Name: megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells
Uploaded: 2017-12-27T14:00:00
Description: Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

著者は、(プロジェクト助成 1012409 BHC にリンク) オーストラリア健康と医学研究評議会のサポートを認めます。

このプロトコルは南東シドニー人間研究倫理委員会で承認され、大学のニュー ・ サウス ・ ウェールズの人間研究倫理委員会によって批准されました。健康なドナーから得られる臍帯血によって、シドニーの臍帯血バンク (シドニー, ニューサウス ウェールズ州, オーストラリア) が提供されました。約 100 mL のボリュームは、この手順で使用されました。
メモ: クラス II...

<ol>
	<li>Nakeff, A., Maat, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+megakaryocytes+from+mouse+bone+marrow+by+velocity+sedimentation.">Separation of megakaryocytes from mouse bone marrow by velocity sedimentation.</a> Blood. 43 (4), 591-595 (1974).</li><li>Zeigler, F. C., 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=In+vitro+megakaryocytopoietic+and+thrombopoietic+activity+of+c-mpl+ligand+(TPO)+on+purified+murine+hematopoietic+stem+cells.">In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells.</a> Blood. 84 (12), 4045-4052 (1994).</li><li>Reems, J. -. A., Pineault, N., Sun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Megakaryocyte+Production+and+Platelet+Biogenesis:+State+of+the+Art.">In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art.</a> Transfus. Med. Rev. 24 (1), 33-43 (2010).</li><li>Choi, E., Nichol, J. L., Hokom, M. M., Hornkohl, A. C., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+generated+in+vitro+from+proplatelet-displaying+human+megakaryocytes+are+functional.">Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional.</a> Blood. 85 (2), 402-413 (1995).</li><li>Perdomo, J., 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=A+monopartite+sequence+is+essential+for+p45+NF-E2+nuclear+translocation,+transcriptional+activity+and+platelet+production.">A monopartite sequence is essential for p45 NF-E2 nuclear translocation, transcriptional activity and platelet production.</a> J Thromb Haemost. 8 (11), 2542-2553 (2010).</li><li>Shim, M. H., Hoover, A., Blake, N., Drachman, J. G., Reems, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profile+of+primary+human+CD34+CD38lo+cells+differentiating+along+the+megakaryocyte+lineage.">Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage.</a> Exp Hematol. 32 (7), 638-648 (2004).</li><li>Feng, Q., 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=Scalable+generation+of+universal+platelets+from+human+induced+pluripotent+stem+cells.">Scalable generation of universal platelets from human induced pluripotent stem cells.</a> Stem cell reports. 3 (5), 817-831 (2014).</li><li>Bruno, S., 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=In+vitro+and+in+vivo+megakaryocyte+differentiation+of+fresh+and+ex-vivo+expanded+cord+blood+cells:+rapid+and+transient+megakaryocyte+reconstitution.">In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells: rapid and transient megakaryocyte reconstitution.</a> Haematologica. 88 (4), 379-387 (2003).</li><li>Iraqi, M., Perdomo, J., Yan, F., Choi, P. Y. I., Chong, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+thrombocytopenia:+antiplatelet+autoantibodies+inhibit+proplatelet+formation+by+megakaryocytes+and+impair+platelet+production+in+vitro.">Immune thrombocytopenia: antiplatelet autoantibodies inhibit proplatelet formation by megakaryocytes and impair platelet production in vitro.</a> Haematologica. 100 (5), 623-632 (2015).</li><li>Lev, P. R., 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=Impaired+proplatelet+formation+in+immune+thrombocytopenia:+a+novel+mechanism+contributing+to+decreased+platelet+count.">Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count.</a> Br. J. Haematol. 165 (6), 854-864 (2014).</li><li>Gandhi, M. J., Drachman, J. G., Reems, J. A., Thorning, D., Lannutti, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+generating+platelet-like+fragments+from+megakaryocytic+cell+lines+and+human+progenitor+cells.">A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells.</a> Blood Cells Mol. Dis. 35 (1), 70-73 (2005).</li><li>Lu, S. -. J., 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=Platelets+generated+from+human+embryonic+stem+cells+are+functional+in+vitro+and+in+the+microcirculation+of+living+mice.">Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice.</a> Cell Res. 21 (3), 530-545 (2011).</li><li>Moreau, T., 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=Large-scale+production+of+megakaryocytes+from+human+pluripotent+stem+cells+by+chemically+defined+forward+programming.">Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.</a> Nature Commun. 7, 11208 (2016).</li><li>De Bruyn, C., Delforge, A., Martiat, P., Bron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+megakaryocyte+progenitor+cells:+cord+blood+versus+mobilized+peripheral+blood.">Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood.</a> Stem Cells Dev. 14 (4), 415-424 (2005).</li><li>Nimgaonkar, M. T., 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=A+unique+population+of+CD34++cells+in+cord+blood.">A unique population of CD34+ cells in cord blood.</a> Stem cells. 13 (2), 158-166 (1995).</li><li>Italiano, J. E., Patel-Hett, S., Hartwig, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+proplatelet+elaboration.">Mechanics of proplatelet elaboration.</a> J Thromb Haemost. 5, 18-23 (2007).</li><li>Matsunaga, T., 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=Ex+vivo+large-scale+generation+of+human+platelets+from+cord+blood+CD34++cells.">Ex vivo large-scale generation of human platelets from cord blood CD34+ cells.</a> Stem cells. 24 (12), 2877-2887 (2006).</li><li>Sullenbarger, B., Bahng, J. H., Gruner, R., Kotov, N., Lasky, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+continuous+in+vitro+human+platelet+production+using+three-dimensional+scaffolds.">Prolonged continuous in vitro human platelet production using three-dimensional scaffolds.</a> Exp Hematol. 37 (1), 101-110 (2009).</li><li>Proulx, C., Boyer, L., Hurnanen, D. R., Lemieux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+ex+vivo+expansion+of+megakaryocytes+from+human+cord+blood+CD34+-enriched+cells+in+the+presence+of+thrombopoietin+and+limiting+amounts+of+stem+cell+factor+and+Flt-3+ligand.">Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.</a> J. Hematother. Stem Cell Res. 12 (2), 179-188 (2003).</li><li>Bornstein, R., Garcia-Vela, J., Gilsanz, F., Auray, C., Cales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+megakaryocytes+do+not+complete+maturation,+as+indicated+by+impaired+establishment+of+endomitosis+and+low+expression+of+G1/S+cyclins+upon+thrombopoietin-induced+differentiation.">Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.</a> Br. J. Haematol. 114 (2), 458-465 (2001).</li><li>Chong, B. H., Choi, P. Y. I., Khachigian, L., Perdomo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+Immune+Thrombocytopenia.">Drug-induced Immune Thrombocytopenia.</a> Hematol Oncol Clin North Am. 27 (3), (2013).</li><li>Stasi, R., 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=Idiopathic+thrombocytopenic+purpura:+Current+concepts+in+pathophysiology+and+management.">Idiopathic thrombocytopenic purpura: Current concepts in pathophysiology and management.</a> Thromb Haemost. 99 (1), 4-13 (2008).</li></ol>

ここで説明されているプロトコルは、MK と臍帯血から文化における血小板の一貫生産に適しています。これらの細胞は、MK 増殖、分化、proplatelet の形成と血小板の生産に及ぼす薬や生物学的活動など様々 なプロセスを研究に使用できます。
文化メディアとサイトカインの組み合わせの様々 な文献で紹介されています。また幹細胞因子、フェルマーの最終定理 3 配位子、...

通常研究室用プライマリ ヒト巨核球 (MK) の十分な数の分離は不可能です、骨髄での低周波による有核細胞1の ~0.01% のアカウントは、彼ら。便利な代替手段は、拡張前のヴィヴォと造血幹・前駆細胞の特定の成長因子の存在下で分化です。巨核芽球の文化の最も効果的な成長および分化因子は、MKs。 トロンボポエチン (TPO) を生産する培養システムの採用 (SCF; c キット リガンド) 幹細胞因子とインターロイキン (IL)-3 IL 11 などサイトカインの数がされています。単独で、または SCF と IL 32などの他のサイトカインが有効であります。TPO は、増殖と MKs2の成熟の両方で発生する幹細胞集団を操作できます。
1000 までは毎日 150-400 x 109/L. 血小板生産の in vitroの血小板数を維持するために形成された proplatelets と呼ばれる細胞質突起から MK プロデュース血小板や、生体内で約 1 × 10 の11の血小板が-これは CD34 を使用して多数の培養条件に上昇を与えているし体内の見積もり3より低いフォールド+を改善する MK と血小板生産体外造血前駆細胞。CD34 の初期ソース+ MK 分化用細胞は、ひと末梢血4だった。骨髄5,6、胚性幹細胞/誘導多能性幹細胞 (ESC/iPSC)7臍帯血 (UCB)8,9,10 などの他の細胞の源.ひと骨髄 CD34+ 11とマウス系統負, 骨髄細胞5生産 MK と血小板体外;人間の骨髄の可用性の欠如が CD34 のソースとしての使用を制限するそれにもかかわらず、+のセル。対照的に、ESC と iPSCの in vitro血小板の生産のための細胞の無制限のソースを表します。これらの細胞からの血小板の生産には、フィーダー細胞マウス OP9 細胞長期培養などが必要です。フィーダー無料条件で派生した血小板は、少ない機能12のように見えます。iPSC 由来血小板が大規模に拡張することができますので、臨床の現場で使用する可能性があります。このプロセスには、転写因子と長期的な細胞文化13レンチウイルスベクターを介した情報伝達が必要です。
UCB は CD34 のアクセス ソース+で容易に使用することができます細胞研究設定。TPO だけではコードの分化を促進することができます血由来 CD34+の血清添加またはフィーダー細胞との共培養を必要とせず非常に純粋な成熟した MKs を生じさせる細胞とこれ。他のサイトカイン SCF UCB CD34 から分化が低下するなど、+細胞、フェルマーの最終定理 3 配位子と IL-11 未熟な巨核球14の生産を推進中。このプロトコルは非常に純粋な臍帯血 CD34 から MK 文化+の生産を記述する無血清条件下で細胞。

<table>
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-013</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM II</td>
			<td>Stem Cell Technologies</td>
			<td>9605</td>
			<td>Serum-free media for CD34+ cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CD34 Isolation Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 MicroBead kit ultrapure</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-453</td>
			<td>This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Alere Technologies</td>
			<td>1114545</td>
			<td>Lymphocyte separation media (density 1.077 g/mL)</td>
		</tr>
		<tr>
			<td>Sterile separation buffer (SB)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td>This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow Cytometry and Cell Staining Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>340669</td>
			<td>Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>PerCP mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>347464</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>PerCP isotype control</td>
			<td>BD Biosciences</td>
			<td>349044</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>FITC Mouse anti-Human CD41a</td>
			<td>BD Biosciences</td>
			<td>340929</td>
			<td>Final antibody concentration 1:5 dilution.</td>
		</tr>
		<tr>
			<td>APC Mouse anti-Human CD42b</td>
			<td>BD Biosciences</td>
			<td>551061</td>
			<td>This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human CD42a</td>
			<td>AbD Serotec</td>
			<td>MCA1227A647T</td>
			<td>Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse Negative Control</td>
			<td>AbD Serotec</td>
			<td>MCA928A647</td>
			<td>Currently distributed by Bio-Rad. Isotype control antibody</td>
		</tr>
		<tr>
			<td>Anti von Willebrand factor rabbit polyclonal</td>
			<td>Abcam</td>
			<td>AB6994</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>V450 mouse anti-humna CD41a</td>
			<td>BD Biosciences</td>
			<td>58425</td>
			<td>1: 20 dilution</td>
		</tr>
		<tr>
			<td>V450 isotype control</td>
			<td>BD Biosciences</td>
			<td>580373</td>
			<td>1:20 dilution</td>
		</tr>
		<tr>
			<td>PAC1-FITC antibody</td>
			<td>BD Biosciences</td>
			<td>340507</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>Anti CD62p mouse monoclonal</td>
			<td>Abcam</td>
			<td>AB6632</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11020</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Ig Isotype Control cocktail-C</td>
			<td>BD Biosciences</td>
			<td>558659</td>
			<td>Isotype control antibody</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C36950</td>
			<td>Counting beads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Smaller and larger columns are also commercially available</td>
		</tr>
		<tr>
			<td>MidiMACS Separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile</td>
			<td>In Vitro technologies</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J3800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIRB inverted microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge</td>
			<td>ThermoScientific</td>
			<td>Cytospin 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser software</td>
			<td>BD Biosciences</td>
			<td>FACS Diva Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell analysis software</td>
			<td>Tree Star</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope software</td>
			<td>Zeiss</td>
			<td>Zen 2 blue edition</td>
			<td></td>
		</tr>
	</tbody>
</table>

megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells

血小板は、骨髄血小板と呼ばれるプロセスで主に発生します。血小板産生、中に造血前駆細胞は巨核球は、proplatelets と呼ばれる長い細胞質プロセスから血小板を解放する区別する末期と呼ばれるフォーム血小板前駆細胞に区別します。巨核球は骨髄に閉じ込められた珍しいセルであり、実験室の使用のための十分な数字で収穫することは困難であるため。ヒト巨核球を効率的に生産することができます達成体外CD34 を養殖+の適した条件下のセルします。ここで詳細なプロトコル CD34 の分離を記述する臍帯血液サンプルから磁気セルソーターによる細胞の+ 。無血清条件下で非常に純粋な成熟した巨核球の生成に必要な手順を示します。巨核球分化の表現型解析および proplatelet の形成と血小板の生産の決定の詳細もあります。巨核球分化および/または抗血小板抗体などトロンボポエチン ヒューマンミメティック最前線の proplatelet の形成に影響を与えるエフェクターは、生物学的機能を調べるための培養細胞に追加できます。

このプロトコルにより、コードから高純度の MK 文化準備血由来 CD34+細胞。CD34 の割合+細胞コード血は約 1.315 (図 1 a)、10 x 90-300 から単核細胞 (ステップ 1.8) 範囲の総数 UCB 単位6 。CD34 の純度 + CD45 陽性細胞分離範囲は 90 から 99% (図 1 b) 後。MK (CD41 として定義されている+細胞...

Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

巨核球の人口は非常に純粋なコードから得られる血由来 CD34+細胞。CD34 のメソッド+細胞分離と巨核球分化をここで説明します。

巨核球分化および人間の血小板形成臍帯血由来 cd34 陽性細胞の+

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

The overall goal of this procedure is to use human cord blood samples to isolate pure CD34-positive cells to study the effects of specific drugs of interest on megakaryocyte differentiation, proplatelet formation, and platelet production. This method can help answer key questions on the megakaryocyte and platelet field, such as what are the effects of a specific treatments on megakaryocyte differentiation and platelet production and function. The main advantage of this technique is that it provides a method for the consistent isolation of viable CD34-positive cells from fresh umbilical cord blood.Through this method can provide insight into megakaryopoiesis, other cell types such as lymphocytes, erythrocytes, and macrophages, can also be studied. Begin by adding 10 milliliters of separation buffer into 150 milliliter conical tube per 10 milliliters of cord blood. Using an 18 gauge blunt needle mounted on a 10 milliliter syringe, transfer 10 milliliters of blood into each tube, followed by the addition of 15 milliliters of lymphocyte separation medium to the bottom of each tube.Separate the cells by density gradient centrifugation, then at the end of the spin, carefully collect five to 10 milliliters of mononuclear cell containing buffy coat layer into one new tube per sample. Bring the total volume in each tube up to 50 milliliters with a fresh separation buffer and centrifuge the cells again. Re-suspend the pellets in five to 10 milliliters of fresh separation buffer and pool the cell samples.Adjust the volume to 50 milliliters with fresh separation buffer for counting and set aside 100 microliters of cells to determine the percentage of CD34 positive cells by flow cytometry. Centrifuge the cells again and re-suspend the pellet in 300 microliters of separation buffer, 100 microliters of FC receptor human IgG blocking reagent, and 100 microliters of CD34 magnetic beads per one times 10 to the eighth cells. Mix the cells gently before placing them at four degrees Celsius for a 30 to 40 minute incubation.During the incubation, place an appropriately sized magnetic bead separation column into the corresponding magnetic holder and wash the column with three milliliters of separation buffer. At the end of the incubation, remove the unbound beads by centrifugation in five to 10 milliliters of separation buffer and re-suspend the magnetic bead and cell pellet in 1.5 milliliters of fresh separation buffer. Load the labeled cells onto the column and collect the unlabeled flow through in a 15 milliliter collection tube.Load the effluent back onto the column and collect the second round of flow through into the same collection tube. Then wash the column three times with three milliliters of separation buffer and remove the column from the magnet. Using the column plunger, gradually flush two milliliters of separation buffer through the column into a new 15 milliliter tube and return the column to the magnet.Load the flushed cells back onto the column. Collect the flow through and wash the column with two milliliters of separation buffer as just demonstrated. After removing the column from the magnet, flush the column with two milliliters of fresh separation buffer to collect the CD34-positive cell fraction as just demonstrated, and collect the CD34-positive cells by centrifugation.Then resuspend the pellet in serum-free medium for CD34-positive cells. For megakaryocyte differentiation, seed five x 10 to the fifth CD34-positive cells per milliliter in 2 milliliters of serum-free medium, supplemented with recombinant human thrombopoietin per well in a 12-well plate for their incubation at 37 degrees Celsius, and 5%carbon dioxide, in a humidified atmosphere. At the appropriate differentiation monitoring time points, harvest the cells from two to three wells per time point without disturbing the cells in the other wells, and stain the collected cells with anti-CD41, and anti-CD42a antibodies to determine the percentage of mature megakaryocytes within the cultures.For microscopic visualization at the cell surface markers, wash the harvested cells in 1 milliliter of separation buffer, and resuspend the pellet in 100 microliters of fresh buffer. Use a cytospin to seed the cells onto a glass slide, and fix the cells to the slide with a 30-second submersion in methanol. After air drying, label the cells with 20 microliters of DAPI-supplemented mounting medium, and cover the cells with a cover slip for visualization by fluorescent microscopy.To count the proplatelets, harvest the cells at day eight or nine of differentiation, and seed the collected cells at one x 10 to the fourth cells per 200 microliters of fresh serum-free medium, supplemented with recombinant human thrombopoietin per well in a 48-well plate. After five days at 37 degrees Celsius and 5%carbon dioxide, count the number of proplatelet-bearing megakaryocytes per well on an inverted light microscope, using the 10 or 20 times objective. To analyze the platelet activation, on day 14 or 15 of culture, gently mix the cells with a Pasteur pipette, and collect 100 microliters of cells.Add 1 milliliter of Tyrode's buffer to the cells and pellet them by centrifugation. Collect the supernatant for centrifugation to pellet the platelet-sized particles, and resuspend the particles in 100 microliters of fresh Tyrode's buffer, then label the particles with anti-PAK1 antibody, activate them with the adenosine diphosphate for 20 minutes at room temperature, and analyze the percentage of PAK1 positive events by flow cytometry. The typical percentage of CD34-positive cells in cord blood is approximately 1.3%and the purity of the CD34-positive CD45-positive cells after isolation ranges from 90 to 99%CD41-positive megakaryocytes are observed early in serum-free CD34-positive cell cultures, with the percentage of mature CD41-positive CD42a-positive megakaryocytes reaching 30 to 40%by day 7, and the highest levels of double positive cells observed between days 10 and 12 of differentiation.Cultured megakaryocytes appear as large, usually multi-nucleated cells that express CD42b Von Willebrand factor, and P-selectin in their cytoplasm. At least three classes of ploidy are also commonly observed in these cell cultures. The percentage of megakaryocytes that bear proplatelets, long beaded fibers that extend from the megakaryocyte body, is typically around 1.3%Most platelets in the cell culture supernatant fall within the analytical gate of peripheral blood platelets, are positive for the CD41 platelet marker, can be activated by platelet agonists like adenosine diphosphate, as demonstrated by an increased binding of the activation specific PAK1 monoclonal antibody.After watching this video, you shall have a good understanding of how to isolate CD34-positive cells from cord blood. You shall also be able to set up megakaryocyte differentiation cultures, count proplatelets, and analyze platelets and megakaryocytes produced in vitro. Don't forget that working with human samples can be hazardous, and precautions, such as working in a class II biological safety cabinet should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

の臨床応用<em&gt;眠れる森の美女</em遺伝的に末梢及び臍帯血由来のT細胞を変更する&gt;と人工抗原提示細胞

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

免疫・感染学

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

単離、分化、および血液からヒト抗体を分泌するB細胞の定量化：体液性免疫の機能読み出しなどのELISpot

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

TGF-βを含むプロトコルを使用したナイーブCD4 + T細胞からのヒトCD4 + FOXP3 +誘導された調節性T細胞（iTregs）のインビトロ分化における

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

通常記憶 B 細胞の長命の形質細胞への分化モデル

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

ヒト幹細胞由来腸腸単層におけるM細胞様細胞の誘導分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

多能性幹細胞からのヒトマクロファージの生成と特性

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...