミリリットル

passaging hues human embryonic stem cell-lines with trypsin.

このビデオでは、我々の研究室は、日常的にトリプシンとの色合いのヒト胚性幹細胞株を通路方法を示します。ヒト胚性幹細胞は、細胞培養の成果物であり、高い人口倍加した核型異常を取得する傾向がある。適切な継代は、健康、未分化、karyotypically正常な色合いのヒト胚性幹細胞培養を維持するために必須です。最初に、拡大培養は残留メディアや細胞破片を除去するためにPBSで洗浄し、次いで細胞は温かい0.05％トリプシン- EDTAの最小限のボリュームでオーバーレイされます。トリプシンは、最大5分間までのセルに残って、その後、細胞を穏やかに2mLの血清学的ピペットで外れています。細胞懸濁液は、その後、細胞を穏やかな遠心分離によって収集され、色相のメディアの大容量で採取し、混合される。不活化トリプシンメディアの混合物を除去し、細胞は、予め温めておいた色合いのメディアに再懸濁されている。適切なスプリット比は、（一般的には1:10〜1:20）に計算されます、そして放射線照射マウス胎児線維芽細胞のフィーダー細胞の単層を含む1から2日目のプレートに再播種した細胞。新たに接種した色合いの培養プレートを48時間静置され、その後メディアはその後、毎日変更されます。これは核型異常を導入するリスクを増加させるとそれは、単一の細胞懸濁液にダウンtrpsinizeことが重要です。

Watch this Scientific Journal Video about Passaging HuES Human Embryonic Stem Cell-lines with Trypsin. at JoVE.com

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

このビデオでは、我々の研究室は、日常的にトリプシンとの色合いのヒト胚性幹細胞株を通路方法を示します。

トリプシンで継代色合いヒト胚性幹細胞、線

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

This is, where did you run The techniques? Oh, where did I teach you? Belcher.Yeah.The first time I did teach you Bel, I spent the summer at my T.Yeah.And that was like, we did like cartilage stuff there. And so I did like social culture there. And then at Hopkins my senior year I worked with different, so I, for like the engineering thing, it was much more about trying, we engineer three dimensional that.

passaging-hues-human-embryonic-stem-cell-lines-with-trypsin

Research

JoVE Journal

Biology

252.0K ビュー.  Harvard. This is, where did you run The techniques? Oh, where did I teach you? Belcher.Yeah.The first time I did teach you Bel, I spent the summer at my T.Yeah.And that was like, we did like cartilage stuff there. And so I did like social culture there. And then at Hopkins my senior year I worked with different, so I, for like the engineering thing, it was much more about trying, we engineer three dimensional that.

ビデオ: Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

ヒト胚性幹（ES）細胞の増殖

生物学

Passaging Human Neural Stem Cells

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

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

ヒト神経幹細胞を継代

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

ヒト胚性幹細胞のミトコンドリア複合体活性の状態調査

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

染色体分析のためのヒト胚性幹細胞の染色体スプレッドの準備

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.