私たちは、Myf5の+ / nLacZマウスを提供するための博士Shahragim Tajbakhshに感謝します。我々はまた、この原稿の重要な読書のためのアレクサンダー·フロン川とマイケルBaumruckerに感謝します。この作品は、筋ジストロフィー協会（MDA）とグレゴリーMarzolfジュニア、MDセンター賞からの補助金によって支えられている。

動物は、SPF環境下で飼育したとミネソタ大学の研究動物資源（RAR）によりモニターした。 1304から30492：動物はすべてのプロトコルは施設内動物管理使用委員会（IACUC、コード番号によって承認されたアベルチン（250 mg / kg）のIP注射で麻酔した後の（CO 2吸入またはKCl注入適切な手段によって安楽死させた）ミネソタ大学の。 マウス骨格筋から単核細胞の1。分離<ol><li>適切に1または2の若い成体マウス（3-8週）を生け贄に捧げる。 <ol><li>鋭いハサミで腹部の皮膚をつまんスリット。完全に（反対方向に皮膚を引っ張って）三頭筋および後肢の筋肉を表示するために皮膚をはがす。 </li><li>ハサミで骨に沿った全ての脚の骨格筋（前脛骨筋、腓腹筋、および大腿四頭筋）と上腕三頭筋を取り除く。その後、10cmプレートに、氷冷、滅菌PBSに筋肉を転送。 </li></ol></li><li>新しい滅菌6cmのプレートにPBS中で筋肉や転送筋肉を血を洗い流します。1プレートを1〜2マウスについて。 </li><li>解剖顕微鏡下で、結合組織、血管、神経束、および脂質生成組織を除去する。 </li><li>眼科用のハサミを使用して、滑らかなパルプ（ 図1Aおよび図1B）内に組織を切断し刻む。彼らは酵素溶液により容易に分解されることはありませんように、大規模な作品を残していないしてみてください。 </li><li> （10％DMEM中でFBS中の0.2％コラゲナーゼタイプ2）ファルコン50 mlのチューブにみじん切り筋肉を転送し、コラゲナーゼ溶液5mlを加える。 60分間37℃でインキュベートする。 </li><li>粉末化し（18 G針で上下）の混合物（ 図1C）を均質化する。次いで、さらに15分間37℃で混合物をインキュベートする。 </li><li>単一細胞懸濁液中に解離する混合物を均質化するために再度粉砕する。アップ単一細胞懸濁液に50mlまでDMEM中の2％FBSを加え、混合するよく。 </li><li>ファルコン50mlチューブ（ 図1D）上にセルストレーナー（70ミクロン）を配置します。セルストレーナーに解離した細胞を含む上清を移し、それが通過するまで上下にフィルター上の細胞懸濁液をピペットで。 </li><li>血球計数器で細胞数をカウントします。 5分間4℃で2,000 rpmで遠心分離し、チューブ;吸引し、上清を捨てる。 </li><li> DMEMで10mlの2％のFBSで再懸濁する。 5分間4℃で2,000 rpmで遠心分離し、チューブ;吸引し、上清を捨てる。 </li><li> DMEM中で2％FBS200μlで再懸濁し、1.5 mlのマイクロチューブに細胞懸濁液を転送します。通常、約2×10 6個の細胞は、1匹のマウスの筋肉から採取しなければならない。細胞を、DMEM、2％FBS100μl中1×10 6細胞の濃度まで希釈される。 </li></ol> 2。抗体染色とMacとの分離次のPRの間にocedures、無菌バッファを使用して無菌状態を維持している。抗体の各ボリュームを添加し、細胞懸濁媒体は、1匹のマウスの全体の筋肉からの細胞について計算される。細胞を、2又はそれ以上のマウスから採取された場合、試薬の量が最適化されるべきである。 <ol><li>細胞懸濁液200μlに1μL、CD31-PE、CD45-PE、SCA-1-PE、およびα7インテグリン抗体をそれぞれ追加します。氷上で30分間インキュベートする。 </li><li>細胞を洗浄：インキュベーション後、1.5mlチューブ中の細胞懸濁液中にDMEM 1ml中の2％のFBSを追加し、3分間、4℃、2,000 rpmで遠心分離する。二回、この手順を繰り返します。 </li><li>吸引し、上清を捨てる。 </li><li> DMEM中で2％FBS200μlで細胞を再懸濁し、そしてアンチPE磁性ビーズ10μlを加える。氷上で30分間インキュベートする。 </li><li>インキュベーション後、1.5 mlのマイクロ遠心チューブ内の細胞懸濁液にMACSバッファーの1ミリリットルを追加し、3分間、4℃で2,000 rpmで遠心分離：細胞を洗浄。この手順TWを繰り返し氷。細胞は磁気カラムで分離される前に、MACSバッファーで洗浄する必要があることに注意してください。 </li><li>吸引し、上清を捨てる。 MACS緩衝液1.0mlで細胞を懸濁します。 </li><li>マグネットボード上のLD列を設定し、MACS緩衝液（ 図1E）の2.0ミリリットルでカラムをすすぐ。 </li><li> LDカラムに細胞懸濁液を移し、1.5mlチューブにフロースルー画分を収集する。この画分は、PE陰性細胞が含まれています。 </li><li> 3分間4℃、2,000 rpmで、吸引で遠心し、上清を捨てる。 </li><li> DMEM中で2％FBS200μlで細胞を再懸濁し、そして抗マウスIgG磁気ビーズの10μlを加える。氷上で30分間インキュベートする。 </li><li>細胞を洗浄：インキュベーション後、1.5mlチューブ中の細胞懸濁液にMACS緩衝液1mlを追加し、3分間、4℃で2,000 rpmで遠心分離する。吸引し、上清を捨てる。二回、この手順を繰り返します。 MACS緩衝液500μlで細胞を懸濁します。 </li><li>マグネットボード上のMSカラムを設定し、MACS緩衝液（ 図1F）を500μlでカラムをすすぐ。 </li><li>転送は、MSカラムに細胞溶液を懸濁し、フロースルー画分（インテグリンα7陰性細胞）を廃棄します。 </li><li> MACSバッファー1mlですすぎ、二回、この手順を繰り返します。 </li><li>すすいだ後、分離器の磁場から列を削除します。カラムをMACS緩衝液1.0mlを適用し、塔頂からシリンジプランジャを押して、1.5mlのマイクロチューブに磁気標識細胞（インテグリンα7陽性細胞）を溶出。 1.5ミリリットルチューブにフロースルーを収集します。 MACSバッファー1.5mlで溶出を繰り返し、フロースルーを収集する。 </li><li> 3分間4℃、2,000 rpmで、吸引で遠心し、上清を捨てる。 </li><li>筋芽細胞培地1mlで精製された細胞を再懸濁（20％FBSは、bFGFとハムのF-10を含まれている）、およびマトリゲル被覆されたを10cmのプレート細胞筋芽細胞中の8ミリリットル（6cmのプレートの5ミリリットル）（ 図1G）でプレート。 1〜2×10 5個の細胞を、潜在的に1無傷マウス筋肉から単離することができることに留意されたい。 </li></ol> 3。メンテナンス<ol><li>筋芽細胞の培地で一日おきに細​​胞を養う。成長筋芽細胞の出現は、それらの核内でのMyoD（ 図2）とのPax7（データは示さず）を発現する小さな丸い形状である。 </li><li>筋芽細胞は、50％のコンフルエンス時または細胞融合を開始する前に継代されるべきである。 PBSで一回洗浄した後、CO 2インキュベーター中で3分間37℃で、0.25％トリプシン溶液で細胞をインキュベートし、筋芽細胞培地で解離した細胞を収集する。遠心分離機細胞（5分間1,000 rpmで）した後、筋芽細胞培地で一時停止し、新しいマトリゲル被覆プレート上に細胞をreplate。コラーゲンコートしたプレートは、流路3の後に使用することができる。一方のプレートは、通常、四時五十七プレートに分割することができる。 </li></ol> 4。Differentiation <ol><li>一日おきに分化培地で再給。 </li><li>分化培地中の1日目では、筋芽細胞は、細胞周期を終了し、ミオシン重鎖（MHC）陽性筋への分化を受ける。これらmyoctyesは多核筋管を生成するために相互に細胞融合を始める。通常、ほとんどの筋芽細胞は、分化培地で一日3〜5により、MHC陽性差別単核筋細胞や筋管（ 図2）になる。 </li></ol>骨格筋再生のためのマウスに5。筋芽細胞移植<ol><li>アベルチン（250 mg / kg）を腹腔内（IP）注射でマウスを麻酔し。 </li><li>前脛骨筋（TA）は、筋肉の皮膚の毛を剃るの周り。二十四時間筋芽細胞移植の前に、10μMのCTX（50μl）を筋肉内に剃毛した皮膚（ 図 31〜Gのインスリンを注射器で筋肉の再生を誘導するためにNOD / SCIDマウスの脛骨筋に注入される60、3）。 </li><li>増殖する筋芽細胞を0.25％トリプシン溶液で解離し、5分間1,000 rpmで遠心分離する。吸引し、上清を捨てる。 DMEM中の2％のFBS50μlの再懸濁、1×10 6個の細胞。 31 Gのインスリン注射器の中に懸濁した細胞を移す。 </li><li>レシピエントマウスは、IP注射によってアベルチン（250mg/kg /日）で麻酔され、1×10 6筋芽細胞（ 図2）は、TA筋肉の再生筋肉に注入される。 </li><li>組織学的分析のために細胞注入後1〜4週までに収穫脛骨筋（ 図3）。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan-4-expressing+muscle+progenitor+cells+in+the+SP+engraft+as+satellite+cells+during+muscle+regeneration.">Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration.</a> Cell Stem Cell. 4, 217-225 (2009).</li><li>Pallafacchina, G., 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=An+adult+tissue-specific+stem+cell+in+its+niche:+a+gene+profiling+analysis+of+in+vivo+quiescent+and+activated+muscle+satellite+cells.">An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.</a> Stem Cell Res. 4, 77-91 (2009).</li></ol>

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このプロトコルでは、静止衛星細胞を容易にコラゲナーゼ消化および表面抗体媒介MACS分離による成体マウスの骨格筋から精製することができる。この方法では、約6時間かかり、このようなFACS装置など高価な設備を必要としません。さらに、この方法は、表面抗体媒介FACS分離に比べて比較的安価である。 FACSレーザ露光分離15の間に細胞死を誘導する傾向があるため、静止衛星細胞の...

筋衛星細胞は、骨格筋線維の基底膜の下に位置する筋原幹細胞の小集団である。それらはのPax7、のPax3、c-Metの、M-カドヘリン、CD34、シンデカン3の発現を特徴とし、カルシトニン、1 - 3。衛星細胞、筋肉幹細胞などの筋再生に関与することが証明されている。大人の筋肉では、衛星細胞は4-8通常、有糸分裂的に静止状態にある。損傷後、衛星細胞は活性化され、MyoDの発現を開始し、そしてその子孫を展開し、細胞周期を入力して、筋原性前駆細胞又は筋芽3と呼ばれる。細胞分裂のいくつかのラウンド後、筋芽細胞は、成熟筋繊維に続いて多核筋管へと分化するために、相互に細胞周期およびヒューズを終了する。大人の筋肉から分離された筋芽細胞は、容易にex vivoで拡張することができます。筋肉を再生させるにしてに筋線維になる筋芽細胞の能力非筋組織における異所性フォーム筋線維は、筋芽細胞移植、デュシェンヌ型筋ジストロフィー（DMD）4のための潜在的な治療アプローチ、泌尿器機能障害9、および心不全10によって利用される。確かに、筋芽細胞は正常にMDX（DMDモデル）マウスおよびDMD患者11から14の両方の筋肉に移植されてきた。注入された筋芽細胞は、正常な、罹患筋の組織学および機能を改善するために、宿主筋線維と融合する。以前の研究は、筋芽細胞の亜集団は、より細胞様幹と筋再生5中に筋肉になった未分化状態のままにしていることを明らかにした。最近の研究は、成体筋から新たに単離されたサテライト細胞、筋肉5-8の再生に、より効率的な生着及び自己再生活性を示す幹細胞様集団を含有することが示されている。従って、成体骨格MUから静止衛星細胞の純粋な集団の精製SCLEは、衛星細胞、筋芽細胞と筋再生の生物学を理解するため、および細胞ベースの治療法の開発のために不可欠である。 しかし、静止衛星細胞の現在の将来の精製方法は、高価な蛍光活性化細胞選別（FACS）1,2,6-8機械の使用を必要とする。また、FACSレーザ露光は、静止衛星細胞15の下部収率を引き起こす分離時に細胞死を誘導する傾向がある。ここでは、成体マウスの骨格筋からの静止衛星細胞の、急速な経済的、かつ信頼性の高い精製のための新しい手法を提案する。この方法では、ソーティング（MACS）、磁気活性化細胞が続く酵素的解離を利用しています。純粋な静止衛星細胞の単離後、これらの細胞は、何代かの継代後の筋芽細胞を大量に得るために培養することができる。我々はまた示すことが、これらの新たに単離された静止衛星細胞またはex VIの筋肉内注射VO展開さ筋芽細胞が心臓毒に移植することができる（CTX）筋線維だけでなく、自己再生活動の検査のための衛星細胞区画への再生へのドナー由来細胞の寄与を調べるために、マウスの骨格筋の再生に誘導される。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Marigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen</td>
			<td>10569010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>100 mg (3-4 mg/ml)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>bFGF, human, Recombinant</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0263</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5611-1G</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F10 Medium</td>
			<td>Gibco-Invitrogen</td>
			<td>11550-043</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco-Invitrogen</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycin</td>
			<td>Gibco-Invitrogen</td>
			<td>15640055</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco-Invitrogen</td>
			<td>14190144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco-Invitrogen</td>
			<td>25200072</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>18G needle with 12cc Syringe</td>
			<td>Fisher Scientific</td>
			<td>22-256-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#956;m)</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 ml tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml disposable pipet</td>
			<td>BD Biosciences</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody-PE</td>
			<td>eBiosciences</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody-PE</td>
			<td>eBiosciences</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca1 antibody-PE</td>
			<td>eBiosciences</td>
			<td>Dec-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;7 antibody</td>
			<td>MBL International</td>
			<td>ABIN487462</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini &#38; MidiMACS Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma Aldrich</td>
			<td>C9759-1MG</td>
			<td>Stock 10 &#956;M in PBS</td>
		</tr>
		<tr>
			<td>31G Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>328438</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge (Microfuge 22R)</td>
			<td>Beckman Coulter</td>
			<td>368826</td>
			<td></td>
		</tr>
		<tr>
			<td>S241.5 Swinging Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368882</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Allegra X-22R)</td>
			<td>Beckman Coulter</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr>
			<td>Nod/Scid immunodeficient mice</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code 394</td>
			<td>Use 2 months old mice</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Name of the reagent</td>
			<td></td>
			<td></td>
			<td>Recipie</td>
		</tr>
		<tr>
			<td>10% and 2% FBS DMEM</td>
			<td></td>
			<td></td>
			<td>DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>0.2% Collagenase solution</td>
			<td></td>
			<td></td>
			<td>Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.</td>
		</tr>
		<tr>
			<td>10% Matrigel solution</td>
			<td></td>
			<td></td>
			<td>Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20&#176;C until use.</td>
		</tr>
		<tr>
			<td>Matrigel-coated plate</td>
			<td></td>
			<td></td>
			<td>Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37&#176;C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20&#176;C for reusing.</td>
		</tr>
		<tr>
			<td>0.01% Collagen solution</td>
			<td></td>
			<td></td>
			<td>Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.</td>
		</tr>
		<tr>
			<td>Collagen-coated plate</td>
			<td></td>
			<td></td>
			<td>Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.</td>
		</tr>
		<tr>
			<td>bFGF stock solution</td>
			<td></td>
			<td></td>
			<td>bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 &#956;l in 500 &#956;l microcentrifuge tubes and kept in -80&#176;C.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td></td>
			<td></td>
			<td>500 mL HAM&#8217;S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 &#956;g of bFGF (20 &#956;l of bFGF stock).</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td></td>
			<td></td>
			<td>500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>10 &#956;M Cardiotoxin stock</td>
			<td></td>
			<td></td>
			<td>1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.</td>
		</tr>
	</tbody>
</table>

isolation, culture, and transplantation of muscle satellite cells

筋衛星細胞は、筋線維におけるsublaminal核の2〜5％を占め、出生後の骨格筋の発達と再生に必要な幹細胞集団である。大人の筋肉では、衛星細胞は通常、有糸分裂的に静止状態にある。損傷後、しかしながら、衛星細胞、筋肉の再生を媒介する筋芽細胞、それらの子孫を生成するために細胞増殖を開始する。衛星細胞由来の筋芽細胞の移植が広く筋ジストロフィー、心不全、および泌尿器機能障害を含むいくつかの疾患のための再生可能な治療法として研究されてきた。ジストロフィー骨格筋、梗塞、心臓、および機能不全、尿管への筋芽細胞移植は、移植された筋芽細胞は、宿主組織内の筋線維に分化し、これらの疾患における部分的な機能改善を表示することができますことを示している。したがって、骨格musclから静止衛星細胞の効率的な精製方法の開発Eだけでなく、衛星細胞由来の筋芽細胞培養および筋芽細胞のための移植法の確立など、衛星細胞の自己再生、活性化、及び分化の背後にある分子メカニズムを理解するために不可欠です。また、筋ジストロフィー、その他の再生の病気のための細胞ベースの治療法の開発は、これらの要因に依存している。 しかし、静止衛星細胞の現在の将来の精製方法は、高価な蛍光活性化細胞選別（FACS）装置の使用を必要とする。ここでは、ソーティング（MACS）、磁気活性化細胞が続く酵素的解離による成体マウスの骨格筋からの静止衛星細胞の、急速な経済的、かつ信頼性の高い精製のための新しい手法を提案する。純粋な静止衛星細胞の単離後、これらの細胞は、何代かの継代後の筋芽細胞を大量に得るために培養することができる。これらの新たに単離した静止衛星細胞またはex vivoで増殖させた筋芽細胞は、心臓毒に移植することができる（CTX）筋線維の再生にドナー由来細胞の寄与を調べるために、マウスの骨格筋の再生に誘導されるだけでなく、自己複製の検査のための衛星細胞区画へ活動。

新たに単離した静止衛星細胞は小さく、丸みを帯びた形状（ 図1G）を表示し、静止衛星細胞のための決定的なマーカーとしてのPax7を発現する。新たに単離した細胞の90％以上がのPax7（ 図1Hおよび1I）を発現する。最も汚染された細胞は、効率的に筋芽細胞の培養条件に従って、インビトロで増殖しない血液細胞からのものである。このように、衛?...

Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Isolation, Culture, and Transplantation of Muscle Satellite Cells

静止衛星細胞の純粋な集団の単離および培養、筋肉幹細胞集団は、筋肉幹細胞生物学および再生、ならびに筋ジストロフィーおよび他の変性疾患における治療のための幹細胞移植の理解に不可欠である。

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

isolation-culture-and-transplantation-of-muscle-satellite-cells

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Unit 1

Fibroblast Transformation and Muscle Stem Cells

SCIENTISTS IN ACTION

41.2K ビュー.  University of Minnesota Medical School. 静止衛星細胞の純粋な集団の単離および培養、筋肉幹細胞集団は、筋肉幹細胞生物学および再生、ならびに筋ジストロフィーおよび他の変性疾患における治療のための幹細胞移植の理解に不可欠である。 

ビデオ: Isolation, Culture, and Transplantation of Muscle Satellite Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

造血幹細胞の分離と移植（造血幹細胞）

生物学

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

ヒト臍帯動脈平滑筋細胞の単離（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

鳥類胚弁膜前駆細胞の単離と培養

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

マウスプライマリ膵臓腺房細胞の単離と培養

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

新生児マウスから肺動脈平滑筋細胞の単離

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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 ...