細胞単離後、プロトコル全体で約2時間かかります。 1。顕微準備<ol><li>標準化されたプロトコル9,12に係るドナー骨髄および単離された亜集団の蛍光標識からの幹細胞の単離を行う細胞は、動物の動作が行われる時間の間休止することを許可する。蛍光標識のために我々はCFDA（カルボキシフルオレセインジアセテートスクシンイミジルエステル）をお勧めします。 </li><li>ケタミン（75 mg / kgで）およびキシラジン（2.5 mg / kg）を用いて体重20〜25gの雄マウ​​スを麻酔。外科的手順および実験プロトコール全体を通して、麻酔補助剤によって維持されるべきである（最初の注射の10％、腹腔内）必要に応じて（通常、毎分30〜60）。 </li><li>そっと右陰嚢の前面だけでなく、右太ももと脚の付け根を剃る。すべては湿らせた綿棒で髪を失って収集しています。プレキシガラスの観察ステージ上でマウスの腹側を上に置き、手数料を修正弾性ドレープを使用してT。ステージは、脚と陰嚢の両方を昇降するためのベースプレートの尾側端でベース板と第2板からなる、カスタムメイドされている。 37℃の体温を維持するために加温パッド上にステージを配置ステージ上で固定後、手術用顕微鏡の下で動物を配置し、10を使用して、以下の操作手順を実行します - 16倍の倍率に。 </li><li>鼠径部にまで右太もも、膝から大腿血管のちょうど前方の皮膚のはさみを使って最初の切開を行います。 </li><li>大腿動脈を特定し、周囲の軟組織から動脈を動員し、近位にそれに従ってください。焼灼器を使用して、左睾丸への大規模なブランチを特定し、カット。その後、左睾丸で表面的に滞在縫合を置く。穏やかな牽引力は、大腿動脈の近位のさらなる動員を促進する。 </li><li>近位の動員が完了した後、識別し、大規模なブランチをカット実行中mediall焼灼器による大腿部のY。その後、右大腿部の遠位部での大腿動脈を結紮。合字にやさしい牽引、さらに準備を支援します。 </li><li>再び近位大腿動脈に今の目を向けるなど近位にできるだけ動脈の周りに縫合糸を配置します。結び目を準備しますが、まだそれを接続しないでください。縫合糸に優しいトラクションを適用し、動脈よじれを作成します。 </li><li>よじれを確立した後、動脈が慎重にmicroscissorを使用している切開。大腿静脈を損傷しないように特に注意してください。 </li><li>切開を介して製造マイクロカテーテル（内径0.28ミリメートル）を挿入します。カテーテルは、（0.9％塩化ナトリウム、NaCl）を脱気し、1 mlシリンジ挿入の前に接続されなければならない。 </li><li>カテーテルの挿入が成功した後、慎重にカテーテルの通過を容易にするために、ねじれを緩めます。動脈血流は、カテーテル内ですぐに表示されるはずです。優しくカテーテルを数ミリメートルを進める前のよじれを超えてS。その後、カテーテルの固定のために準備合字を縛り付ける。 </li><li>そっと正しい位置を確認するために、カテーテル（0.9％NaCl）にして吸引をフラッシュします。さらに運用段階にカテーテルを固定するためにテープの一部を使用してください。 </li><li>十分な固定後、精巣挙筋の次の準備ステップのために取ってカテーテルを入れた。血栓形成を防止するため、定期フラッシングとカテーテルごとに5〜10分間の吸引を適用します。 </li><li>右陰嚢に今注目を回して、右陰嚢の腹側面上の皮膚および筋膜を切断することによって切開する。どんな機器とその下の組織に触れないように注意してください。 </li><li>陰嚢の先端に鼠径倍と尾側まで切開を拡張します。 </li><li>それに触れることなく、精巣挙袋上記の基本的なソフトで結合組織を慎重に分離します。精巣挙の先端部を特定し、SLにここに滞在縫合を配置ightly筋肉を伸ばす。優しく尾側と前方の筋肉を引っ張って筋肉の袋の下に今残っている軟組織を切除。 </li><li>結合組織から完全に分離した後、ハ​​サミで頭蓋端に1点で精巣挙袋を開きます。筋肉の先端部に、この開口部から下に切開を延長する。 </li><li>その後、はさみを使って筋肉と精巣の間の残りの接続を切る、焼灼器を使用して睾丸と精巣挙をつなぐ小さな容器を密閉する。 </li><li>顕微鏡分野のうち、睾丸を脇に置きます。 </li><li>開かれた精巣挙は今、ステージの上に平らに産む。精巣挙筋組織を広げるために、組織の端に4 6-0プロレン滞在縫合糸を配置し、弾性ドレープでそれらを修正。これから連続的に1mlシリンジ（ 図1）を用いてリン酸緩衝生理食塩水（PBS）で組織を湿ら。 </li><li>今15分間休む調製を可能にする。その後注入微小血管のコントラスト強調のため左大腿動脈を介して、1％ローダミン-デキストラン0.05〜0.1 mlである。任意のケモカインや炎症剤がアプリケーションのために計画されている場合は、ここで局所的に1ミリリットルの注射器を使用して、それぞれの試薬で筋肉を湿らすべきである。対照実験の場合には、PBSで湿らせ続ける。 </li></ol> 2。生体顕微鏡検査<ol><li>操作ステージに固定されたマウスを維持し、落射照明用に変更し、CCD（電荷結合素子）ビデオカメラに接続され、蛍光顕微鏡下で移動する。大腿動脈カテーテルを脱臼しないよう特に注意してください。 </li><li>水浸対物レンズを用いて実験を行う。 </li><li>精巣挙微小循環の十分な視覚化のために、細胞の注射の前に目標を調整した後、確率的にしっかりした幹細胞の接着の後の分析のために6毛細血管細静脈を定義します。 </li><li>蛍光標識された幹細胞を投与し、大腿動脈カテーテルを利用して5つの連続した注射剤との合計1mlシリンジで、注射当たり0.4×10 6個の細胞で、200μlのPBSに溶解した。慎重に最初の注射前に細胞サンプルを振る。 </li><li>レコードの幹細胞は、それによって典型的なセルラと組み合わせた内皮内層に沿った細胞の速度の50％以上の減少として「ローリング」を定義する、細胞注射の直後にローリング」棒と解放」運動を。ローリング幹細胞の計数およびすべての単一の注射後の観察の1分の間に事前定義された細静脈の距離を通過するすべての幹細胞を実行します。幹細胞ローリングの量は全て通過する細胞の割合として表される。ローリング細胞のようなランダムな簡単なテザリング現象を示す細胞をカウントされません。 </li><li>最後のセルの注射後の計数でしっかりと接着性幹細胞の数を決定し、目の計算された内皮表面に関連して金額を表現接着細胞/ mm 2のようなE 6事前定義された細静脈（直径×長さ×π）。まだ細胞運動が30秒間検出されないときに強固な接着が考えられる。 </li><li>生体顕微鏡が完了した後、慎重に精巣挙筋を取り外し、後で分析（免疫組織化学や分子生物学）に対応して保管してください。頚椎脱臼により動物を生け贄に捧げる。幹細胞のローリングと企業内皮接着、ならびに赤血球速度（100mm /秒）、血管長さ（μm）、血管径（μm）を、壁のシェア率などの微小血管血行動態の定量化のための生体顕微鏡記録の解析（秒-1）;画像処理ソフトを利用し、それぞれの実験組織処置を知らされていない研究者がオフラインで実行する必要があります。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., 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=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, 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=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. 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この原稿のために行った全ての動物実験は、地元の動物管理使用委員会によって承認されている。 著者は、彼らが競合する経済的利益を持っていないことを宣言します。

<html>生体内蛍光顕微鏡検査は、直接視覚化および幹細胞、内皮相互作用の定量的な分析を可能にする。顕微露光および生体内視覚化技法はよく白血球 - 内皮相互作用および種々の化学成分の研究の間に選択的に単純な表面灌流技術によって標的組織に適用することができる確立されたので、精巣挙筋モデルは、特に有用である。による動きアーチファクトおよび重度画像ノイズが存在しない?...

本資料の目的は、末梢骨髄幹細胞の移動の直接観察および分析のためのマウス精巣挙筋の微小循環に塗布生体顕微鏡（IVM）の技術を記述することである。 幹細胞に基づく組織および臓器再生の現在の概念は、損傷組織1に骨髄由来幹細胞のホーミングを含む。成功した幹細胞の移動のための重要なステップは、経内皮移動、最終的には、臓器移植の2に続いて負傷した臓器内のローカル内皮と細胞の相互作用幹が含まれています。これらの幹細胞の生体内分析-内皮細胞相互作用は、 インビボで異なる病態生理学的な設定での幹細胞に基づく再生応答の定量のために理想的なパラメータを形成する。さらに、それは、考えられる思われる、将来的には、特定の幹細胞pの遊走能の生体内分析標準化された微小環境内opulationsは、さらなる臨床試験をこれらの集団を進める前に適用される可能性があります。 生体顕微鏡は、最初は炎症研究3分野におけるin vivoでの白血球-内皮細胞相互作用を観察し、定量化のために開発されました。生体顕微鏡研究の最初の成功の記録は、光学顕微鏡4の下にカエル」舌や腸間膜を勉強し、19世紀にはすでにコーンハイムから報告された。その最初の使用IVMは、驚異的な技術開発、今日を受けているので、ある種の必須成分は、定量IVMを行うために前提条件を形成は、（i）組織の光アクセスを可能にする製剤、（ii）は、顕微鏡によって検出できる分子プローブ、（三）検出システム、画像データから関心対象のパラメータを抽出することができる（iv）のコンピュータベースの分析システムに接続された顕微鏡4を設定します。 組織標本の様々な腸間膜およびマウスおよびラット5、マウス6、ハムスター、ウサギの耳といくつか例を挙げると、ハムスターの頬袋の背側皮下脂肪チャンバーの肝臓を含むIVM研究のために導入されている。 準備と可視化が十分に標準化された外科的手技によって可能であり、一般的には運動の成果物に問題が発生しないようにただし、次の年には、生体内の観察のための理想的な組織を代表する、マウスの精巣挙筋に焦点を当てます。オープン精巣挙筋標本はバエズや同僚7によって1970年に初めて実施された。当初のラットについて記載し、それはマウス8にも正常に採用されている。これまでの研究は、主に血管壁と白血球の相互作用に焦点を当てた後、私たち自身のグループは最近、評価引当として、マウス精巣挙筋標本を導入直接可視化し、定義微小環境9内の幹細胞内皮相互作用の定量分析のためのBLEツール。様々な幹細胞亜集団は、マウスのc-kit +骨髄幹細胞および間葉系幹細胞、ならびにヒトCD 133 +骨髄幹細胞を10〜12を含む、このモデルを用いて研究されている。ドナー骨髄および可視化のための蛍光標識からの細胞単離後に、幹細胞は、選択的に遠隔の臓器内の任意の細胞の捕捉を回避し、大腿動脈を介して動脈注入を利用して精巣挙筋の微小循環に適用される。潜在的に、それぞれの設定値内の局所幹細胞の遊走を媒介する種々のケモカインは、単純な局所灌流法により標的組織に適用することができるので、精巣挙筋モデルが特に有用である。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>C1157</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G (1%)</td>
			<td>Sigma-Aldrich, Munich, Germany</td>
			<td>83697</td>
		</tr>
		<tr>
			<td>Ketamin (Ketanest)</td>
			<td>Pfizer, Berlin, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Xylacin (Rompun)</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate - buffered saline (PBS)</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
		</tr>
	</tbody>
</table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

再生細胞治療の文脈における血管細胞のアプリケーションプロトコルの時代において、nonmarrow組織への幹細胞の遊走の基礎となるメカニズムは完全に解明されていない。ここでは、インビボで周辺の骨髄幹細胞の遊走の分析のためのマウスの精巣挙筋の微小循環に適用される生体顕微鏡の技術を記載している。 M.の精巣挙の生体顕微鏡は、以前の血管内皮と白血球の相互作用を直接観察するための炎症研究の分野で導入されている。十分な末梢幹細胞および前駆細胞の移動は、それを裏打ちする内皮に接着沿ってしっかりと圧延同様の初期ステップを含んでいるので内皮とintravasculary投与幹細胞の相互作用の観察および定量化のためM.精巣挙モデルを適用することが考えられる。様々な化学成分を選択的に標的tに適用することができるように初期の幹細胞の動員、骨髄外、生体内の場所を取るための単純な灌流技術による問題は、それが、本質的な微小環境の前提条件を確立することが可能である。

一般的には、精巣挙筋の微小循環内の血管内皮と直接注入幹細胞または前駆細胞の相互作用はまれな事象であると毛細血管細静脈（直径30〜80ミクロン）で独占的に発生します。による蛍光標識圧延しっかりと付着幹細胞に明らかに観察細静脈（ 図2）における内因性白血球の循環から分離して定量化することができる。 血流速度と壁剪断速度で表される微小...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

マウスのM.精巣挙微小循環の生体顕微鏡は、ユニークと周辺の骨髄幹細胞の移動を解析するための生体モデルでよく標準化されています。

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Research

JoVE Journal

Biology

15.1K ビュー.  University Rostock. マウスのM.精巣挙微小循環の生体顕微鏡は、ユニークと周辺の骨髄幹細胞の移動を解析するための生体モデルでよく標準化されています。 

ビデオ: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

微小循環の生体内イメージングのためのマウス精巣挙筋の準備

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

生物学

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

微小循環の生体内蛍光イメージングのためのMicroiontophoresisとマイクロマニピュレーション

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

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 ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

ヒト骨格筋からの血管由来の多能前駆体の単離

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNAシーケンシング解析によりEML造血前駆細胞における自己再生および分化を調節する重要な要因の同定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

ラット精巣挙筋の細動脈における無細胞層の可視化と定量化

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP顕微鏡によるmiRNAのギャップジャンクション依存的転写の解析

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>乳腺の生体内細胞内顕微鏡検査

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

乳腺の生体内細胞内顕微鏡検査

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

ゼブラフィッシュ脳に侵入する膠芽腫細胞における微小管動態のライブイメージング

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...