Name: isolating mesangiogenic progenitor cells (mpcs) from human bone marrow
Uploaded: 2016-07-15T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

著者は、特に骨髄サンプルと人間の骨前駆細胞の彼の専門知識を提供するための、ピサの大学、博士パオロ・パーチ、外科、医療・分子病理学とクリティカルケア医学の部門に感謝したいと思います

注：書面による同意後、HBMサンプルは人工股関節置換のための整形外科手術中に得られました。直ちに大腿骨頚部骨切り術の後にヘパリンの500 UIを含む20 mlシリンジをリーマ大腿前に、新鮮なBMを吸引するために使用されました。プロトコルは、任意のBM源に広く適用可能と考えられるべきです。 ヒト骨髄単核細胞（HBM-多国籍企業）の1の単離<ol><li>ダルベッコのリン酸緩衝生理食塩水（D-PBS）を適用し、50ミリリットルに新鮮なBMの10ミリリットル、反転により混合する - 5を希釈します。同様に、二つの新しい50mlコニカルチューブに25ミリリットルを配布し、各チューブにD-PBSの25ミリリットルを追加し、反転により混合します。 </li><li>管は、溶液からの鉱物骨片と脂肪の分離のために、室温で10分間放置します。 </li><li>慎重に滅菌パスツールピペットで浮遊脂肪を除去し、ミネラル骨片ペレットを乱すことなく、70μmのフィルターを通して細胞懸濁液をフィルタリングします。 </li> &lt;リー&gt;は不連続密度勾配遠心分離のための培地を15ml（1.077グラム/ ml）を用いて4 50mlチューブを設定します。この媒体は、室温であることを確認します。 </li><li>密度勾配媒体の上に希釈されたBMの25ミリリットル - 静かに20を置きます。層の混合を防止するために、チューブ壁に細胞懸濁液のトリクルをさせる、慎重にこの操作を実行します。 </li><li>無効ブレーキを室温で30分間、400×gでの密度勾配遠心分離を行います。 </li><li>滅菌パスツールピペットを使用して、2つの相の間に位置する細胞の白っぽいリングを収集し、新しい50 mlチューブに移します。 </li><li>新鮮な培養培地で細胞を洗浄する：フェノールレッドを含まない、低グルコース（1,000mgの/ l）のダルベッコ改変イーグル培地（DMEM）、10％（v / v）のプールしたヒトAB型血清（PhABS）、2mMのL-グルタミンおよび抗生物質（DMEM / 10％PhABS）。 5分間400×gで遠心分離します。 </li><li>新鮮なDMEM / 10％PhABSの10ミリリットル - 5で上清を吸引し、再サスペンドペレット。 </li><li>細胞数に進んでください。トリパンで1希釈：1で白血球の数を決定します。血球計数器にこれを適用し、位相差顕微鏡下で観察します。細胞数から小さく、完全に丸みを帯びた赤血球およびブルー染色死細胞を除外します。 注：非常MPC回復でのパフォーマンスのためPhABSバッチをスクリーニングすることをお勧めします。異なる起源のほとんどの血清がMSC様細胞の割合が高いとMPCの文化をもたらした米国源からPhABSは、最良の結果を与えています。 </li></ol> HBM-多国籍企業からのMPCの2の単離<ol><li>新鮮なDMEM / 10％PhABS 15mlで疎水性のT-75フラスコを設定し、pH及び温度を30分間、5％CO 2中37℃でプレインキュベートすることにより平衡化させ。 </li><li>シード4 -フラスコあたり6×10 7 HBM-多国籍企業とは48時間、5％CO 2中で37℃でインキュベートします。 </li><li>フラスコから培地および非接着細胞を吸引し、廃棄します。新鮮なDMEM / 10％PhABSの15ミリリットルを加え、37℃でインキュベート5％のCO 2。培地を48時間ごとに変える、8日 - 6のための文化を維持しています。 オプション：MPC収量を増加させるために、2.3から非接着細胞を新しい培養フラスコに再播種し、一次培養物について記載されるように維持することができます。 </li><li>吸引し、は、フラスコから培地を捨て、新鮮なDMEMで洗浄し、溶液を取り外す動物自由プロテアーゼの2ミリリットルを追加します。 （長時間のインキュベーションを避けるため）15分 - 5 37℃でインキュベートします。 </li><li> 5分間400×gで細胞懸濁液と遠心分離機を吸引、新鮮なDMEM / 10％PhABSの10ミリリットルを追加します。 </li><li>吸引し、上清を廃棄し、1にペレットを再懸濁 - 新鮮なDMEMの2ミリリットル/ 10％PhABS。ステップ1.10で説明したように細胞数に進んでください。 注：試薬を取り外すように、トリプシン/ EDTAを使用しないでください。 MPCが耐性トリプシンされています。文化の形態学的スクリーニングは、細胞収穫前に、非常に紡錘状MSC様細胞の存在を評価するために推奨されます。 MSC様細胞の場合、かなりの量でdはetected、選択的に汚染された細胞を除去することにより、細胞生成物の純度を高めます。そうするために、トリプシン消化は、2分間のトリプシン/ EDTA 2mlの0.05％を添加することにより、前のMPCの収穫に行うことができます。 DMEM / 10％PhABSの5ミリリットルで二回洗浄培養は、その後、上記のように治療をプロテアーゼに進みます。 </li></ol> 3.セル特性評価<ol><li>フローサイトメトリー<ol><li>洗浄液中に10 5の新たに分離した細胞の二連のサンプルを設定する：D-PBS、0.5％（v / v）のウシ血清アルブミン（BSA）および0.02％（w / v）のアジ化ナトリウムを補充しました。 5分間400×gで遠心分離します。 注意：アジ化ナトリウムは有毒です。 </li><li>洗浄溶液の200μl中のペレットを再懸濁し、蛍光色素（ &quot;TEST&quot;）と結合した抗CD90、抗CD45、抗CD73および抗CD31抗体を追加します。並列にアイソタイプコントロール（「CTRL」）を設定します。 注：染色する抗体の量は、滴定またはアコーによって決定されるべきです製造元の指示に鼎。 </li><li> 4℃で30分間サンプルをインキュベートします。 </li><li> 5分間400×gで遠心分離します。洗浄液500μlの中で細胞を再懸濁し、7 9 10サイトメータ多色の流れに少なくとも5×10 4のイベントを取得します。 </li><li>ドットプロットで結果を分析し、象限を設定するには、「CTRL」記録されたイベントを使用します。 注：MPC文化としての文化を定義するためにCD73 陰性 CD90の陰性の CD45 + CD31 +細胞の割合が95％の上にあるべきです。 98％ -いくつかの特定のアプリケーション、 すなわち 、遺伝子発現解析のために、回復カットオフは97に増加されなければなりません。 </li></ol></li><li>ネスチン検出およびF-アクチンの組織分析<ol><li>培養チャンバースライド上のプレート新たに単離したMPC（2万/ cm 2）でした。細胞は5％CO 2中で37℃で一晩インキュベートすることによって接着することを可能にします。 </li><li>洗浄液とFiの中で細胞を洗浄4％×15分間、室温で（w / v）のパラホルムアルデヒド。固定液を除去するために、洗浄液を追加し2分間インキュベートし、注ぎます。 </li><li>二回の洗浄を繰り返します。 </li><li> D-PBSで透過化細胞を室温で15分間、0.05％（v / v）のトリトンX-100を補いました。 </li><li>タンパク質を含まないシグナルエンハンサー（室温で30分）または標準ブロッキング溶液によって反応を停止する（D-PBSで3％を補った（w / vで）BSA）。 </li><li>ブロッキング溶液/シグナルエンハンサーを削除します。 </li><li> 7 / mlの追加抗ヒトネスチン一次抗体の（w / vの）と加湿チャンバー内で4℃で一晩インキュベートします。並行して、特定のない蛍光信号を評価するために、アイソタイプコントロール抗体を使用しています。 </li><li> D-PBSを加えることによって、スライドを洗浄し、2分間のままにして注ぎます。二回繰り返します。 </li><li>蛍光色素結合二次抗体を2μg/ mlの（w / vの）を追加し、暗所で1時間4℃でインキュベートします。 </li><li>ウォッシュは、上記のようにスライドします。 </li><li>蛍光ファロイジン（5 UI / ml）を追加し、30メートルをRTで残します暗闇の中でとD-PBSで3回洗浄します。 </li><li>チャンバ壁を除去し、核を検出するための退色防止試薬および4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）を補充した水性マウンティング培地中でスライドをマウントします。イメージング7 9 10に進んでください。 </li></ol></li></ol> MPCの4 Mesengenic分化<ol><li>プレート2×10 4 / cm 2のTC処置T75培養フラスコ中で新たに単離したMPC、細胞を5％CO 2中、37℃でDMEM / 10％PhABSに一晩接着させ。 </li><li>間葉系幹細胞の増殖（MSC-RS媒体）用に設計された標準的な減少血清培地、約200マイクロリットル/ cm 2と、を含むDMEM / 10％PhABSを交換してください。通常、誘導から7〜10日から、（P1-MSCを）コンフルエンスまで細胞を増殖させます。 2日ごとに培地リフレッシュ。 </li><li>吸引し、は、フラスコから培地を捨て、新鮮なMSC-RS培地で洗浄し、溶液を取り外す動物自由プロテアーゼの2ミリリットルを追加します。 37インキュベート76; 5のためのC - 15分（長時間のインキュベーションを避けるため）。 </li><li> 、新鮮なMSC-RS培地10mlを加え、5分間400×gで細胞懸濁液と遠心分離機を吸引</li><li>新鮮なMSC-RS培地2ml - 1で上清を吸引し、再サスペンドペレット。ステップ1.10で説明したように細胞数に進んでください</li><li> 5×10 3細胞/ cm 2から3を播種することによって、それらのサブカルチャーに進んでください。 （P2-MSCを）コンフルエンスまで細胞を増殖させます。 </li><li>ステップ4.3から説明したように、プロテアーゼ消化によって細胞を回収し、4.5に進み、第3章で説明したように特性評価に進みます。 注：CD73 + CD90 + CD45 陰性 CD31の陰性細胞の割合よりも低い95％が部分的にしか分化を示し、さらに培養継代を必要とするであろう。 </li><li> 6ウェルTC処理プレート中の2×10 4個 / cm 2でプレートP2-MSCおよびMSC-RS培地でコンフルエントになるまで成長します。 </li><li> 「いいえサ &quot;としてマーク2つのウェルとMSC-RS媒体をリフレッシュ。 </li><li>マーク2も「骨」などの、具体的にMSCの分化のために設計され、標準的な骨形成培地200μl/ cm 2の培地を交換してください。 </li><li>マーク「脂肪」として、2つのウェル、具体的にMSCの分化のために設計され、200μlの標準/脂肪生成培地のcm 2の培地を交換してください。 </li><li>メディア全体をそれぞれ48時間を変更することにより、5％CO 2中で37℃で培養を維持します。 注：非常にテスト再現するための標準的な市販の差別メディアを使用することをお勧めします。細胞内の脂肪滴の蓄積は、脂肪生成誘導された細胞において明らかである間に差別条件の下2/3週間後、カルシウム沈着は、骨形成誘導培養に表示されます。 </li><li> D-PBSで洗浄後、培養培地を吸引し、廃棄します。 </li><li>室温で15分間、パラホルムアルデヒド（w / v）の4％の1ミリリットルを追加することにより、文化を修正しました。 </li><li>固定液を除去するためにD-PBSを追加し、2分間インキュベートし、注ぎます。繰り返し洗濯二回。 </li><li> 2つの「骨」と1「いいえ差分を「染色ヒドロキシアパタイト特定の蛍光溶液中に井戸をマークされ、2つの「脂肪」と1「いいえ差分は「200 nMのナイルレッド溶液中に井戸をマークしました。室温11,12で30分間インキュベートします。 </li><li>染色ソリューションを削除し、二回D-PBSで洗浄します。 </li><li> D-PBSを削除し、D-PBSを追加50％（v / v）のグリセリンを補充し、イメージング7 9 10に進みます。 </li></ol> 5. MPCスフェロイド萌芽アッセイ<ol><li> 3Dスフェロイドを生成するには、シャーレのふたの内側に新たに単離したMPC懸濁液（1.5×10 4細胞/ドロップ）の20μlの滴を置きます。 注：取扱滴が自分の破裂につながる可能性としては、それが過剰でそれらを置くことは非常にお勧めです。 </li><li>慎重に吊り蒸発を低下防止するために、D-PBSを含むペトリ皿を要約するために蓋を使用します。 5％CO 2で37℃でインキュベート一晩の細胞が3Dスフェロイドに集約することを可能にします。 </li><li>プレ冷蔵24ウェル培養プレート中で標準的なECMタンパク質の300μlのアリコートを加えることにより、ネズ​​ミの細胞外マトリックス（ECM）タンパク質の厚いゲルをセットし、37℃で30分間インキュベートします。 </li><li>慎重にペトリ皿のふたを転倒し、静かに滅菌パスツールピペットを用いて、スフェロイドを拾います。 </li><li> 、ECMタンパク質ゲルにスフェロイドを置く標準VEGF豊富な内皮細胞増殖培地の700μlのアリコートを添加し、5％CO 2中、37℃でインキュベートします。 </li><li> 24時間後と7日間の培養で、4倍​​の倍率での3D培養物の写真を撮ります。最後の侵入細胞と回転楕円体のエッジとの間の半径方向の距離を測定することにより、画像解析ソフトウェアを適用するスフェロイドから発芽評価します。少なくとも20の異なる方向に沿って対策を繰り返します。平均距離は、ときに50ミクロン以上陽性とみなされます。 </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+heterogeneity+of+mesenchymal+stem+cell+populations:+clues+to+their+therapeutic+efficacy.">Biochemical heterogeneity of mesenchymal stem cell populations: clues to their therapeutic efficacy.</a> Cell Cycle. 6 (23), 2884-2889 (2007).</li><li>Phinney, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+mesenchymal+stem+cells:+implications+for+cell+therapy.">Functional heterogeneity of mesenchymal stem cells: implications for cell therapy.</a> J Cell Biochem. 113 (9), 2806-2812 (2012).</li><li>Tolar, J., Le Blanc, K., Keating, A., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+hitting+the+right+spot+with+mesenchymal+stromal+cells.">Concise review: hitting the right spot with mesenchymal stromal cells.</a> Stem Cells. 28 (8), 1446-1455 (2010).</li><li>Corselli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tunica+adventitia+of+human+arteries+and+veins+as+a+source+of+mesenchymal+stem+cells.">The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells.</a> Stem Cells Dev. 21 (8), 1299-1308 (2012).</li><li>Bieback, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+alternatives+to+fetal+bovine+serum+for+the+expansion+of+mesenchymal+stromal+cells+from+bone+marrow.">Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.</a> Stem Cells. 27 (9), 2331-2341 (2009).</li><li>Watson, L., Elliman, S. J., Coleman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+isolation+to+implantation:+a+concise+review+of+mesenchymal+stem+cell+therapy+in+bone+fracture+repair.">From isolation to implantation: a concise review of mesenchymal stem cell therapy in bone fracture repair.</a> Stem Cell Res Ther. 5 (2), 51 (2014).</li><li>Pacini, S., Petrini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+MSCs+angiogenic+cells?+New+insights+on+human+nestin-positive+bone+marrow-derived+multipotent+cells.">Are MSCs angiogenic cells? New insights on human nestin-positive bone marrow-derived multipotent cells.</a> Front Cell Dev Biol. 2, 20 (2014).</li></ol>

The authors have no competing financial interests or other conflicts of interest.

In the last decades, MSCs have been extensively researched and pre-clinically evaluated for possible application in the treatment of various bone/articular, immunological, neurological, cardiovascular, gastrointestinal and hematological disorders14,15. The easy and inexpensive isolation of multipotent MSCs, from many different tissues, together with their lack of significant immunogenicity16, contribute to make these cells one of the most interesting cell population to be applied in cell based therapies. Nonetheless, the very low frequency in the tissue of origin represents a great limitation to the MSCs application in clinics, forcing the expansion of these cells, in vitro, before the infusion or transplantation. 
Expanded MSC cultures have revealed high grades of heterogeneity and variability17-19 making it difficult to reach a consensus about MSC production and characterization protocols. Moreover, recent investigations suggested the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to culture heterogeneity10,20. In fact, it has been proposed that particular culture conditions possibly select or simply promote specific sub-populations of MSCs progenitors present, in various percentages, in "crude" and unprocessed samples like bone marrow (hBM-MNCs) or adipose tissues (stromal vascular fraction)2. Thus, the variability in MSC-initiating cell populations together with the great number of different enrichment/isolation and culture protocols applied, represent a great obstacle to the definition of feasible MSC-based therapies.
A crucial factor affecting heterogeneity of MSC cultures is serum supplementation21. In our hands replacement of FBS with PhABS in primary cultures from hBM-MNCs, combined with high density seeding on hydrophobic plastics, led to the isolation of a novel highly adherent cell population with distinct biological features named MPCs5,6. We observed that the addition of small percentages of PhABS to FBS primary cultures also allowed MPC isolation, suggesting the presence of MPC inducing agents in the human serum6. At the moment, the MPC isolation/characterization protocol is a unique method available to obtain almost pure MPCs. The protocol has been carefully adjusted and it is highly reproducible for quality screening of MPC preparations before further applications.
MPCs could be used as a source for MSC production, thus limiting the variability introduced by use of unfractionated starting material. The precise definition of the multiple steps characterizing MPC mesengenic differentiation reported9 would allow synchronized mesenchymal cell expansion. Nonetheless, this latest condition could be realized exclusively applying highly purified MPC population, as a consequence the characterization of the cell products obtained by the protocol described here, results of crucial importance. This isolating method has been reported allowing MPC recovery with purity generally around 95%. However, donor/patient variability together with the variability related to the different batches of human pooled serum applied, could lead to a significant percentage of MSC-like cells co-isolated together with MPCs, under selective conditions. 
It is not clear if these "contaminating" MSC-like cells could arise from the other different in vivo progenitors described in bone marrow22 or from uncontrolled and spontaneous MPC differentiation. In any case, a consistent percentage of MSC-like cells in the MPC products nullify the possibility to applying these cells as homogeneous starting material for the MSC expansion. Thus, here it has been suggested a simple and inexpensive method, based on the MPC resistance to trypsin digestion, increasing the purity of the MPC products. Similar or even better results in purifying MPC cultures could be achieved by fluorescent or magnetic cell sorting performing CD73 and/or CD90 depletion, but significantly prolonging the process time and increasing the costs.
Moreover, MPCs showed expression of pluripotency-associated markers and Nestin, all rapidly lost during mesengenic differentiation7. Sprouting assay revealed MPC ability to invade murine ECM protein gel. Taken together these results indicate that MPCs have to be considered a more immature progenitor, retaining angiogenic potential. Nonetheless, the initial enthusiasm about mesodermal differentiation potential of MPCs is actually waning. In fact, after more than 7 years of studies on MPCs, mesengenic and angiogenic potential have been extensively described5-9, but differentiation toward any other cells of mesodermal origin is still lacking. Thus, here we propose a new, and more rigorous, definition of these cells as "Mesangiogenic Progenitor Cells", maintaining the acronym MPCs.
We also believe that most controversies about MSC angiogenic potential could be related to the heterogeneous composition of expanded cultures consisting of sub-populations of MPCs and MSCs in variable percentages23.
Finally, MPCs could also play a crucial role for the implementation of CBMPs applicable for tissue reconstruction, as these cells could also support the neo-vascularization. In fact, future studies on regeneration should take in consideration that the newly formed tissue growth should be supported by concomitant neo-angiogenesis. The co-existence of mesengenic and angiogenic potential in MPCs could significantly improve the regeneration potential of new therapeutic approaches that involve these interesting cells.

間葉系幹細胞（MSC）は、それらの多系列分化能及び増殖因子/サイトカインを分泌するだけでなく、免疫調節1において役割を果たすことが造血を支持する能力、に関連する臨床的価値があります。 MSCベースの治療用細胞の生産とアプリケーションの定義では医薬品（CBMP）治療3ベースのセルの安全性と有効性のための具体的な国際規制に特に注意して、大規模な臨床および前臨床研究2の対象となっています。ヒトMSCは、ウシ胎児血清（FBS）、およびウシトリプシンのような動物由来のサプリメントおよび試薬を含む培地中で広範囲に培養されます。したがって、細胞操作に関連した感染リスクと並んで、患者はまた、プリオンの暴露ならびに細胞収穫とtransplan後も持続可能性タンパク質、ペプチドまたは動物由来の他の生体分子に結合された免疫学的なリスクに直面してテーション4。 この問題を回避するために、我々は、プールされたヒトAB型血清（PhABS）でFBSを置き換え、ヒト骨髄（HBM）動物を含まない培地中で由来MSCを培養しました。これらの条件下では、成長のMSCと一緒に、我々は、新規な細胞集団を同定しました。これらの細胞は、形態学的およびMSCからの表現型に異なっていたと独特の遺伝子発現プロファイルと同様に特徴的な成長/接着特性を示しました。彼らはmesengenicと潜在的な血管新生の両方に保持され、したがって、 中胚葉前駆細胞 （MPCは）5と命名しました。その後、我々は純度6の高品位でのMPCを生成するために、選択的かつ再現可能な培養条件を定義することができました。 我々はさらにのMPCの形態学的および生物学的特性を調べました。 MPCが、正ネスチンサイクリングで遅い、のKi-67陰性、および長いテロメア5によって特徴付け染色体であることを示しました。彼らはplurを表明しましたipotency関連転写は、因子Oct-4およびNanogのではなく、MSCのマスターレギュレータのRunx2とのSox9 7。間葉系マーカーCD73、CD90、CD166を欠く表現型が、MPCのは、MSCよりも低いレベルでエンドグリン（CD105）を発現しました。 MPCはまた、具体的ポドソーム状構造8を維持する接着PECAM（CD31）の一貫性の発現を特徴とする分子、インテグリンαL（のCD11a）、αM（CD11bの）、αX（CD11cの）だけでなく、インテグリンβ2（CD18）の特徴的なパターンを示しました。標準的なMSC増殖培地では、MPCが速やか9シグナリング Wnt5 /カルモジュリン細胞の活性化を特徴と中間段階を介してのMSCに分化しました。ネズミの細胞外マトリックス（ECM）タンパク質の三次元培養におけるスフェロイドから発芽する能力によって実証されるようのMPCはまた、血管形成特性を保持していました。血管新生の可能性は急速にmesengenic系統に沿ってMPCの分化後に失われました。 ここでは、プロの提示しますトコールは、単離するとHBMの血液サンプルから高度に精製されたMPCを特徴付けるために最適化されました。 MPC mesengenicと血管新生の分化のための再現性のあるプロトコルも記載されています。

<table><tbody><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Bioscience (San Jose, CA-USA)</td><td>354230</td><td>Murine ECM proteins&lt;br/&gt; Stock Concentration: 100% (9 - 12 mg/ml)&lt;br/&gt; Final Concentration: 100%</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (D-PBS)</td><td>Sigma (St. Louis, MO, USA)</td><td>D8537</td><td></td></tr><tr><td>70 &amp;mu;m Filters</td><td>Miltenyi Biotec (BergischGladbach, Germany)</td><td>130-095-823</td><td></td></tr><tr><td>Ficoll-Paque PREMIUM&amp;nbsp;</td><td>GE Healthcare (Uppsala, Sweden)</td><td>17-5442-03</td><td>medium for discontinuos density gradient centrifugation</td></tr><tr><td>Pooled human AB type serum (PhABS)</td><td>LONZA (Walkersville MD-USA)</td><td>14-490E</td><td>Final Concentration: 10%</td></tr><tr><td>Glutamax-I</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>35050-038</td><td>Stabilized L-Glutamine&lt;br/&gt; Stock Concentration: 100x&lt;br/&gt; Final Concentration: 2 mM</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td>Stock Concentration: 7.5%&lt;br/&gt; Final Concentration: 0.5%</td></tr><tr><td>Sodium Azide</td><td>Sigma (St. Louis, MO, USA)</td><td>S8032</td><td>Final Concentration: 0.02%</td></tr><tr><td>Penicillin/Streptomycin (Pen Strep)</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td>Antibiotics&lt;br/&gt; Stock Concentration: 5,000 UI/ml penicillin, 5,000 &amp;#956;g/ml Streptomycin&lt;br/&gt; Final Concentration: 50 UI/ml penicillin, 50 &amp;#956;g/ml Streptomycin</td></tr><tr><td>T-75 culture flask for suspension cultures</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658 190</td><td></td></tr><tr><td>T-75 culture flask TC treated</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658170</td><td></td></tr><tr><td>TrypLE Select</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12563-011</td><td>Animal-free proteases detaching solution&lt;br/&gt; Stock Concentration: 1x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Trypsin/EDTA</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>15400-054</td><td>Phenol red free&lt;br/&gt; Stock Concentration: 0.5%&lt;br/&gt; Final Concentration: 0.25%</td></tr><tr><td>anti-CD90 APC antibody&amp;nbsp; (CD90)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-402</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD45 APC-Vio770 antibody (CD45)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-609</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD73 PE antibody (CD73)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-182</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD31 PE Vio-770 antibody (CD31)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-105-260</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 APC antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-846</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG2a APC Vio770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-637</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-845</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE Vio-770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-563</td><td>Final Concentration: 1:40</td></tr><tr><td>Low Glucose Dulbecco&#039;s Modified Eagle Medium&amp;nbsp; (DMEM)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>13-1331-82</td><td>Phenol red-free minimal essential medium&lt;br/&gt; Stock Concentration: 1,000 mg/L glucose</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp; (FBS)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>10500</td><td>Stock Concentration:0.2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Prolong Gold antifade reagent with 4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>P-36931</td><td>Aqueous mounting medium + DAPI&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Paraformaldehyde</td><td>Sigma (St. Louis, MO, USA)</td><td>P6148</td><td>Fixative&lt;br/&gt; Final Concentration: 4%</td></tr><tr><td>LAB-TEK two-well chamber slides</td><td>Sigma (St. Louis, MO, USA)</td><td>C6682</td><td></td></tr><tr><td>Anti-Nestin antibody [clone 10C2]</td><td>Abcam (Cambridge, UK)</td><td>ab2035</td><td>Stock Concentration: 1 mg/ml&lt;br/&gt; Final Concentration: 7 &amp;mu;g/ml</td></tr><tr><td>Alexa Fluor 555 Phalloidin</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A34055</td><td>Stock Concentration: 200 UI/ml&lt;br/&gt; Final Concentration: 5 UI/ml</td></tr><tr><td>Triton X-100</td><td>Euroclone (Milan, Italy)</td><td>EMR237500</td><td>Final Concentration: 0.05%</td></tr><tr><td>MesenPRO RS Medium&amp;nbsp; (MSC-RS medium)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12746-012</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse SFX kit</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A31619</td><td>Goat anti-mouse secondary antibody + Signal enhancer&lt;br/&gt; Stock Concentration: 2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Pasteur Pipette</td><td>Kartell Labware&amp;nbsp; (Noviglio (MI), ITALY )</td><td>329</td><td></td></tr><tr><td>StemMACS AdipoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-679</td><td></td></tr><tr><td>StemMACS OsteoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-678</td><td></td></tr><tr><td>Osteoimage Bone mineralization Assay</td><td>LONZA (Walkersville MD-USA)</td><td>PA-1503</td><td>Hydroxyapatite specific fluorescent staining solution</td></tr><tr><td>50 ml Polystyrene conical tube</td><td>Greiner bio-one (Kremsm&amp;uuml;nster&lt;br/&gt; Austria)</td><td>227261</td><td></td></tr><tr><td>Nile Red</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>N1142</td><td>Fluorescent staining solution for lipids&lt;br/&gt; Stock Concentration: 100 mM&lt;br/&gt; Final Concentration: 200 Nm</td></tr><tr><td>Glycerin</td><td>Sigma (St. Louis, MO, USA)</td><td>G2289</td><td>Final Concentration: 50%</td></tr><tr><td>Polistirene Petri dishes</td><td>Sigma (St. Louis, MO, USA)</td><td>P5606</td><td></td></tr><tr><td>24-well plates TC-treated</td><td>Greiner Bio-one GmbH (Frickenhausen, Germany)</td><td>662160</td><td></td></tr><tr><td>Endothelial Growth Medium, EGM-2 BulletKit&amp;nbsp; (EGM-2)</td><td>LONZA (Walkersville MD-USA)</td><td>CC-3162</td><td>VEGF-rich endothelial cell growth medium</td></tr><tr><td>Leica Qwin Image Analisys Software</td><td>Leica (Wetzlar, Germany)</td><td></td><td>Image analysis software</td></tr></tbody></table>

isolating mesangiogenic progenitor cells (mpcs) from human bone marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

ここで説明する選択的培養条件はHBM-多国籍企業の1.0％として、新規な接着性と、ほぼ単型細胞集団の単離を可能にしている（0.5から2.0×10 5から6 HBM-多国籍企業-新鮮BMサンプルの10ミリリットル）5,6 。 MPCは5のように、静止状態、のKi-67陰性細胞を丸め、 -私たちは、これらの大（直径60μmで40）を同定しました。形態学的には、それらはより高い倍率（ 図1 A.1中の白矢印）での糸状仮足の多くを示すフラットな薄い周囲に囲まれた厚いコア領域と独特の目玉焼き-形状によって特徴づけられます。外側のセル境界の極性の伸長は、多くの場合（ 図1 A.1の黒矢印）が観察されます。このような形態は、標準的なMSC培養で報告された典型的な紡錘状間葉系間質細胞の外観とは明らかに異なります。フローサイトメトリーメートルしばらくCD31およびCD45を発現するために新たに単離したMPCの95％以上を示しました。esenchymal関連マーカーCD90とCD73 13は検出不能であった（ 図1 A.2）。私たちは、MPCのための4つの抗原のこの制限されたセットが示すものとして考えています。 MPCのさらなる顕著な特徴は、細胞内で検出されないポドソームのような構造（ 図1 A.3における赤）とネスチンの強烈な表現（ 図1 A.3で緑）、の数を明らかに点線のF-アクチン分布していますアイソタイプ対照抗体で染色した（データは示さず）。 メディア指数関数的に成長しているMSC様細胞（ 図1 B.1）への迅速な分化MSC拡張の結果を得るために設計された標準のRSでのMPCを培養します。 2継代細胞は、最終的には、CD73 + CD90 + CD45 陰性 CD31の陰性 （ 図1 B.2）にCD73 陰性 CD90 陰性 CD45 + CD31 +からその表現型を切り替えた後。プロセスにおいて、MPCは再orgaがストレスファイバーにnize F-アクチンネスチン発現は、いくつかのまれな細胞（ 図1 B.3）に閉じ込めなるが。明確なMSC様表現型へのMPC mesengenic分化は異なる細胞形態によって明らかにされた二つの別個のステップを介して行われます。 MSC RS培地で1週間MPC様細胞の残留集団後も（ 図2（a）におけるP1-MSCの）平坦な多角形の多分岐細胞のコンフルエントな層内で検出可能です。さらに、通路は、（ 図2のAにP2-MSCの）紡錘状MSC様細胞のほぼ単相培養物を得るために必要とされます。従って、それらのMSCの性質を確認し、少なくとも2週間選択培地に移したとき、これらの指数関数的に増殖する細胞は容易に骨芽細胞や脂肪細胞に分化することができます。骨形成誘導培養物において、カルシウム沈着は、測色アリザリンS染色または特異的な蛍光色素（ 図2 Bで緑）のいずれかによって検出することができます。脂肪生成誘導細胞の後（ 図2 Bに赤）のいずれかの比色オイルレッドや蛍光ナイルレッド染色によって明らかにされたように脂肪滴の蓄積を示しています。 MPCタイプは、血管新生アッセイを発芽によって確認しました。 MPCが24時間のVEGF-刺激（ 図2 C）後の3Dスフェロイドから（マイクロメートル50以上）は、マウスのECMタンパク質ゲルに侵入する能力を示しました。 600μmの距離 - 細胞に侵入し、一週間後に300で検出されました。逆に、侵入能力はmesengenic分化（ 図2 D）後にP2-MSCの中で失われました。 <img alt="図1" src="/files/ftp_upload/54225/54225fig1.jpg" /> 図1. 新たに単離したMPCが特徴的な機能を持っている。DMEM / 10％PhABSで培養HBM-多国籍企業の7日間は、MSCから容易に区別できる静止状態のMPC（A）の人口（B）を生じさせます</stronグラム&gt;形態（A.1、B.1、スケールバー= 100μm）で、表現型（A.2、B.2）、F-アクチン分布（A.3で赤、B.3）の観点から、とネスチン表現（A.3、B.3、スケールバーの緑色の= 20ミクロン）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54225/54225fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54225/54225fig2.jpg" /> 図2 のMPCは 、in vitroでの 標準MSCおよび表示萌芽血管新生に分化する 。MSCの拡張のために設計された市販のRS媒体を含むDMEM / PhABSの交換はMPCはのmesengenic誘導をトリガします。文化の中で1週間後、いくつかの残留のMPCはまだMSC-RS媒体のさらなる通路がコンフルエントMSC様細胞の集団（P2-MSCは、スケールバー= 100μm）とにつながりながら、検出可能な（P1-のMSC）です。 Pカルシウム沈着（Bにおける緑）、脂肪滴の蓄積（Bで赤、スケールバー= 200μm）とによって明らかにされたように2-MSCは末端それぞれ、適切な刺激下での骨細胞や脂肪細胞に分化します。 MPCがP2-のMSC（D、スケールバー= 1.0ミリメートル）との違いで、マウスのECMタンパク質ゲル（C）におけるスフェロイドから発芽一貫性を示している。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54225/54225fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

ヒト骨髄から単離することMesangiogenic前駆細胞（MPCは）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

isolating-mesangiogenic-progenitor-cells-mpcs-from-human-bone-marrow

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.1K ビュー.  University of Pisa. Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

ビデオ: Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

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.

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 ...

発生生物学

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

ヒト心臓組織からの血管周囲多能前駆細胞集団の単離

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

ひと臍帯血由来の血管内皮前駆細胞の分離

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

分離、カルチャ、および骨髄間質細胞とマウスから破骨細胞前駆細胞の分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

生物学

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

免疫・感染学

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

生物工学

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

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

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

ヒト骨格筋からの血管由来の多能前駆体の単離

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

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

医学

ラット顎骨髄間葉系幹細胞のニッチベースの単離

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

著者スポットライト:組織工学における高純度顎骨髄間葉系幹細胞の応用

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

ラット顎骨髄間葉系幹細胞の単離および培養技術

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...