VVPMDLに健康賞R01 HL105734の国立研究所によってサポートされていましたこの作品は、部分的には、ヘルスリサーチのキャリア賞K08 HL094748の国立研究所によって、サポートされています。 VVPは、付与11IRG900384でサポートされている米国心臓協会の革新的な研究である。

必要なソリューションと機器の準備1。 <ol><li>まず、15分のためにそれらをオートクレーブで手術器具（ストレート形状鉗子、湾曲した形状の鉗子、ストレート形状のはさみ、湾曲形状はさみ）を殺菌する。 121℃。組織をミンチに使用される1組の心と他のセットを解剖するために使用されている楽器、2セットを準備します。 </li><li>滅菌濾過し、水900mlでMCDB153培養培地の1パケットを溶解し、粉末が溶解するまでゆっくり撹拌する。溶解するまで調製し、撹拌している培地の最終容量1リットルのために、[w / vの7.5％] 1.2グラムの重炭酸ナトリウム、または重炭酸ナトリウム溶液15.7 mlを加え。その後、組織培養フード（バイオセーフティキャビネット）において無菌条件下で0.2μmのボトルトップフィルターを介してメディアを注ぐ、1N HClまたは1N NaOHのいずれかを追加することによって、7.2にpHを調整する。その後、メディアを収集し、4℃で保管してください。 </li><li>サプリメントへの準備を培養培地（材料リスト）に追加する。サプリメントは、メディアが細胞をプレーティングするために必要とされる直前に培地に添加されるべきである。 </li><li> 10％FBSを加えた抗生物質（ペニシリンおよびストレプトマイシン）とのDPBSを準備します。 </li><li> DPBSを加えた抗生物質（ペニシリンおよびストレプトマイシン）100mlに、次に、トリプシン、0.25gの（USBウシ膵臓、活性&gt; 2,500 USP単位/ mg、超高純度）を添加することによって0.25％トリプシン溶液を調製。 </li><li>この手順を完了するために必要なその他の必要な機器は、実体顕微鏡、ペトリ皿（10センチ）、50 mlのコニカルチューブ、40μmのセルストレーナー、DPBSを加えた抗生物質、60 mmの培養皿および35mmの培養皿が含まれています。 </li></ol>新生児マウスの仔から2分離ハーツ<ol><li>まず、P2新生仔マウスにP0を取得した（n = 6〜8）。 EYFP対立遺伝子またはZ / EGアレルラベルを付ける：R26Rどちらが探しこれは、ヘテロ接合性である雄とDCT-Creをホモ接合の雌の繁殖によって生成された子犬の使用をお勧めしますメラノサイト様細胞。両R26R：EYFPおよびZ / EGマウスは、ジャクソン·ラボラトリーズ（それぞれ株式数006148と003920）から入手可能である。 </li><li>次に、新生児仔マウスを安楽死させるアルコール綿で胸を拭く、次にDPBSで10センチのペトリ皿に子犬を置く。 </li><li>滅菌ピンセットとはさみで胸と胸骨の上に皮膚をカットした後、実体顕微鏡下で慎重に胸を開きます。新生児仔マウスの心は非常に小さいので、添付の心房と心臓全体を削除してくださいするように注意してください。 </li><li> DPBSで心を洗い、新しい10cmのペトリ皿に移す。 </li></ol> 3酵素消化<ol><li>組織培養フード（バイオセーフティキャビネット）に切除した心でペトリ皿を持参してからフード内で無菌技術を使用して以下の手順に従ってください。 </li><li>まず、無菌DPBSプラス抗生物質に心を3回洗浄する。 </li><li>でた（n = 6-8）の心を置きます60 mmの培養皿と皿に0.5％トリプシン溶液6-8 mLを加え。 </li><li>次に、（サイズが1ミリメートル未満の）小片に心を切断し、5％CO 2雰囲気中で30分間37℃でそれらをインキュベートする。 </li><li>インキュベーション期間が完了した後、滅菌50 mlコニカルチューブに心臓組織と皿からトリプシンの得られた溶液を注ぐ。 </li><li>その後、すぐにかつ慎重に、10 mlの滅菌ピペットを用いて、50mlのコニカルチューブ内で30〜50回のソリューションを粉砕する。 </li><li>ろ液を収集するために、新しい、清潔な50mlのコニカルチューブの上に40μmのセルストレーナーを通して得られた溶液をろ過する。 </li><li>次に、室温で5分間卓上遠心機で240×gで50 mlチューブを遠心する。その後、静かに上清を吸引し、滅菌DPBS 6-8中で残ったペレットを懸濁します。このステップをさらに2回繰り返します。このステップは、心房筋細胞や心臓のメラノサイトをペレットが、第がダウンすることはありません線維芽細胞からの芽細胞は小さく、上清中に残ります。 </li></ol> 4。メッキ心臓メラノサイト様細胞<ol><li>遠心分離後の第3のすすぎに続いて、慎重にドレインまたは吸引洗浄溶液（DPBS）オフやサプリメントが追加された先のMCDB培地6mlにペレットを再懸濁します。 </li><li>さて、無菌トランスファーピペットに再懸濁させた溶液を描き、35 mmディッシュ中で、あるいは6ウェルプレートのウェル中の細胞を置き、1 35-mmディッシュに3新生児マウスから単離された心臓細胞をプールかよく。 </li><li> 、5％CO 2雰囲気下で37℃で一晩皿をインキュベートあらゆる付着していない細胞と一緒に培養培地をオフに吸引した後、DPBSで一度プレートをすすぐ。その後、プレートに新鮮な培地を追加し、2日ごとにメディアを交換。細胞は成体聞くから単離され、一方、新生児または胚の心臓から単離された細胞は、最大で3週間培養物中に維持することができるtsが最大10日間培養物中に維持することができる。 </li></ol> 5。成体マウス心臓から心臓メラノサイト様細胞を単離する<ol><li>まず、マウスにヘパリン100 Uの腹腔内に投与し、それらを安楽死させた後（3-4週齢、n = 3）の成熟したマウスから心を取り除くミッドライン胸骨切開を通して。それは、この手順のためにマウスを安楽死させるために、ペントバルビタールの使用をお勧めしますが、この薬は、すべての場所で​​は使用できません。 </li><li>次に、組織培養フード（バイオセーフティキャビネット）中の抗生物質を含む滅菌DPBS中に切除心を洗う。そこから残りの血液を除去した後、DPBSでもう一度心をすすぐために湾曲した解剖用鉗子で少し心を絞る。 </li><li>心臓から心房と房室弁輪を取り外します（これらの構造はメラニン細胞様細胞が含まれている）、および35-mmディッシュで切除した組織標本を置く。 </li><li>湾曲したハサミと力を用いて小片に心をカットpsの、その後皿に0.5％トリプシン溶液6-8 mLを加え。その後、45〜60分間37℃で料理をインキュベートする。 </li><li>さて、以降のステップ3.6から始まる上記の手順から手順に従ってください。 </li></ol>

<ol>
	<li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

私たちは、開示することは何もない。

心臓メラノサイトは、一般的に心房性不整脈のトリガーを引き起こす心臓内の解剖学的位置に存在している。これらの領域は、肺静脈、房室弁輪、後部左心房と卵円孔（ 図4）が挙げられる。これらの細胞は不整脈原性に果たす役割を理解するために、私たちはそれらを分離し、その生理機能を研究するための技術を開発する。しかし、心臓のメラノサイトの健康でより大量に生...

私たちは最近、メラニン合成酵素を発現するマウスとヒトの中心部に新たな細胞集団を同定し、形態学的に皮膚のメラノサイトに似ている。当社は、これらの細胞の心臓メラノサイト」と呼ばれ、彼らは心房性不整脈が頻繁に人間に由来トリガするの解剖学的位置に存在しているが見つかりました。また、心臓のメラノサイトは、DCTの生殖細胞欠失を有するマウスでは心房性不整脈に貢献した。心臓のメラノサイトは、まばらにそれらが全ての心房細胞の0.1％未満を含み、マウスの心房全体に分散されている。心臓のメラノサイトの電気的および生物学的特性を調査するために、私たちは、Cre誘導性メラノサイト特異的マーカーを用いて、マウスの心臓からそれらを分離するための手順を開発しました。当初の手順は、新生児マウスの心筋細胞を単離するために使用されるものから開発され、パッチクランプ記録またはPCR分析に適した少数細胞を得た。しかし、癒すを改善する目と心臓のメラノサイトの生存率は、より詳細な電気生理学的解析や生化学的研究のために、私たちは皮膚のメラニン細胞を単離するために設計されたものから洗練された手順を開発しました。このレビューに記載され、実証された手順は、純粋な集団として、または心筋細胞でメッキすることができる健康な心臓のメラノサイトを生成するという利点を有している。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>MCDB 153</td>
 <td>Sigma-Aldrich</td>
 <td>M7403</td>
 <td>Dissolve in: Water</td>
 </tr>
 <tr>
 <td>DPBS, no calcium no magnesium</td>
 <td>Invitrogen</td>
 <td>14190136 </td>
 <td>Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate, or Sodium Bicarbonate Solution, 7.5% </td>
 <td>Sigma-Aldrich </td>
 <td>S5761 or S8761 </td>
 <td>Water 
 Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140163 </td>
 <td>Stock Conc.: 100X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technology</td>
 <td>Certified</td>
 <td>Working Conc.: 4%</td>
 </tr>
 <tr>
 <td>TPA</td>
 <td>Sigma-Aldrich</td>
 <td>P1585</td>
 <td>DMSO 
 Stock Conc.: 3.2 &mu;M 
 Working Conc.: 32 nM</td>
 </tr>
 <tr>
 <td>Transferrin</td>
 <td>Sigma-Aldrich</td>
 <td>T8158</td>
 <td>DPBS 
 Stock Conc.: 0.1 mg/ml 
 Working Conc.: 1 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Dibutyryl cyclic AMP</td>
 <td>Sigma-Aldrich</td>
 <td>D0627</td>
 <td>DPBS 
 Stock Conc.: 0.5 mM 
 Working Conc.: 5 &mu;M</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>10 mM 
 acetic acid 
 Stock Conc.: 1 mg/ml 
 Working Conc.: 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Basic FGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Water 
 Stock Conc.: 4 ng/&mu;l 
 Working Conc.: 4 ng/ml</td>
 </tr>
 </tbody>
</table>

isolating primary melanocyte-like cells from the mouse heart

私たちは、マウスでの心房性不整脈のトリガーに貢献し、マウスおよびヒトの心の中に（また、心臓メラノサイトとも呼ばれます）メラニン細胞様細胞の新規集団を同定した。心臓のメラノサイトの電気的および生物学的特性を調査するために私たちは、新生児マウスの心筋細胞を単離するために設計されたものに由来するマウスの心臓からそれらを分離するための手順を開発しました。より広範なパッチクランプまたは生化学的研究に適して健康的な心臓メラノサイトを得るために、私たちは、もともと皮膚のメラニン細胞を単離するために設計されたものに基づいて心臓のメラノサイトを分離し、メッキする洗練された手順を開発しました。洗練された手順は、このレビューで実証し、純粋な集団として、または心筋細胞でメッキすることができる健康的なメラニン細胞様細胞のより大きな数字を生成する。

この資料に記載されたプロトコルは、私たちが以前にマウスの心臓から心臓メラノサイトを分離するために公開1と比較して改善された手法である。この手順では、心臓のメラニン細胞様細胞を標識するためにメラニン合成酵素特異的にCre-リコンビナーゼにより駆動される蛍光マーカーの使用を採用する。新たに単離された胚性または新生児の細胞を扱うとき心房筋細胞から心臓のメラノサ?...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

このプロトコルでは、W eは心房性不整脈に貢献したマウスとヒトの心の中に（また、心臓メラノサイトとも呼ばれます）メラニン細胞様細胞の新規な集団を同定したマウスでトリガします。

マウスの心臓から初代メラノサイト様細胞を単離する

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

isolating-primary-melanocyte-like-cells-from-the-mouse-heart

Research

JoVE Journal

Biology

10.4K ビュー.  University of Pennsylvania. このプロトコルでは、W eは心房性不整脈に貢献したマウスとヒトの心の中に（また、心臓メラノサイトとも呼ばれます）メラニン細胞様細胞の新規な集団を同定したマウスでトリガします。 

ビデオ: Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

ソフト筋骨格組織から幹細胞を単離する

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

生物学

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

大人の線維芽細胞から再プログラムヒト人工多能性幹細胞のコロニーを選択し、分離

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

途上胚性マウス心臓弁地域からのタンパク質の単離

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

マウス網膜色素上皮からの一次電池文化

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...