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

The authors acknowledge the support of the Australian Health and Medical Research Council (project grant 1012409 linked to BHC).

This protocol was approved by the South Eastern Sydney Human Research Ethics Committee and ratified by the University of New South Wales&#39; Human Research Ethics Committee. Umbilical cord blood obtained from healthy donors was provided by the Sydney Cord Blood Bank (Sydney, NSW, Australia). Volumes of approximately 100 mL were used for this procedure.
NOTE: Work in a Class II biosafety cabinet using aseptic technique. Decontaminate the exterior of the cord blood bag with 70% ethanol. Use ste...

<ol>
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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>

The protocol described here is suitable for consistent production of MK and platelets in culture from umbilical cord blood. These cells can be used to study various processes such as the effect of drugs or biological activities on MK proliferation, differentiation, proplatelet formation, and platelet production.
A variety of culture media and cytokine combinations have been presented in the literature. Addition of cytokines such as stem cell factor, Flt-3 ligand, IL-3, and IL-6 supports CD34<s...

Isolation of adequate numbers of primary human megakaryocytes (MK) for regular laboratory use is not feasible due to their low frequency in the bone marrow, where they account for ~0.01% of nucleated cells1. A convenient alternative is the ex vivo expansion and differentiation of hematopoietic stem and progenitor cells in the presence of specific growth factors. A number of cytokines including stem cell factor (SCF; c-kit ligand) and interleukin (IL)-3 and IL-11 have been employed in culture systems to produce MKs. Thrombopoietin (TPO) is the most effective growth and differentiation factor for megakaryocytic cultures and is effective alone or with other cytokines, such as SCF and IL-32. TPO can act on stem cell populations to result in both the proliferation and maturation of MKs2.
MK produce platelets from cytoplasmic protrusions called proplatelets and, in vivo, approximately 1 x 1011 platelets are formed daily to sustain platelet counts of 150 - 400 x 109/L. Platelet production in vitro is up to 1000-fold lower than the in vivo estimates3, and this has given rise to numerous culture conditions using CD34+ hematopoietic progenitor cells to improve MK and platelet production in vitro. The initial source of CD34+ cells used for MK differentiation was human peripheral blood4. Other cell sources include bone marrow5,6, embryonic stem cells/induced pluripotent stem cells (ESC/iPSC)7, and umbilical cord blood (UCB)8,9,10. Human bone marrow CD34+ 11 and mouse lineage negative bone marrow cells5 produce MK and platelets in vitro; nevertheless, the lack of availability of human bone marrow limits its use as a source of CD34+ cells. In contrast, ESC and iPSC represent an unlimited source of cells for in vitro platelet production. Platelet production from these cells requires feeder cells such as murine OP9 cells and longer culture periods. Platelets derived in feeder-free conditions appear to be less functional12. iPSC-derived platelets are likely to be of use in clinical settings since they can be expanded to a large scale. This process requires lentiviral-mediated transduction of transcription factors and long-term cell culture13.
UCB is an accessible source of CD34+ cells that can be readily used in research settings. TPO alone can promote differentiation of cord blood-derived CD34+ cells and this gives rise to highly pure, mature MKs without the need for serum supplementation or co-culture with feeder cells. Other cytokines such as SCF may decrease differentiation from UCB CD34+ cells, while Flt-3 ligand and IL-11 promote the production of immature megakaryocytes14. This protocol describes the production of highly pure MK cultures from cord blood CD34+ cells in serum-free conditions.

<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

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

This protocol allows the preparation of highly pure MK cultures from cord blood-derived CD34+ cells. The percentage of CD34+ cells in cord blood is approximately 1.3%15 (Figure 1A) and the total number of mononuclear cells (step 1.8) ranges from 90 - 300 x 106 per UCB unit. The purity of CD34+/CD45+ cells after isolation ranges from 90 to 99%&#160;(Figure 1B). MK (defined a...

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 (Video) | JoVE

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

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

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

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Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells (Video) | JoVE

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 (Video) | JoVE

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 ...

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

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

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus (Video) | JoVE

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood (Video) | JoVE

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

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

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood (Video) | JoVE

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÂ¯ve CD4+ T Cells Using a TGF-ÃŽÂ²-containing Protocol (Video) | JoVE

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

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue (Video) | JoVE

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

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells (Video) | JoVE

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 ...