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

저자 (그랜트 1012409 제너 시스에 링크 된 프로젝트) 호주 보건 및 의료 연구 위원회의 지원을 인정 한다.

이 프로토콜 남쪽 동부 쪽 시드니 인간의 연구 윤리 위원회에 의해 승인 하 고 대학의 뉴 사우스 웨일즈의 인간 연구 윤리 위원회에 의해 비준 되었다. 탯 줄 혈액 건강 한 기증자 로부터 얻은 의해 시드니 코드 혈액 은행 (시드니, NSW, 호주)를 제공 했다. 볼륨 약 100 mL의이 절차를 위해 사용 되었다.
참고: 클래스 II biosafety 무 균 기술을 사용 하 여 캐비닛에서 작동 합니다. 70% 에...

<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>

여기에 설명 된 프로토콜은 MK와 문화 탯 줄 혈액에서 혈소판의 일관 생산에 적합 합니다. 이러한 세포 MK 확산, 감 별 법, proplatelet 형성 및 혈소판 생산에 마약 또는 생물 학적 활동의 효과 같은 다양 한 프로세스를 연구에 사용할 수 있습니다.
다양 한 문화 미디어와 cytokine 조합 문학에서 제시 되었다. + 줄기 세포 요소, Flt 3 ligand, 일리노이-3, 일리노이-6 지원 CD34 등 cyto...

일반 실험실 사용에 대 한 기본 인간의 없으며 (MK)의 충분 한 숫자의 절연은 ~0.01% nucleated 세포1의 담당 그들은 골 수에서 낮은 주파수로 인해 가능 합니다. 편리한 대안 ex vivo 확장 및 조 혈 줄기와 조상 세포 특정 성장 요인의 존재의 차별화 이다. Megakaryocytic 문화에 대 한 가장 효과적인 성장과 차별화 요소는 MKs. Thrombopoietin (TPO) 생산 문화 시스템에서 고용 cytokines 줄기 세포 요소 (SCF; c-kit ligand), IL-11 및 인터 루 킨 (IL)-3 등의 숫자 되었습니다. 혼자 또는 다른 cytokines, SCF 및 IL-32등 효과적 이다. TPO 확산 MKs2의 성숙에 결과 줄기 세포 인구에 작동할 수 있다.
Proplatelets 라는 세포질 돌출부에서 MK 생산 혈소판, vivo에서, 약 1 x 1011 혈소판은 최대 1000는 150-400 x 109/L. 혈소판 생산에서 생체 외에서 혈소판 수를 유지 하기 위해 매일 형성 -배는 vivo에서 견적3, 그리고이 에겐 CD34를 사용 하 여 여러 문화 조건 보다 낮은+ MK와 혈소판 생산에서 체 외에서개선 하기 위해 조 혈 조상 세포. CD34의 초기 소스+ 세포 MK 차별화 사용 했다 인간 주변 혈액4. 다른 셀 소스 등이 골5,6, 배아 줄기 세포/유도 만능 줄기 세포 (ESC/iPSC)7, 탯 줄 혈액 (UCB)8,9,10 . 인간의 골 CD34+ 11 와 마우스 리니지 부정적인 골 수 세포5 생산 MK와 혈소판 시험관; 인간의 골의 가용성의 부족 CD34의 근원으로 그것의 사용을 제한 하는 그럼에도 불구 하 고,+ 세포. 대조적으로, ESC, iPSC 생체 외에서 혈소판 생산을 위한 세포의 무제한 소스를 나타냅니다. 이 세포에서 혈소판 생산 murine OP9 셀 등 오랜 문화 피더 세포를 필요합니다. 급지대 무료 조건에서 파생 된 혈소판 기능 덜12나타납니다. iPSC 파생 혈소판 때문에 그들은 큰 규모로 확장 될 수 있다 임상 설정에 사용 될 가능성이 있다. 이 프로세스에서는 녹음 방송 요인과 장기 세포 문화13lentiviral 중재 변환 합니다.
UCB는 CD34의 액세스할 수 있는 소스+ 세포에 쉽게 사용할 수 있는 설정 연구. TPO 혼자 코드의 차별화를 홍보할 수 있는 혈액 유래 CD34+ 세포와이 생겨나게 혈 청 보충 또는 급지대 셀과 공동 문화에 대 한 필요 없이 매우 순수 하 고, 성숙한 MKs. 다른 cytokines SCF UCB CD34에서 차별화를 저하 될 수 있습니다와 같은+ 세포, Flt 3 ligand 그리고 IL-11 미 숙 없으며14의 생산을 촉진 하는 동안. 이 프로토콜에 설명 합니다 매우 순수한 MK 문화 코드 혈액 CD34에서에서+ 의 생산 세럼 무료 조건에서 세포.

<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

혈소판 생산 thrombopoiesis로 알려진 프로세스에서 골 수에서 주로 발생 합니다. Thrombopoiesis, 동안 조 혈 조상 세포 형태 혈소판 선구자 라는 없으며, 말기 출시 되 나 proplatelets 긴 세포질 프로세스에서 혈소판 분화를 구분 합니다. 없으며 희귀 세포 골 수에 국한 되며 따라서 실험실 사용을 위해 충분 한 숫자에 수확 하기가. 인간의 없으며의 효율적인 생산 수 달성 생체 외에서 CD34 경작+ 적당 한 조건 하에서 세포. 여기서 설명 하는 프로토콜 CD34의 격리에 설명 합니다+ 탯 줄 혈액 샘플에서 정렬 하는 자기 셀에 의해 세포. 매우 순수 하 고, 성숙한 없으며 혈 청 무료 조건 하에서 생산 하는 필요한 단계를 설명 합니다. Megakaryocyte 차별화의 phenotypic 분석 및 proplatelet 형성 및 혈소판 생산의 결정도 제공 됩니다. 이펙터 megakaryocyte 차별화 및 항 혈소판 항 체 등 thrombopoietin mimetics proplatelet 형성에 영향을 주는 생물 학적 기능 검사 경작된 한 세포에 추가할 수 있습니다.

이 프로토콜 수 있습니다 매우 순수 MK 문화 코드에서의 준비 혈액 유래 CD34+ 세포. CD34의 비율+ 세포 탯 줄 혈액은 약 1.315 (그림 1A)와 단 세포 (단계 1.8) 범위 90-300 x 10의 총 수 UCB 단위 당6 . CD34의 순도 + CD45 + 세포 격리 범위 90에서 99% (그림 1B) 후. MK (CD41 정의+ 세포) 혈 청 무료 CD3...

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

매우 순수한 인구의 없으며 코드에서 얻어질 수 있다 혈액 유래 CD34+ 세포. CD34 방법+ 셀 고립과 megakaryocyte 차별화는 여기에 설명 된.

Megakaryocyte 차별화와 인간에서 혈소판 형성 코드 혈액 파생 CD34+ 세포

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

임상 신청<em&gt; 잠자는 숲속의 미녀</em유전자 주변과 배꼽 탯줄 혈액에서 T 세포를 수정하는 셀을 제시&gt;과 인공 항원

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 ...

연구

면역학 및 감염병학

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

라이브 마우스의 혈관 염증과 혈전 형성하는 동안 활성화 내피에 Heterotypic 혈소판 - 호중구 상호 작용의 실시간 영상

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

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

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

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

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

분리는 차별화 및 정량화 인간의 혈액에서 B 세포를 항체가 분비 : ELISPOT을 체액 면역의 기능 판독으로

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 함유 프로토콜을 사용하여 나이브 CD4 + T 세포에서 인간의 CD4 + Foxp3의 + 유도 규제 T 세포 (iTregs)의 체외 분화

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

절연, 확장, 및 Adipogenic CD34 + CD31 + 인간의 Omental 및 피하 지방 조직에서 내 피 세포의 유도

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

수명이 긴 플라즈마 세포에 인간의 정상적인 메모리 B 세포의 체 외 분화 모델

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

인간 줄기 세포 유래 Ileal 장용 단층에서 M 세포 유사 세포의 분화 유도

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

다능성 줄기 세포에서 인간 대식세포의 생산 및 특성화

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