著者らは、フローサイトメトリーとの彼女の優れた技術支援のためのアリソンロガールに感謝したい。この作品は、カザフスタン共和国（AS）の国防総省（JH）、ヘンリー·J·マンキン寄附（JH）、および文部科学省科学からの助成金によってサポートされていました。 CWCは、米国心臓協会の博士号を取得する前のフェローシップ（11PRE7490001）によって部分的にサポートされていました。 M.Corselliは再生医療の訓練助成金のためのカリフォルニア工科大学（TG2-01169）によってサポートされていました。

1筋生検の処理<ol><li>ダルベッコ改変イーグル培地、5％ウシ胎児血清（FBS）を補足し（DMEM）中で、氷上で、ヒト骨格筋生検を維持する1％ペニシリン - ストレプトマイシン輸送時（P / S）。 </li><li>筋生検の受領後、輸送容器から検体を除去し、無菌条件下で、2％抗生物質 - 抗真菌溶液（A / A）を添加したリン酸緩衝生理食塩水（PBS）で二回洗浄する。 </li><li> 2％A / Aを添加したDMEM中の無菌のハサミとピンセットで付属脂肪および結合組織を除去します。 </li><li>解剖顕微鏡下で大血管を削除し、その後に小さな断片に筋肉標本をカット（&lt;サイズが1cm 2）。 </li><li> 20ミリリットル保存培地におけるカット筋片（&lt;5グラム）を維持（PMを、DMEM、10％FBSおよび1％P / Sを補充した）、5日間まで4℃で。 </li></ol> 2マッスルDissociatイオンと細胞単離<ol><li>細胞単離の日に、PMから筋肉片を除去し、PBSで2回洗って、2％P / Sを補充した。消化溶液を作るために、新鮮に、PMにI型、II型、及びIV型コラゲナーゼ（100 mg / mlの）を追加します。 </li><li>細かくみじん切りにし、機械的に解決策のない凝固で10mlの血清学的ピペットを通過するまで、PM少量のペトリ皿中の滅菌ハサミや鉗子で筋肉の部分をミンチ。このプロセスの間に残留脂肪および結合組織を除去します。大人の筋肉の少なくとも8〜10グラムまたは各細胞を単離するための胎児の筋肉の2グラムを使用してください。 </li><li> 10ミリリットルの血清学的ピペットを用いて消化溶液ofthe（10ミリリットル中にまたはミンチ胎児筋の2グラム）20mlに刻んだ大人の筋肉4-5グラムの転送します。 80rpmで - 70のオービタルシェーカー上で37℃で60分間 - 50のためのダイジェスト。それに応じて量に基づいて、消化時間および/または撹拌速度を調整することにより、細胞収量および表面抗原の保存を最適化組織の。濁りがほぼ透明になるまで消化の状態ごとに15〜20分を観察します。 </li><li>激しく消化された組織を10ミリリットルの血清学的ピペットで3〜5回ピペット。反応を停止した後、4分間400×gで遠心分離するPMの等しい量を追加します。 </li><li>注意深く上清を除去し;洗浄するために10ミリリットルのPMでペレットを再懸濁した後、100μmのセルストレーナーで濾過した。 </li><li> 4分間400×gで遠心分離し、上清を注意深く取り除きます。赤血球溶解緩衝液（155 mMの塩化アンモニウム、10mMのKHCO 3、0,1mM EDTA）に再懸濁ペレットを、単一細胞懸濁液を得るために、70μmのセルストレーナーでろ過した後、室温で10分間インキュベートする。いずれの沈殿が観察された場合には、再度70-mセルストレーナーを通して濾過する。 </li><li> 4分間400×gで遠心し、0.5 mlのPBS中に細胞ペレットを再懸濁します。セルの数をカウントします。細胞選別のために少なくとも5万個の細胞の総数を取得します。 SINGLを希釈染色のためPBSで1mlあたり5未満百万個の細胞に電子細胞懸濁液。対照染色（非染色コントロール、ネガティブコントロール、および単一色陽性対照）のための11のチューブに細胞懸濁液とスプリットの50〜100μLを取り出します。 </li></ol> 3細胞標識およびソート<ol><li>必要に応じて目的を阻止するためのマウス血清（PBS中に希釈1:10）での4℃で10分間の単一細胞懸濁液をインキュベートする。 </li><li>単一細胞懸濁液にし、4℃で20分間インキュベート：CD34-APC、CD45-APC-Cy7で、CD56-PE-Cy7で、CD144-PE、およびCD146-FITC（100すべて1）を追加します。ネガティブコントロールのために、APC-、APC-Cy7-、PE-Cy7-、PE-およびFITC結合アイソタイプのIgG抗体の同等の濃度を追加し、4℃で20分間インキュベートする。単色陽性対照のために、個別に各チューブに、CD34-APC、CD45-APC-Cy7で、CD56-PE-Cy7で、CD144-PE、およびCD146-FITCと同等の濃度を追加し、4℃で20分間インキュベートする。 </li><li>インキュベーション後、4分間400×gで遠心分離器は、洗浄する。 5％FBSおよび1％P / Sを補充した2mlのDMEM中1 - で細胞ペレットを再懸濁する。細胞懸濁液の最終濃度は、1ml当たり5未満万細胞であるべきである。 7-AAD（1：100）を追加し、死細胞の排除のために室温で15分間インキュベートする。陰性対照および単一色陽性対照のために0.5mlのDMEMで再懸濁した細胞ペレットを、5％FBSおよび1％P / Sを補充した。 </li><li>試験管フローサイトメトリーの丸底ポリスチレンフローにすべての細胞懸濁液を転送します。細胞収集チューブを準備し（：ACSの交流媒体のMECのためのMPM; EGM-2 PC用それぞれのチューブは、適切な培養液500μlで予め充填されている）。セルソーターに氷上で細胞懸濁液を輸送する。 </li><li>非染色対照、陰性対照、単一色の陽性対照、および主細胞懸濁液の順にセルソーター上で実行細胞懸濁液セルpを最大にするために厳密にゲーティングレーザー強度、チャネル補償、および細胞集団を調整しながらurity（フローサイトメトリーの詳細についてはJoveの及びその他の学術誌によって発表された論文を参照してください）​​。 </li><li>適切な採血管において所望の細胞集団を収集します。 4℃で保存収集された細胞はすぐに播種されていない場合。 </li></ol> 4。後の仕分け細胞培養<ol><li> I型コラーゲンでプレコートしたプレート上にMPMイン/ cm 2で&lt;10,000細胞たてのソートのMEC（P0）をシード。のMECのその後の継代のために、3,500シード-でプレコーティングプレート/フラスコ上にMPMに4,000細胞/ cm 2の I型コラーゲン。 </li><li>たて0.2％ゼラチンでコーティングしたプレート上にEGM-2の&lt;20,000細胞/ cm 2でのたてソートのPC（P0）をシード。 PC培地中で3比P2まで定期的なポリスチレン培養プレート上に（20％のFBSおよび1％P / Sを補充したDMEM）：パソコンのその後の継代のために、1で細胞を分割する。 P3から以降は、種子細胞における6,500 -正規ポリスチレン培養プレート/フラスコ上にPC培地中7,000細胞/ cm 2。 </li><li>定期的なポリスチレン培養プレート上に（20％のFBSおよび1％P / Sを補充したDMEM）AC培地中で&lt;20,000細胞/ cm 2で新たにソートのAC（P0）をシードする。定期的なポリスチレン培養プレート/フラスコの上にAC培地中7,000細胞/ cm 2 - 6,500で、後続のACの継代、種子細胞について。 </li></ol>

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J.H. received remuneration from Cook MyoSite, Inc. for consulting services and for royalties received from technology licensing during the period that the above research was performed. All other authors have no conflict of interest to disclose.

hBVSC亜集団の同定および精製は、MSC個体発生の理解における大きな進歩を表している。 MSCの血管周囲起源および組織特異的前駆細胞および血管22-25の間の関連性を示す証拠が増えている。また、hBVSCsの均質な亜集団を単離するための能力は、さらに、MSCの不均一性および血管細胞生物学26の理解を助ける。 過去数年間で、のMEC、PC、およびACSは、個別に独...

ヒト骨格筋の幹/前駆細胞の臨床的に魅力的な供給源と考えられてきた。骨格筋は、コミットされた筋原前駆細胞、骨格筋芽細胞だけでなく、衛星細胞や筋肉由来幹細胞（MDSCを）1を含むプリミティブ筋原幹細胞だけでなく、含まれています。ヒト筋肉由来の幹/前駆細胞、自己または同種異系の使用は、再生医療において広範に前臨床動物モデルおよび臨床試験で検討されている。筋肉幹細胞/前駆細胞の再生アプリケーションは、デュシェンヌ型筋ジストロフィー（DMD）患者におけるジストロフィー再生筋肉から心臓発作の患者で負傷心臓を修復するまでの範囲である。 骨髄由来多能性成体前駆細胞（MAPCを）および脂肪由来幹細胞（ADSCの）、成体幹/を含む間葉系幹/間質細胞（MSC）と他の多能性前駆細胞集団の発見以来、前駆細胞は、広範囲の日付1-9に研究されてきた。それにもかかわらず、 その場での母国のアイデンティティと局在は遡及的単離法によって隠されています。最近では、蛍光活性化細胞選別（FACS）を使用して、私たちは他のグループは、将来に向かって特定されてきた血液ヒト骨格筋内の血管や他のいくつかの器官から精製された3多能前駆細胞集団：筋原内皮細胞（のMEC）、周皮細胞（パソコン）、および外膜細胞（ACS）10。内膜、中膜、および外膜：ヒト血液·血管由来幹細胞（hBVSCs）これらの3つの亜集団は、それぞれの血管の3の構造層で見つけることができます。 ACSは、より大きな動脈および静脈の外膜層に局在しながらより具体的には、のMECとPCは、微小血管および毛細血管で検出される。のMEC（C：各前駆細胞サブセットは、細胞表面抗原の独特な組合せを表すD34 + / 56 + / 144 + / 45 - ）、パソコン（CD146 + / 34 - / 45 - / 56 - ）、およびACS（CD34 + / 31 - / 45 - / 56 - / 146 - ）。 これらhBVSCサブセットのさらなる特徴付けは、すべての3つの前駆細胞集団は、骨格筋、骨、軟骨、および脂肪生成を含む典型的なMSCは、中胚葉に類似発達ポテンシャルを有することが明らかになった。すべてhBVSCサブセットはまた、CD44、CD73、CD90、およびCD105、たてや文化などの古典的なMSCのマーカーを呈する。集合的証拠のこれらの作品は、MSCの血管起源を支持した。また、のMEC、パソコン、およびACSの治療能力は、最近独立した研究で実証されている。成人の筋肉生検から選別のMECは、負傷したとジストロフィー骨格筋や修理負傷した心筋よりも効率的に骨格筋芽細胞と血管エンドを再生成することが示されたthelial細胞（EC）。異なるヒト器官から精製されたパソコンにも/修復し、損傷したとジストロフィー骨格筋を再生し、衛星細胞プール13-16に寄与することが示されている。ごく最近、私たちはヒト骨格筋から得られたPCを効果的に間接的なパラクリン作用と直接的な細胞間相互作用17を介して梗塞心筋を修復することを実証した。 ACSは、一方で、直接外植された血管から単離されているかまたはヒト脂肪組織および骨格筋からFACSによって精製した。のACの顕著な血管新生促進効果は、マウス後肢虚血モデル19で実証された。また、ACSはまた、虚血性組織修復20で、ACSの堅牢な治療の可能性を示して、より効率的に、従来のMSCより梗塞心筋を修復することが示されている。 同時現在の精製プロトコルの助成金、のMECの将来の精製、PC、および単一のヒト骨格筋生検の血管系からのAC。これは、私たちは勉強し、および/または個別の治療目的のための最適なhBVSC亜集団を選択することができます。さらに、この新しい手法は、更に、その再生医療のための多能性前駆細胞の理想的な供給源作り、ヒト骨格筋に由来することができる幹細胞/前駆細胞のレパートリーを拡大する。

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 1</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C5894</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 2</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1764</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 4</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1889</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD34 APC</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">555824</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD45 APC-Cy7</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">557833</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD56 PE-Cy7</td>
			<td style="width:99px;">BD Pharmingen&#160;</td>
			<td style="width:148px;">557747</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD144 PE</td>
			<td style="width:99px;">Beckman Coulter</td>
			<td style="width:148px;">A07481</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-human CD146 FITC</td>
			<td style="width:99px;">AbD Serotec</td>
			<td style="width:148px;">MCA2141F</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">FACSAria II Flow Cytometer</td>
			<td style="width:99px;">Becton-Dickinson</td>
			<td style="width:148px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">EGM-2 Complete Medium</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:148px;">CC-3162</td>
			<td style="width:193px;">For culturing PCs (only P0)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11965</td>
			<td style="width:193px;">For culturing PCs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, with sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11995</td>
			<td style="width:193px;">For culturing MECs and ACs</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">10437-028</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated horse serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">26050-088</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15140-122</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15240-062</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Trypsin-EDTA 0.5%(10X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15400-054</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Dulbecco&#8217;s PBS without calcium and magnesium</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Chick embryo extract</td>
			<td style="width:99px;">Accurate Chemical</td>
			<td style="width:148px;">CE650T-10</td>
			<td style="width:193px;">Filter before use</td>
		</tr>
	</tbody>
</table>

isolation of blood-vessel-derived multipotent precursors from human skeletal muscle

間葉系幹/間質細胞（MSC）の発見以来、ネイティブのアイデンティティおよびMSCの局在は、培養中の彼らの回顧分離によって隠されています。最近では、蛍光活性化細胞選別（FACS）を用いて、われわれおよび他の研究者が前向きにヒト骨格筋の血管系に関連付けられた多能性前駆細胞の三亜集団を同定および精製された。これらの3つの細胞集団：内膜、メディア、および外膜：筋原内皮細胞（のMEC）、周皮細胞（パソコン）、および外膜細胞（ACS）は、血管の3の構造層のそれぞれに局在​​している。すべてのこれらのヒト血管由来幹細胞（hBVSC）集団は古典のMSCマーカーを発現するだけでなく、典型的なのMSCに類似した中胚葉の発達ポテンシャルを有していないだけの。以前は、のMEC、PC、およびACSは別個のプロトコルを使用して単離され、その後独立した研究で特徴づけられている。現在の分離PROTocol、選択的な細胞表面マーカーの分離プロセスへの修正や調整を通じて、私たちは同時に単一のヒト筋生検からFACSによる3つのすべてのhBVSC亜集団を精製することができます。この新しい方法は、複数のBVSC亜集団の単離を効率化するだけでなく、明確な治療目的のためのhBVSCsの将来の臨床応用が容易になるだけでなく、。

FACSパラメータは、最初の未染色対照、陰性対照、及び単色陽性対照から得られたデータに基づいて補正される。死細胞を除外した後、蛍光標識された細胞懸濁液は、負および正の細胞表面マーカーの一連の選択に供される。まず、CD45 +細胞は、CD56 +前にゲートされており、CD56 -フラクション-細胞がCD45から分離される。 CD56 +画分は、さらに、CD34-CD144...

Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

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

ヒト骨格筋内の血管は、再生アプリケーションに最適ですいくつかの多系列前駆体集団を保有する。内膜から筋原内皮細胞、培地から周皮細胞、および外膜から外膜細胞：この分離法は、血管の三つの構造層からそれぞれ3多能性前駆細胞集団の同時精製を可能にする。

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 ...

isolation-blood-vessel-derived-multipotent-precursors-from-human

Research

JoVE Journal

Biology

11.6K ビュー.  University of Pittsburgh. ヒト骨格筋内の血管は、再生アプリケーションに最適ですいくつかの多系列前駆体集団を保有する。内膜から筋原内皮細胞、培地から周皮細胞、および外膜から外膜細胞：この分離法は、血管の三つの構造層からそれぞれ3多能性前駆細胞集団の同時精製を可能にする。 

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

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 ...

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨格筋からのミトコンドリア単離

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

高解像度の呼吸計測測定のための差動遠心分離することにより骨格筋から無傷ミトコンドリアの単離

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

ラット骨髄からの破骨細胞前駆体の分離・精製・分化

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

ビブラートムカット心筋切れ切り切れからヒト心室心筋細胞を分離

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...