作者は感謝して子供の病院、その培養にhNSPCsと初期命令を提供するためのオレンジカウンティ研究所の国立ヒト神経幹細胞資源の博士フィリップH.シュワルツを認めます。

注 ：週に一度私たちのhNSPCsのルーチン培養するために、我々は一日おきに、通常は通過して1時02分メディアの50〜100％に変更。 F12ベースのメ​​ディア：文化メディアは20％のBIT - 9500 DMEMで、1X抗生物質/抗真菌剤、および成長因子（EGF、FGF、およびPDGF 40 ng / mlの時それぞれ）が含まれています。 コー​​トフラスコを準備し、セルの関連付け解除<ol><li> 37℃の組織培養インキュベーターで、継代細胞のために新しいフラスコを準備するには、EMEMで10μg/ mlのヒトフィブロネクチンでコーティングT25フラスコに4時間、または一晩。右の細胞を継代する前に、フィブロネクチン溶液を除去し、PBSでフラスコを洗う。 </li><li>新しいフィブロネクチンでコートフラスコ（各フラスコのためのメディアの半分最後のボリュームが&quot;エアコン&quot;メディアとなるよう）に継代する細胞から古い&quot;エアコン&quot;メディアを転送します。これらの培養条件は、その未分化状態でこれらの細胞を保つのを助ける。 </li><li>一度にすべてのメディアの継代する細胞から削除され、PBSで細胞を1回すすいでください。細胞を乾燥しないように注意してください。 </li><li>直接細胞（T25フラスコ用1.0〜1.5ミリリットル）に細胞解離バッファー（CDB）を追加します。室温で約5分間、バッファーで細胞をインキュベート。静かにフラスコをタップすると細胞剥離をスピードアップに役立ちます。 </li></ol> 、遠心分離し再懸濁し、そしてめっきセル<ol><li>剥がれた細胞をフラスコに（CDBの〜3倍のボリュームを使用）：血清含有培地を（F12 10と％熱不活化ウシ胎児血清DMEM）を追加します。 15 mlコニカルチューブにメディアとセルを削除する。フラスコを洗浄し、残りの細胞を回収するために新鮮な血清含有培地の小さなボリュームを使用してください。 </li><li> 5分間で1000rpm（〜200xg）でメディアと細胞を遠心分離します。 </li><li>血清含有培地オフ吸引し、均一な細胞懸濁液を作るためにピペッティングし、新鮮培地で細胞を懸濁します。 </li><li> 1：02分割のそれぞれの新しいフラスコに再懸濁した細胞の半分を譲渡する。フラスコの新しい道の番号に注意してください。 </li></ol>

<ol>
	<li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Nethercott, H. N., Maxwell, H., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+culture.">Neural stem cell culture.</a> Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Palmer, T. D., Schwartz, P. H., Taupin, P., Kaspar, B., Stein, S. A., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture.+Progenitor+cells+from+human+brain+after+death.">Cell culture. Progenitor cells from human brain after death.</a> Nature. 411, 42-43 (2001).</li><li>Schwartz, P. H., Bryant, P. J., Fuja, T. J., Su, H., O'Dowd, D. K., Klassen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+post-mortem+human+cortex.">Isolation and characterization of neural progenitor cells from post-mortem human cortex.</a> J Neurosci Res. 74, 838-851 (2003).</li></ol>

我々は、このプロトコルはhNSPCsの信頼性の高い文化を提供することを見出した。我々の細胞の一つの重要な要因は、彼らはかなり密培養物として維持されると細胞が疎であることがポイントに継代することができる必要があるということです。私たちの手では、疎な文化は非常にゆっくりと成長または完全に分割することを中止。文化は非常に密集しているときにこのような理由から、我々は、通常私たち?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BIT-9500 (BSA, Insulin, Transferrin)</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>09500</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic-Antimycotic</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>DMEM:F12 (1X)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>FGF, human basic recombinant</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-18B</td>
<td>&nbsp;</td>
<tr>
<td>PDGF-AB</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-00AB</td>
<td>&nbsp;</td>
<tr>
<td>EGF, human recombinant</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40052</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Cell Culture Flask, 25 cm2</td>
<td>Tool</td>
<td>Corning</td>
<td>10-126-28</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fibronectin, human, natural</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40008A</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Human Neural Stem/Precursor Cells</td>
<td>Cells</td>
<td>National Human Neural Stem Cell Resource http://www.nhnscr.org/default.htm </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell Dissociation Buffer</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>13150-016</td>
<td>&nbsp;</td>		</tbody>
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passaging human neural stem cells

in vitroでのヒト神経幹/前駆細胞（hNSPCs）を操作する能力は、治療目的のための細胞移植としてのユーティリティを調査するだけでなく、人間の神経発生と病理学の多くの基本的なプロセスを探索する手段を提供します。このプロトコルは、この手法を標準化し、ヒト幹細胞研究の再現性を高めることを望んで培養し、継代hNSPCsの簡単な方法を提示。我々が使用するhNSPCsは国立ヒト神経幹細胞資源で死体生後脳の皮質から単離し、フィブロネクチン（パーマーら、2001;シュワルツら、2003。）でコーティングしたフラスコ上に付着培養物として増殖させた。 DMEMで我々の文化は私たちのhNSPCs：F12無血清培地はEGF、FGF、およびPDGFを補充し、通過それらの1時02分、約7日ごと。これらの条件を使用して、培養中の細胞の大半は、バイポーラの形態を維持し、未分化な神経幹細胞（そのようなネスチンとSox2など）のマーカーを発現する。

Watch this Scientific Journal Video about Passaging Human Neural Stem Cells at JoVE.com

遺伝子発現 |生物学ビデオコレクション

Passaging Human Neural Stem Cells

in vitroでのヒト神経幹/前駆細胞（hNSPCs）を操作する能力は、治療目的のための細胞移植としてのユーティリティを調査し、人間の神経発生を探索することができます。このプロトコルは、ヒト幹細胞研究の増加再現性を期待して培養し、継代hNSPCsの方法を提示。

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 ...

Research

JoVE Journal

Biology

14.5K ビュー.  University of California, Irvine (UCI). in vitroでのヒト神経幹/前駆細胞（hNSPCs）を操作する能力は、治療目的のための細胞移植としてのユーティリティを調査し、人間の神経発生を探索することができます。このプロトコルは、ヒト幹細胞研究の増加再現性を期待して培養し、継代hNSPCsの方法を提示。

ビデオ: Passaging Human Neural Stem Cells

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

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 ...

生物学

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

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofectorでヒト神経幹細胞をトランスフェクト

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

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 ...

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.

免疫細胞：ヒト神経幹細胞

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

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 ...

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

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 physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

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 ...

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 ...

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 ...