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 border="0" cellpadding="0" cellspacing="0" width="1201">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:327px;">BD Bioscience (San Jose, CA-USA)</td>
			<td style="width:149px;">354230</td>
			<td style="width:416px;">Murine ECM proteins 
			Stock Concentration: 100% (9-12 mg/ml) 
			Final Concentration: 100%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS)</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">D8537</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">70 &#956;m Filters</td>
			<td style="width:327px;">Miltenyi Biotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ficoll-Paque PREMIUM&#160;</td>
			<td>GE Healthcare (Uppsala, Sweden)</td>
			<td style="width:149px;">17-5442-03</td>
			<td>medium for discontinuos density gradient centrifugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pooled human AB type serum (PhABS)</td>
			<td>LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">14-490E</td>
			<td style="width:416px;">Final Concentration: 10%</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Glutamax-I</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">35050-038</td>
			<td style="width:416px;">Stabilized L-Glutamine 
			Stock Concentration: 100X 
			Final Concentration: 2 mM</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">A8412</td>
			<td style="width:416px;">Stock Concentration: 7.5% 
			Final Concentration: 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Sodium Azide</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">S8032</td>
			<td>Final Concentration: 0.02%</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:309px;">Penicillin/Streptomycin (Pen Strep)</td>
			<td style="width:327px;">Gibco (Grand Island, NY, USA)</td>
			<td style="width:149px;">15070-063</td>
			<td style="width:416px;">Antibiotics 
			Stock Concentration: 5,000 UI/mL penicillin, 5,000 ug/mL Streptomycin 
			Final Concentration: 50 UI/mL penicillin, 50 ug/mL Streptomycin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">T-75 culture flask for suspension cultures</td>
			<td style="width:327px;">Greiner Bio-one&#160; (Frickenhausen, Germany)</td>
			<td style="width:149px;">658 190</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">T-75 culture flask TC treated</td>
			<td style="width:327px;">Greiner Bio-one&#160; (Frickenhausen, Germany)</td>
			<td style="width:149px;">658170</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">TrypLE Select</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">12563-011</td>
			<td style="width:416px;">Animal- free proteases detaching solution 
			Stock Concentration: 1X 
			Final Concentration: 1X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Trypsin/EDTA</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">15400-054</td>
			<td style="width:416px;">Phenol red free 
			Stock Concentration: 0.5% 
			Final Concentration: 0.25%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD90 APC antibody&#160; (CD90)</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-402</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD45 APC-Vio770 antibody (CD45)</td>
			<td>MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-096-609</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD73 PE antibody (CD73)</td>
			<td>MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-182</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD31 PE Vio-770 antibody (CD31)</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-105-260</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 APC antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-846</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG2a APC Vio770 antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-096-637</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 PE antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-845</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 PE Vio-770 antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-563</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Low Glucose Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">13-1331-82</td>
			<td style="width:416px;">Phenol red-free minimal essential medium 
			Stock Concentration: 1&#39;000 mg/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Fetal Bovine Serum&#160; (FBS)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">10500</td>
			<td style="width:416px;">Stock Concentration:0.2 mg/mL 
			Final Concentration: 2 &#956;g/mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Prolong Gold antifade reagent with 4&#8217;,6-diamidino-2-phenylindole</td>
			<td style="width:327px;">Invitrogen (Waltham, MA,&#160; USA)</td>
			<td style="width:149px;">P-36931</td>
			<td style="width:416px;">Aqueous mounting medium + DAPI 
			Final Concentration: 1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Paraformaldehyde</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">P6148</td>
			<td style="width:416px;">Fixative 
			Final Concentration: 4%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">LAB-TEK two-well chamber slides</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">C6682</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Anti-Nestin antibody [clone 10C2]</td>
			<td style="width:327px;">Abcam (Cambridge, UK)</td>
			<td style="width:149px;">ab2035</td>
			<td style="width:416px;">Stock Concentration: 1 mg/ml 
			Final Concentration: 7 &#956;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Alexa Fluor 555 Phalloidin</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">A34055</td>
			<td style="width:416px;">Stock Concentration: 200 UI/ml 
			Final Concentration: 5 UI/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Triton X-100</td>
			<td style="width:327px;">Euroclone (Milan, Italy)</td>
			<td style="width:149px;">EMR237500</td>
			<td style="width:416px;">Final Concentration: &#39;0,05%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">MesenPRO RS Medium&#160; (MSC-RS medium)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td>12746-012</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Alexa Fluor 488 anti-mouse SFX kit</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">A31619</td>
			<td style="width:416px;">Goat anti-mouse secondary antibody + Signal enhancer 
			Stock Concentration: 2 mg/ml 
			Final Concentration: 2 &#956;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Pasteur Pipette</td>
			<td style="width:327px;">Kartell Labware&#160; (Noviglio (MI), ITALY )</td>
			<td>329</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">StemMACS AdipoDiff&#160; Media</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-091-679</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">StemMACS OsteoDiff&#160; Media</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-091-678</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Osteoimage Bone mineralization Assay</td>
			<td style="width:327px;">LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">PA-1503</td>
			<td>Hydroxyapatite specific fluorescent staining solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">50mL Polystyrene conical tube</td>
			<td style="width:327px;">Greiner bio-one (Kremsm&#252;nster 
			Austria)</td>
			<td style="width:149px;">227261</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Nile Red</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">N1142</td>
			<td style="width:416px;">Fluorescent staining solution for lipids 
			Stock Concentration: 100 mM 
			Final Concentration: 200 Nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Glycerin</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">G2289</td>
			<td>Final Concentration: &#39;50%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Polistirene Petri dishes</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">P5606</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">24-well plates TC-treated</td>
			<td style="width:327px;">Greiner Bio-one GmbH (Frickenhausen, Germany)</td>
			<td style="width:149px;">662160</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Endothelial Growth Medium, EGM-2 BulletKit&#160; (EGM-2)</td>
			<td style="width:327px;">LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">CC-3162</td>
			<td>VEGF-rich endothelial cell growth medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Leica Qwin Image Analisys Software</td>
			<td style="width:327px;">Leica (Wetzlar, Germany)</td>
			<td style="width:149px;"></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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Research

JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.0K 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

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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 ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

成体ラットおよびヒト脊髄の脳室周囲領域から神経幹/前駆細胞の単離

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

ヒト人工多能性幹細胞由来の錐体ニューロンからの樹状突起棘の三次元定量

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

骨髄ニッチに造血細胞の生着のダイナミクスを研究する遺伝的モデルと生体内蛍光顕微鏡 (IVFM) を組み合わせること

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

潜在的細胞質置換療法のための骨髄間葉系幹細胞からの血漿膜小胞の調製

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

成熟シュワン細胞の発生源としての骨髄由来前駆細胞の低酸素プレコンディショニング

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...