Teitellラボでヒト胚性幹細胞研究は再生医療のためのカリフォルニア工科大学（CIRM）種子助成RS1 - 00313でサポートされています。我々は、UCLA、特に博士Amanderクラーク博士ジェロームザック、そして我々の研究の支援のためのUCLAのブロード研究所の幹細胞の中核施設のメンバーで、再生医療と幹細胞研究のブロードセンターのメンバーに感謝。

マウス胚性繊維芽細胞（MEF）上に播種した分割するヒト胚性幹細胞（ヒトES） 通常コンフルエントhESCのプレートは、特定のhESCのラインに応じて、1:10に1時06分を分割することができます。分割板が分割後に再度5〜7日間コンフルエントになります。 <ol><li>分割の前に2日間、0.1％ゼラチン溶液を用いてプレートをゼラチン状にする。 6ウェルプレートの場合は、一晩37℃、5％CO 2組織培養インキュベーターで各ウェルでインキュベートプレートに2ミリリットルを追加します。 </li><li>ゼラチン6ウェルプレート上にプレートMEFのあなたが分割でヒトES細胞を計画する前に、一日。 （注：MEFのは3日以前の必要に応じて、できるだけ多くのようにめっきすることができる〜3日後、MEFのは平坦とスプレッドアウトよりとMEFのできるヒトES細胞だけでなく、新鮮を維持できないとなる。。）G -照射またはマイトマイシンのバイアルテイクCは37℃の水浴中で2分間液体窒素と雪解けから、5-6 × 10 6個の細胞を含む、CF1 MEFSを扱う。 50ミリリットルファルコンチューブに暖かいMEFの培養培地で細胞を1回洗浄し、暖かいMEF培地の最終容量に再懸濁します。 5〜6 × 10 6個の細胞では、これは十分な板二、三、6ウェルプレートになります。 </li><li> MEFのが洗浄されている間、ゼラチン化プレートを取り出して、井戸からすべてのソリューションを削除し、ウェルあたりMEF培地の2.5 mlを加える。 </li><li>各ウェルの最終容量として3ミリリットル達成するために、ウェルあたりMEFの懸濁液の0.5mlを追加。 MEFのは均等に分散して戻って解決するために一晩培養器内の場所の板されていることを確認します。 </li><li>ヒトES細胞の分裂の日に、1mg/mlで新鮮なまたは使用&lt;2週齢の無菌コラゲナーゼIV溶液を調製。分割したいhESCの井戸からすべてのメディアを削除し、2ミリリットルあたりも暖かい1 × PBS、pH7.4で一回洗浄し、コラゲナーゼIV液を1ml加える。 37℃で5〜10分間インキュベートする。インキュベーターのうち6ウェルhESCのプレートを取ると、各ウェルにESメディア（bFGFを除く）の1mlを加える。各ウェル内の溶液を用いて、1mlのピペットを使用してメディアを吸い上げるとプレートの幹細胞を吹き飛ばす。各ウェルについて、これは約5〜10回の繰り返しがかかります。その後、50ミリリットルのファルコンチューブに懸ヒトES細胞を譲渡する。分割されるすべてのウェルに対して、この手順を実行一50ミリリットルファルコンチューブに結合する。ペレットは、5分間200グラムで、室温でヒトESおよびbFGFを欠くESメディアで1回洗う。 </li><li>ヒトES細胞を洗浄されている間、、MEFのプレートを取り出す井戸からすべてのメディアを削除し、1回洗浄滅菌、暖かい1 × PBS、pHを7.4にしてESメディア2.5mlの（現在は10 ng / mlのbFGFを添加）あたりを追加するうまく。 </li><li> 10ng/mlのbFGFを添加したESメディアの適切な量のペレットヒトES細胞を、懸濁します。 （注意：。。ヒトES細胞が再懸濁するとき、彼らはほぼ均一なコロニーの大きさや形があるはずの再懸濁のボリュームが分割比に依存する）ピペットはhESCのサスペンションを上下に数回コロニーは小さく、より均一にするため、しかし、そうでもない、単一の細胞または非常に小さなコロニーが生成されていること。各ウェルの最終容量として3mlを達成するためにMEFプレートにウェル当たりhESCの懸濁液0.5mlを追加します。視覚的にヒトES細胞を沈降一晩インキュベーターにバックプレートを配置する前に均等に分散されていることを確認してください。メッキした後、それは通常、コロニ​​ーが彼らの特徴的な形状と境界線の外観に反映するために数日かかります。 </li></ol>

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science . 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. 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=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nature Biotechnology . 19, 971-974 (2001).</li></ol>

このビデオでは、マウス胚性線維芽細胞（MEF）フィーダー細胞上でヒト胚性幹細胞（ヒトES）を成長させる方法を示しています。メッキ前の最後のステップでは、ヒトES細胞が再懸濁するとき、彼らはほぼ均一なコロニーの大きさと形状でなければならない。慎重にコロニーが小さく、より均一にするため、上下に数回hESCの懸濁液をピペットではなく、あまり単一の細胞または非常に小さいコロニーのよう?...

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<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
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<td>Reagent</td>
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<td>631107</td>
<td>&nbsp;</td>
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<td>Gibco</td>
<td>25030-081</td>
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<td>Fisher</td>
<td>BP176-100</td>
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<td>R&D Systems</td>
<td>233-FB-025</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
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<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>17105-041</td>
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<td>Reagent</td>
<td>Gibco</td>
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<td>Santa Cruz Biotechnology</td>
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<td>FAB1435P</td>
<td>&nbsp;</td>
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<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
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from mefs to matrigel i: passaging hescs in the presence of mefs

このビデオでは、マウス胚性線維芽細胞（MEF）フィーダー細胞上でヒト胚性幹細胞（ヒトES）を成長させる方法を示しています。

Watch this Scientific Journal Video about From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs at JoVE.com

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

このビデオでは、マウス胚性線維芽細胞（MEF）フィーダー細胞上でヒト胚性幹細胞（ヒトES）を成長させる方法を示しています。 3のパート1。

MEFの存在下で継代ヒトES細胞：MEFをから私をマトリゲルする

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

from-mefs-to-matrigel-i-passaging-hescs-in-the-presence-of-mefs

Research

JoVE Journal

Biology

12.6K ビュー.  University of California, Los Angeles. このビデオでは、マウス胚性線維芽細胞（MEF）フィーダー細胞上でヒト胚性幹細胞（ヒトES）を成長させる方法を示しています。 3のパート1。

ビデオ: From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

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 ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

ソックス- 2の強制発現によってMEFSからiPS細胞を生成し、OCT - 4、c - Mycの、とKLF4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

マトリゲル上のMEFから分割ヒトES細胞：MEFのからマトリゲル2〜

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

マトリゲル上にマトリゲルからの継代ヒトES細胞：MEFのからマトリゲル3〜

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

腫瘍細胞のVasculogenic活性を評価するためにマトリゲルベースの​​チューブ形成アッセイ

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

エピジェネティックリプログラミングによるヒトES細胞からMyospheresの生成

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

ハイスループットマイクロプレート呼吸測定のためのマウスの骨格筋の最小量からミトコンドリアの単離

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

天然化合物の存在下でそのまま β 細胞のミトコンドリア機能を測定するための高解像度の呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...