私たちは、原稿上で貴重な議論のためにイ·ラボのメンバーに感謝したいと思います。李研究室での作業は、細胞研究基金（TEDCO）を幹ニューヨーク幹細胞財団のロバートソン研究者賞からメリーランド州からの補助金によって支えられている。

96ウェルプレートの1の調製<ol><li> 9μlの単一細胞溶解溶液に単セルDNase1の1μLを混ぜる。 </li><li> 96ウェルPCRプレートの各ウェルに10μlの混合溶液を入れた。 </li></ol> FACS精製のために2。の切り離したhESC <ol><li>ヒトESメディアで中和し、37℃で20分間、1ミリリットルをAccutase 60 mmディッシュ、、からOCT4 :: EGFP ES細胞株を取り外します。 </li><li> FACS緩衝液1ml中に細胞集団を調製し、1×10 6細胞/ mlに細胞を調節する。 </li><li> 35μmのセルストレーナーキャップチューブを介して細胞試料を渡す。 </li><li>細胞選別の前に氷の中にチューブに保管してください。 </li></ol> 96ウェルプレートの各ウェルにおけるFACS精製した単一細胞の3。溶解<ol><li>訓練されたオペレータとのセルソーターにEGFP陽性細胞のためのサンプルをソートします。 96ウェルPCRプレートの単一のセルLysis/DNase1液に単一のセルを置く。必要に応じて、9サンプル6ウェルプレートは一月未満-80℃の冷凍庫に保存することができる。 </li><li>細胞溶解を室温でサンプルを5分間インキュベート。 </li><li>溶解反応を停止させる停止溶液1μLを加える。 </li><li>室温で2分間インキュベートする。 </li></ol> 4。逆転写<ol><li> 20μMのSMA-T15、SMA-Aの各0.5μlのアリコートに追加します。 </li><li>各ウェルに4μL5Xバッファー、2μlのDTT、1μL逆転写酵素、および1μlのdNTPを追加します。 </li><li>サーマルサイクラーに逆転写を行う。 <ol><li> 90分×42℃の熱プログラムを設定し、85℃×5分で逆転写酵素を不活性化する。 </li></ol></li></ol> 5。増幅<ol><li>各逆転写されたサンプルにExoSAP-IT試薬4を添加する。 <ol><li> ExoSAP-IT試薬を不活性化する15分間、15分間および80℃で、37℃でサンプルをインキュベートする。 </li></ol></li><li> PCR反応混合物Wを準備するSMA-P2（2 nM）のi番目</li><li>各逆転写されたサンプルにPCR反応混合物10μlを追加します。 </li><li>変性の20サイクル（30秒94°C）からなる増幅を行う、アニーリング（30秒、57°C）、及び（10分間68°C）伸長。 </li></ol> 6。定量RT-PCR性能<ol><li>各ウェルに2倍のSYBR Green PCRマスターミックス、1μLの増幅したcDNA、2 nMのプライマー、および7μlの水を10μlを加える。 <ol><li> ×40サイクル30秒、3秒、95℃に続いて、プログラムを設定し、60℃。 </li></ol></li><li>技術的なエラーのために二重に行う。 </li></ol>

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著者らは、開示することは何もありません。

単一細胞遺伝子プロファイリングは、単一のセルまたは集団全体の機能性を予測するための主要なツールである可能性があります。技術的な制限により、全遺伝子プロファイリング解析は、人口の平均値に制限されています。個々の細胞亜集団との間の遺伝子発現パターンおよびレベルの変動は、誤った解釈を引き起こすことが提案されている。このような多様な細胞の側面はヒトES細胞で?...

ほとんどの高等真核生物の個体数は、プールされた人口の分析をこのように不均一であり、それはそれらの細胞の機能を解釈することは困難であることが多い。集団内の個々の細胞は微妙に異なっていてもよく、これらの違いは、全人口1,2の性質及び機能に重要な影響を有することができる。特に、ヒト胚性幹細胞（hESC）は微妙に独特のやり方3、4系統の仕様に多能性と多様な電位の異なるレベルを引き起こす、不均一であることが知られている。例えば、異なる細胞表面抗原は、未分化の多能性幹細胞を分類するために使用することができ、5オースティンスミス基は、それらの形態、分化傾向と経路6シグナルの依存性をもとにして、マウス胚性幹細胞における多能性の異なるレベルを提案した。この現象は、ヒト胚に仮定されたnicの幹細胞を7。全体的な研究は、異なる幹細胞株ではなく、個々の単一の幹細胞の中で行われたのに対し、それは潜在的に全ての体細胞系統に向かって分化能力に影響を与える単一細胞レベルで多能性の異なるレベルを分析することは非常に興味深いかもしれない。 細胞および分子不均一性は、 &#39;単一細胞トランスクリプトーム」と呼ばれる、転写プロファイリングによって決定し、遺伝子発現レベルを8月10日を定量化するための新しいアプローチを強調している可能性があります。個々の細胞における遺伝子発現レベルの分析のために、我々は、単一細胞の定量的RT-PCRの、単純で堅牢なプロトコルを開発した。我々は最小限の技術的変動および相違点で、その結果、各単一細胞溶解物の半分だけでなく、ヒトES細胞の段階希釈した全RNAを比較することによって、我々のプロトコルの有効性と実現可能性を確認した。さらに、我々は均質なPを分離するために遺伝的レポーターラインを使用ジーンターゲッティングシステムを使用してhESCのopulation。 OCT4遺伝子座（OCT4-2A-EGFP-PGK-ピューロ構築物）およびTALENプラスミドの対を標的化するためのドナーベクター11を使用した。ドナーベクターとTALENプラスミドのペアは、私たちのクレオを使用してヒトES細胞（H9、WA09）に導入し、ヒトES細胞のクローン選択プロトコルおよびメンテナンスは私たちの日常的プロトコル12に基づいて実施された。我々は、この遺伝的レポーターラインは、OCT4にOCT4発現のために、EGFPを表現:: EGFPのhESCを確認した。 私たちの結果は（OCT4によって並べ替え:: EGFP強陽性細胞）は、個々のヒトES細胞は、OCT-4の発現レベルが高いが、Nanog発現の異なるレベルを保持することを実証している。したがって、我々の単一細胞の遺伝子発現アッセイは、多能性幹細胞の集団の不均一性を研究するのに有用であるべきである。

<table border="0" cellpadding="0" cellspacing="0" width="807">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">96 wll PCR Plate</td>
			<td style="width:123px;">USA scientific</td>
			<td style="width:119px;">1402-8900</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Ambion Cell Lysis Kit</td>
			<td style="width:123px;">Life Technologies</td>
			<td align="right" style="width:119px;">4458235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SMARTScribe Reverse Transcriptase</td>
			<td style="width:123px;">Clonetech</td>
			<td align="right" style="width:119px;">639536</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ExoSAP-IT</td>
			<td style="width:123px;">USB</td>
			<td align="right" style="width:119px;">78200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">11304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">10mM dNTP Mix, PCR Grade</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">18427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SYBR Universal 2X Master Mix</td>
			<td style="width:123px;">Kapa biosystem</td>
			<td style="width:119px;">KR0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>S-1100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FACS buffer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td>45 ml PBS, 5 ml a-MEM, 100 ul DNase, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:123px;">BD Biosciences</td>
			<td align="right" style="width:119px;">352235</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling individual human embryonic stem cells by quantitative rt-pcr

幹細胞集団の不均一性は、異なる系統に向けてその分化の傾向として、幹細胞生物学の詳細な理解を妨げる。単一細胞トランスクリプトーアッセイは、個人差を解剖するための新しいアプローチすることができます。我々は、単一細胞のqRT-PCR法を開発し、この方法は、いくつかの遺伝子発現プロファイルにうまく機能することを確認した。単一細胞レベルでは、OCT4並び順各ヒト胚性幹細胞、:: EGFP陽性細胞は、OCT4高発現であるが、NANOGの発現の異なるレベルを有する。我々の単一細胞の遺伝子発現アッセイは、不均質集団に質問するのに有用であるべきである。

効率的かつ堅牢な単一細胞RNA増幅 ヒトES細胞の中で転写変動を最小限に抑えるために、我々は、OCT4を使用:: FACS精製用のEGFPのhESCクローン。 SMA-T15（GACATGTATCCGGATGTTTTTTTTTTTTTTTT）プライマーを用いてSMA-A（ACATGTATCCGGATGTGGGで固定96ウェルプレートにOCT4 :: EGFP陽性細胞を選別した後、各細胞を溶解緩衝液で溶解し、完全長cDNAにポリ（A）+ RNAに変換...

Watch this Scientific Journal Video about Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR at JoVE.com

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

単一細胞の遺伝子発現アッセイは、幹細胞の不均質性を理解するために必要とされる。

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

profiling-individual-human-embryonic-stem-cells-by-quantitative-rt-pcr

Research

JoVE Journal

Biology

11.5K ビュー.  Johns Hopkins University School of Medicine. 単一細胞の遺伝子発現アッセイは、幹細胞の不均質性を理解するために必要とされる。 

ビデオ: Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

磁気共鳴イメージングのためのヒト胚性幹細胞のin vitroでラベリングの

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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 ...