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

Los autores reconocen el apoyo de la salud australiano y el Consejo de investigación médica (proyecto grant 1012409 ligado a CSF).

Este protocolo fue aprobado por el Sur Oriental Sydney humano investigación Comité de ética y ratificado por el Comité de ética de investigación humana de la Universidad de New South Wales. Sangre del cordón umbilical obtenida de donantes sanos fue proporcionada por el Sydney cable de banco de sangre (Sydney, NSW, Australia). Se utilizaron volúmenes de 100 mL aproximadamente para este procedimiento.
Nota: Trabajo en un gabinete utilizando una técnica aséptica de bioseguridad clase 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>

El protocolo descrito aquí es conveniente para la producción constante de MK y plaquetas en la cultura de la sangre del cordón umbilical. Estas células se pueden utilizar para estudiar varios procesos tales como el efecto de drogas o actividades biológicas sobre la proliferación de MK, diferenciación, formación proplatelet y producción de plaquetas.
Una variedad de medios de cultivo y combinaciones de citoquinas se han presentado en la literatura. Además de las citoquinas como el fac...

Aislamiento de suficiente número de megacariocitos primarias humanas (MK) para uso en laboratorio regular no es factible debido a su baja frecuencia en la médula ósea, donde representan el ~0.01% de las células nucleadas1. Una alternativa conveniente es la expansión ex vivo y diferenciación de vástago de hematopoietic y células progenitoras en la presencia de factores de crecimiento específicos. Un número de citoquinas como factor de la célula de vástago (SCF; ligando c-kit) y el interleukin (IL) -3 y IL-11 ha sido empleadas en sistemas de cultivo para producir MKs. Thrombopoietin (TPO) es el factor más eficaz de crecimiento y la diferenciación megakaryocytic culturas y es eficaz solo o con otras citoquinas, como SCF y IL-32. TPO puede actuar sobre las poblaciones de células madre para dar lugar a la proliferación y maduración de MKs2.
Plaquetas de productos MK de protuberancias citoplasmáticas llamadas proplatelets y, en vivo, aproximadamente 1 x 1011 plaquetas formado diariamente para mantener las cuentas de plaqueta de 150-400 x 109plaquetas /L. producción en vitro es hasta 1000 -pliegue más bajo que el en vivo estima3, y esto ha dado lugar a numerosas condiciones de cultivo usando CD34+ células progenitoras hematopoyéticas para mejorar MK y plaquetas producción en vitro. La fuente inicial de CD34+ las células utilizadas para diferenciación de MK era sangre periférica4. Otras fuentes de células incluyen médula ósea5,6, inducido células madre embrionarias pluripotentes células madre (ESC/iPSC)7y cordón umbilical sangre (UCB)8,9,10 . Médula ósea humana CD34+ 11 y ratón linaje negativo médula células5 productos MK y las plaquetas in vitro; sin embargo, la falta de disponibilidad de médula ósea humana limita su uso como fuente de CD34+ las células. Por el contrario, ESC y iPSC representan una fuente ilimitada de células para la producción de plaquetas en vitro . Plaquetas la producción de estas células requiere células de alimentador como células murinas de OP9 y períodos más largos de la cultura. Plaquetas en condiciones libres de alimentación parecen ser menos funcional12. iPSC-derivado de las plaquetas están probable que sean de uso en contextos clínicos puesto que puede ser ampliados a gran escala. Este proceso requiere transducción mediada por lentivirales de factores de transcripción y a largo plazo de la célula cultura13.
UCB es una fuente accesible de CD34+ las células que se pueden usar en ajustes de la investigación. TPO solo puede promover la diferenciación de cable derivadas de sangre CD34+ las células y esto da lugar a MKs altamente puro, maduro, sin la necesidad de suplementos de suero o cocultivo con células de alimentador. Otras citocinas tales como SCF puede disminuir la diferenciación de UCB CD34+ de las células, mientras que el ligando de Flt-3 y IL-11 promoción la producción de megacariocitos inmaduros14. Este protocolo describe la producción de cultivos altamente puros de MK de sangre CD34+ las células en condiciones libres de suero.

<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

Producción de plaquetas se produce principalmente en la médula ósea en un proceso denominado Trombopoyesis. En la Trombopoyesis, células progenitoras hematopoyéticas distinguen a precursores de plaquetas de forma llamados megacariocitos, que terminal para liberar las plaquetas de procesos citoplásmicos largo denominados proplatelets. Megacariocitos son las células raras que limitan a la médula ósea y por lo tanto son difíciles de cosechar en número suficiente para uso en laboratorio. Producción eficiente de megakaryocytes humanos puede ser alcanzado en vitro por cultivo CD34+ de las células bajo condiciones convenientes. El protocolo detallado aquí describe el aislamiento de CD34 de las células+ por célula magnética clasificación de muestras de sangre de cordón umbilical. Se describen los pasos necesarios para producir los megakaryocytes altamente puros y maduros en condiciones libres de suero. También se proporcionan detalles de análisis fenotípico de la diferenciación de megacariocitos y determinación de la producción proplatelet de formación y de la plaqueta. Determinantes que influyen en la diferenciación de megacariocitos o formación de proplatelet, tales como anticuerpos antiplaquetarios o miméticos de la trombopoyetina, se pueden agregar a las células cultivadas para examinar la función biológica.

Este protocolo permite la preparación de cultivos altamente puros de MK de cable derivadas de sangre CD34+ las células. El porcentaje de CD34+ las células de cordón de sangre es de aproximadamente 1,3%15 (figura 1A) y el número total de las gamas de células mononucleares (paso 1.8) de 90-300 x 106 por unidad UCB. La pureza de CD34 + / CD45 + células después de rangos aislamiento de 90 a 99% (<st...

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

Una población altamente pura de megacariocitos puede obtenerse de cable derivadas de sangre CD34+ las células. Un método para CD34+ aislamiento y del megakaryocyte diferenciación se describe aquí.

Diferenciación de megacariocitos y plaquetas formación de humanos CD34 derivadas de sangre de la cuerda+ de las células

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

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Investigación

Inmunología e Infección

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