This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

注：腸骨稜からのヒト骨髄吸引は、ルツェルン州の倫理委員会の承認を得てドナー同意から採取した。犬の所有者のconsent.Human（約20ml）およびイヌ（約10ml）を骨髄穿刺液を直ちに中止後に3.8％クエン酸ナトリウムを15mlの添加により抗凝固して腸骨稜からイヌ骨髄吸引物を採取した手術室で。試料を回収と同日処理する実験室の環境に移した。 ウロキナーゼの調製（従来1回目使用すること） <ol><li>製造業者の説明書に従って、滅菌リン酸緩衝生理食塩水（PBS）を使用してウロキナーゼを再：10 mlシリンジを用いてセプタム入口フラスコに10mlのPBSを加える。ミリリットル50,000射出ユニット（U）を得かき混ぜることによって乾燥物質を溶解する。 </li><li> Sに500μlのアリコート（25000 Uそれぞれ）を準備terileチューブ。ストアアリコート-20℃と、少なくとも6ヶ月間に必要なときにそれを使用しています。 </li></ol> 2.取手順（従来すべての骨髄治療へ） <ol><li>凝固した骨髄穿刺液あたりウロキナーゼの一アリコート（25000 U）を解凍し（25 mlまで吸引ボリュームが正常25000 Uの単一のアリコートを使用して治療されている）。 </li><li> 37℃の水浴または振盪インキュベーターを予熱。 </li></ol>血餅の3酵素消化物<ol><li>処理中の骨髄試料の微生物汚染を避けるために、層流生物学的安全キャビネット内ですべての作業を行う。感染の可能性の人間の材料を使用する場合は、保護手袋の使用だけでなく、慎重な取り扱いをお勧めします。 </li><li>無菌の50ミリリットルチューブの上に100μmのセルストレーナーを置きます。慎重にストレーナを傾けたり、凝固材料の周りに移動する、セルストレーナーを通して骨髄穿刺液を注ぐ。のための滅菌ピペットチップを使用して、フィルターメッシュを通してより良い流れ。 注：PBSで凝血塊を洗浄することにより、例えば 、血餅のすべての希釈は避けてください。ウロキナーゼは、間接的に作用し、効果的なダイジェストのための生検からの血清の成分（ すなわち 。、プラスミノーゲン）が必要です。凝固材料と手順を継続しながら、ろ液をステップ3.6においてさらに使用するまで室温で保つことができる。 </li><li>滅菌ピンセットを使用して、空の細胞培養皿に骨髄クロット材料を転送します。滅菌メスを用いて約2ミリメートル3の小片に破片をカット。 </li><li>滅菌ピンセットを用いた50ml反応管に血餅の小片を転送する。ミンチ血栓が濡れ外観を有することを確認してください。それが乾燥しているように見える場合は、湿らせるステップ3.1からのろ液の一部を使用。 </li><li>ピペッティング5ミリリットルマイクロピペットを用いて、5回によって凝血塊を粉砕する。サンプルにウロキナーゼの1分量を追加します。水浴中、またはのいずれかで37℃で30分間インキュベート揺れ（優しく）インキュベータ。 </li><li> 5ミリリットルピペットを用いて5回上下にピペッティングとによって粉砕物。バイオセーフティキャビネットに、この手順を実行します。 37℃でさらに30分間インキュベートする。インキュベーション後5mlのピペットを使用して再度5回粉砕する。ステップ3.2からの濾液とプールする新鮮さ100μmのセルストレーナー上を通過。 </li><li>遠心分離機500×gで10分間、周囲温度で細胞懸濁液。上清を捨てる。以下に記載またはMSC増殖培養のためのあなたの研究室の標準的な手順に従って、メディアで細胞ペレットを再懸濁。 </li></ol>拡張細胞培養およびCFUプレート4.シード<ol><li> α-MEM、10％ウシ胎児血清（FBS）、100 U / mlペニシリン、100mg / mlのストレプトマイシンおよび2.5mg / mlのアンホテリシンBを補充した：基礎培地50ml中の（赤血球および単核細胞からなる）細胞を再懸濁。ノイバウアーchambeを使用してトリパンブルー溶液で1:10に希釈した細胞を数えるR。 </li><li> 5×10 7細胞/ cm 2の密度で細胞培養フラスコ中の細胞をプレーティングし、37℃の加湿インキュベーター中でインキュベートする。利用可能な場合は、低酸素（5％酸素）の条件を使用しています。 CFUアッセイの制御のために、直径10cmの細胞培養皿に10 9細胞をプレー。 </li><li>他の細胞は懸濁状態のままで、培養の3日後に、間葉系幹細胞は、細胞培養皿に付着している。 5 / mlの塩基性線維芽細胞増殖因子（bFGF）を補充した基礎培地でメディアを変更。 CFUアッセイ制御のために、同様に細胞培養皿を扱う。 </li><li>メディア週3回の交換を、2週間の合計のための細胞培養を続けます。細胞が80％コンフルエントを超えた場合、トリプシン処理によって分割。 CFUアッセイ制御のために、同様にメディアを交換しますが、2週間のために細胞を分割しないでください。 </li></ol> CFUアッセイ5.ギムザ染色<ol><li> DIL：皿当たりギムザ-溶液10mlを準備UTE原液（グリセロール中で7.6グラム/リットルギムザ：メタノール）滅菌水で1:10（常に新鮮なワーキング希釈液を調製）。 </li><li>細胞培養皿から培地を除去し、PBSで細胞を洗浄。ペトリ皿に液体を適用するときに非常に警戒して、細胞を洗浄をオフにしないようにしてください。 </li><li> RTで5分間純メタノールで細胞を固定してください。メタノールを捨てる。ギムザ溶液を添加し、37℃の加湿インキュベーター中で60分間インキュベートする。 </li><li> PBSで2回洗浄する。空気は、ペーパータオル上で第一の板状のヘッドを乾燥させる。手動でコロニーを数える。これが最善のプレートの背面にマーカーペンを使用して達成される。 </li></ol> 6. MSCの分化<ol><li>犬MSCの分化 注：犬MSCは、4週間の適切なメディアでの刺激により軟骨、骨形成および脂肪生成系統に分化した。 </li><li>のために 脂肪細胞分化、4×10 5 CEでの単層における培養のMSC次のように二つの異なる培養条件を交互LLS / cm 2で、 <ol><li> DMEM +グルタ、3％FBS、100単位/ mlのペニシリン、100mg / mlのストレプトマイシン、2.5 / mlのアンホテリシンBおよび170 mMのインスリンを含有する脂肪生成維持培地で培養。 </li><li> 3％FBS、5％ウサギ血清、1μMデキサメタゾン、500μMの3-イソブチル-1-メチルキサンチン、33μMビオチン、5μMロシグリタゾンおよび17μMパントテン酸16を補足した維持培地と脂肪生成誘導メディア文化。 </li><li>オイルレッドOで染色することにより脂肪滴を楽しむ。簡単に述べると、10％ホルムアルデヒドで細胞を固定し、PBSで洗浄し、イソプロパノール中0.35％オイルレッドOで染色。 </li></ol></li><li> 7×10 3細胞/ cm 2と以下に刺激で単層中の骨形成分化培養MSCの場合： <ol><li>アドバンストDMEM（GIBCO）+グルタ、5％FBS、100単位/ mlのペニシリン、100mg / mlのストレプトマイシン、2.5 / mlのアンホテリシンB、501; M L-アスコルビン酸-2-リン酸、10mMのSS-グリセロリン酸および100nMデキサメタゾン。 </li><li>フォン·コッサ染色（5％のAgNO 3）で鉱化預金を特定します。簡単に説明すると、蒸留水5％のAgNO 3を含むPBS汚れで洗浄し、10％ホルムアルデヒドで細胞を固定する。 </li></ol></li><li>軟骨細胞分化<ol><li>スポンジ状の医療機器からカットキューブ（側面あたり3 mm）は、凍結乾燥されたI型コラーゲンから構成され、セル17を支持する足場材料として用いる。 </li><li> 4×10 6細胞/ ml（〜7万細胞/立方体）の濃度の立方体の上にシードのMSC。 </li><li>培地の添加の前に、細胞を30分間キューブに付着させる。 </li><li> DMEM / F12 +グルタ、2.5％FBS、100単位/ mlのペニシリン、100mg / mlのストレプトマイシン、2.5 / mlのアンホテリシンB、40 / mlのデキサメタゾン、50μg/ mlのアスコルビン酸からなる軟骨形成培地中のMSC-コラーゲン構築物を維持する酸2リン酸、を50μg/ mlのL-プロリン、1×インスリン - トランスフェリン - セレンX、および増殖因子β1を形質転換を10ng / mlである。 </li><li>構文セクションにプロテオグリカンの蓄積を可視化するためにアルシアンブルー染色を使用してください。簡単に言えば、0.01％のH 2 SO 4および0.5M塩酸グアニジンに溶解した0.4％アルシアンブルーのセクションO / Nを染色する。次に、洗浄のセクションでは、40％DMSOおよび0.05MのMgCl 2中で30分間洗浄した。細胞を核ファーストレッドで対比染色した。 </li></ol></li></ol>

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The authors have nothing to disclose.

患者は少しだけ追加作業が手術室に人員によって行わなければならない利点と、（我々の場合、主に手術を背骨）手術を受けている間に定期的に、我々は、骨髄を​​サンプリングする。サンプルはすぐに撤退した後、クエン酸ナトリウムと混合されるにもかかわらず、彼らは、処理のために研究室に到着したとき、多くのサンプルが部分的に凝固しただった。この段階では、凝固した標本?...

間葉系幹細胞（MSC）は、再生医療及び組織工学における主要な役割を果たしている。彼らは彼らに自家療法の2,3のための理想的な候補をレンダリングしている、様々な細胞タイプ1に分化し、生着、移行することができます。最近、骨および軟骨修復のためのMSCを用いた臨床試験は、宿主病または心臓病、移植片対4を立ち上げた。これらのMSCは、臍帯または脂肪組織から採取することができるが、最も有望な結果は、骨髄由来の幹細胞5から得られた。 腸骨稜は、骨髄のかなりの量を収集することができ、したがって、吸引6の主な部位として働く。しかし、吸引の品質を回収骨髄の体積の増加に伴って減少する。骨髄穿刺液の最初の5mlを、高品質のMSCを含み、一方、より大量の撤退は、末梢血fの吸引物の希釈につながる高度に血管骨7を挿入します。抗凝固剤を使用しない場合は、そのため、本巨核球及び血小板の、骨髄吸引物は、凝固する傾向がある。しかし、たとえ抗凝固剤で、血栓が発生することがあります。 骨髄では、MSCは、全細胞プール8のごく一部を表し、ほとんどの組織工学または治療用途4培養中で増殖しなければならない。そのような文化が大きくすなわち 、初期の細胞プールに依存している。、多様性と高始動番号9の品質。引き出しからMSCの低い数字は、部分的にドナーの変動によって説明することができる。一方、低品質のサンプルからのMSCは、所望の細胞数に到達するために培養においてより長い時間を延長継代を必要とする。いずれの場合も、拡張継代は、細胞老化の源であり、10の潜在的分化の喪失につながる可能性がある。したがって、セルyを最大化することができるプロトコルを最適化ieldと有害な影響を防ぐには、11,12を開発しなければならない。 私たちは、犬のMSCとの仕事を始めたとき、私たちは幸いに凝固したヒトサンプル（10人に1）はあまり頻繁であったが約3〜4におけるイヌの骨髄サンプルは、血栓が含まれているのを見て驚いた。一方で、それは我々が凝固したサンプルからのMSCのはるかに低い収量を観察していることを、全く驚きませんでした。凝固したサンプルの定期的な問題を解決するために、我々は代わりにリサンプリングの血栓溶解薬ウロキナーゼを使用してプロトコルを開発しました。 血栓溶解療法は、心臓発作、脳卒中ため、または不要な凝固の塞栓症の原因となる血管の閉塞などの生命を脅かす状況に対抗することができます。彼らは、プラスミンおよび酵素プラスミノーゲン活性化因子によるフィブリンの酵素的切断を通して血栓の分解によって働く。患者の治療のための幅広い使用にもかかわらず、ごく少数の出版物は、その利用血栓溶解活性を存在実験室でのアプリケーションは、凝固したサンプルを救出するために、主にリンパ球に焦点を当てた。 1987年、肉じゃがら 。 13と4年後の機能的なリンパ球になり、血栓を溶解するためのストレプトキナーゼの使用を記載し、デ·ヴィスら 。フローサイトメトリーアプリケーション14のための血液および骨髄からの白血病細胞を単離するためのストレプトキナーゼの使用を拡張する。より最近の刊行物は、癌の診断15アルテプラーゼの使用を示唆している。同じ酵素アプローチを使用している間、私たちのプロトコルは、多能MSCの分離に焦点を当てて、幹細胞分野の研究者のためのツールを提供するために、骨髄を形成する。

<table><tbody><tr><td>Basal Medium Components</td><td></td><td></td><td></td></tr><tr><td>PenStrep 100X</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>MEM Alpha w/ Nucleoside, w/ stable Glutamine</td><td>Amimed</td><td>1-23S50-I</td><td></td></tr><tr><td>FBS Heat Inactivated</td><td>Amimed</td><td>2-01F36-I</td><td></td></tr><tr><td>Amphotericin B</td><td>Applichem</td><td>A1907</td><td></td></tr><tr><td>Adipogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>DMEM-HAM F12 + GlutaMAX</td><td>Amimed</td><td>1-26F09-I</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma</td><td>I5500</td><td></td></tr><tr><td>Rabbit serum&amp;nbsp;</td><td>Gibco</td><td>16120099</td><td></td></tr><tr><td>Dexamethasone</td><td>Applichem</td><td>D4902</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>Biotin&amp;nbsp;</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>Rosiglitazone&amp;nbsp;</td><td>Sigma</td><td>R2408</td><td></td></tr><tr><td>Pantothenate&amp;nbsp;</td><td>Sigma</td><td>P5155</td><td></td></tr><tr><td>Oil Red-O&amp;nbsp;</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>Osteogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>&amp;szlig;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>Silver nitrate (AgNO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S6506</td><td></td></tr><tr><td>Chondrogenic Medium Components</td><td></td><td></td><td></td></tr><tr><td>Biopad - sponge shaped medical device&amp;nbsp;</td><td>Euroresearch</td><td></td><td></td></tr><tr><td>L-proline&amp;nbsp;</td><td>Sigma</td><td>P5607</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium X</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>Human transforming growth factor-&amp;beta;1&amp;nbsp;</td><td>Peprotech</td><td>100-21</td><td></td></tr><tr><td>Alcian Blue 8GX</td><td>Sigma</td><td>A3157</td><td></td></tr><tr><td>Nuclear fast red</td><td>Sigma</td><td>N8002</td><td></td></tr><tr><td>Generic</td><td></td><td></td><td></td></tr><tr><td>Tri-Sodium citrate dihydrate</td><td>Applichem</td><td>A3901</td><td></td></tr><tr><td>PBS</td><td>Applichem</td><td>964.9100</td><td></td></tr><tr><td>Urokinase</td><td>Medac</td><td>1976826</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Gibco</td><td>15400054</td><td></td></tr><tr><td>Giemsa stain</td><td>Applichem</td><td>A0885</td><td></td></tr><tr><td>Formaldehyde</td><td>Applichem</td><td>A0877</td><td></td></tr><tr><td>Sulfuric acid (H2SO4)</td><td>Applichem</td><td>A0655</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Applichem</td><td>A1584</td><td></td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Applichem</td><td>A3618</td><td></td></tr><tr><td>Guanidine hydrochloride</td><td>Applichem</td><td>A1499</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>50 ml reaction tube</td><td>Axygen</td><td>SCT-50ML-25-S</td><td></td></tr><tr><td>10 ml syringe</td><td>Braun</td><td>4606108V</td><td></td></tr><tr><td>Sterican needle (22G)</td><td>Braun</td><td>4657624</td><td></td></tr><tr><td>1.7 ml Microtubes</td><td>Brunschwig</td><td>MCT-175-C</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Falcon</td><td>6.05935</td><td></td></tr><tr><td>sterile forceps</td><td>Bastos Viegas, SA</td><td>489-001</td><td></td></tr><tr><td>sterile scalpel</td><td>Braun</td><td>5518059</td><td></td></tr><tr><td>Primaria cell cuture dish</td><td>Falcon</td><td>353803</td><td></td></tr><tr><td>C-Chip Neubauer Improved</td><td>Bioswisstech</td><td>505050</td><td></td></tr><tr><td>cell culture flask - Flask T300</td><td>TPP</td><td>90301</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Microbiological biosafety cabinet class II</td><td>Skan</td><td>82011500</td><td></td></tr><tr><td>water bath</td><td>Memmert</td><td>1305.0377</td><td></td></tr><tr><td>Stripettes Serological Pipette 5ml</td><td>Corning</td><td>4487-200ea</td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>humidified incubator Heracells 240</td><td>Thermo scientific</td><td>51026331</td><td></td></tr><tr><td>Heraeus Multifuge 1S-R</td><td>Thermo scientific</td><td>75004331</td><td></td></tr></tbody></table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

と一緒に、彼らは私たちの研究室（ 図1A）に到着したとき、犬の骨髄サンプルの74パーセント（N = 54）は血栓を含んでいたことの事実は、私たちはMSCの相当数が、血栓内に捕捉されたと信じていた、これらのサンプルからMSC利回りの低下。実際に、切片凝固材料の単純なDAPI染色は、高密度（ 図1B）中の有核細胞の存在を確認する。これは最終的にウロキナーゼを使用...

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酵素法は、凝固した骨髄試料からの間葉系幹細胞を救出する

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.2K ビュー.  Swiss Paraplegic Research. Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

ビデオ: An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

ヒト間葉系幹細胞の単離および多孔質骨基質上での栽培

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

発生生物学

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

分離、シーケンスおよびヒト間葉系幹細胞からの細胞のマイクロ Rna を分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

遺伝学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

ラット間葉系幹細胞（MSC）とMSCを派生シングルコロニーの分離の分離と濃縮

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

生物学

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

自家条件で間葉系幹細胞の培養および配信：整形外科アプリケーションのためのスマートなアプローチ

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

生物工学

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

ヒト脂肪由来間葉系幹細胞の単離・増殖のための酵素フリー法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

医学

胸腺および骨髄からの間質細胞単離の最適化

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

著者スポットライト:シングルセルシーケンシングのための間質細胞単離を用いた造血研究の推進

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

造血器官からの間質細胞の単離

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

酵素法は、凝固した骨髄試料からの間葉系幹細胞を救出する

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造血器官からの間質細胞の単離

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