Name: Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells (Video) | JoVE Journal
Uploaded: 2017-12-27T14:00:00.000+00:00
Duration: 9 min 46 s
Description: Learn megakaryocyte differentiation and platelet formation from human cord blood-derived CD34+ cells. This method enables researchers to study proplatelet production and analyze platelet activation in vitro.

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5 minutes

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 sterile instruments (scissors, tweezers) for this procedure.
1. Cord Blood Cell Preparation and Isolation of CD34+ Cells
<ol>
	<li>Prepare sterile separation buffer (SB) with phosphate buffered saline (PBS), at pH 7.2, and containing 0.5% bovine serum albumin and 2 mM EDTA.</li>
	<li>Dispense 10 mL of SB into a 50 mL conical tube (one tube per 10 mL of blood is required). Using a G18 blunt needle mounted on a 10 mL syringe, draw 10 mL of blood from the bag and dispense into the 50 mL tubes containing 10 mL of SB.</li>
	<li>Add 15 mL of lymphocyte separation media (see Table of Materials) to the bottom of the tube containing the diluted blood, creating two layers. 
	NOTE: Dispense media slowly at the bottom of the tube to avoid mixing the different layers.</li>
	<li>Centrifuge tubes at 1,200 &#215; g for 30 min at room temperature (RT) with no break and no acceleration.</li>
	<li>Transfer the layer near the center of the tube containing mononuclear cells (approximately 5 - 10 mL from each tube) into a new 50 mL tube using a Pasteur pipette. Add SB to each tube to a total volume of 50 mL.</li>
	<li>Centrifuge at 400 &#215; g for 10 min at RT. Discard the supernatant.</li>
	<li>Resuspend cell pellets carefully with a Pasteur pipette in 5 - 10 mL of SB. Combine the suspended cells and bring volume to 50 mL with SB.</li>
	<li>Count cells using Trypan blue staining and a hemocytometer or automated cell counter (see Table of Materials). To determine the percentage of CD34+ cells in the sample, resuspend 2.5 &#215; 105 cells in 100 &#181;L of SB and stain by adding 10 &#181;L of anti-CD34-PE antibody for 15 - 30 min at 4 &#176;C. Analyze by flow cytometry (Figure 1A).</li>
	<li>Centrifuge tubes at 400 &#215; g for 10 min at 4 &#176;C. Discard supernatant. 
	NOTE: If not required immediately, mononuclear cells can be frozen at this stage.</li>
	<li>Resuspend cells in 300 &#181;L of SB per 108 cells. Add 100 &#181;L of FcR human IgG (blocking reagent, see Table of Materials) and 100 &#181;L of CD34 magnetic beads per 108 cells. Mix gently and incubate at 4 &#176;C for 30 - 40 min. 
	NOTE: A single cell suspension is required (if necessary, pass the cells though a 30 &#181;m filter before adding reagents). Use the reagent volumes described here for 108 cells or less. For more than 108 cells, scale up the reagents (SB, blocking solution, and CD34 magnetic beads) accordingly.</li>
	<li>Prepare the LS separation column during the incubation period by placing the LS column in a magnetic holder and washing with 3 mL of SB. Discard the effluent. 
	NOTE: LS separation columns can be loaded with up to 2 &#215; 108 total cells. Smaller and larger columns are available.</li>
	<li>Add 5 - 10 mL of SB to the cell mixture and centrifuge at 400 &#215; g for 10 min. Discard the supernatant.</li>
	<li>Resuspend cells in 1.5 mL of SB and load the cell suspension (1.5 mL) onto the LS column. Collect the flow through containing the unlabeled cells in a 15 mL collection tube (negative fraction #1). Let the liquid drain and wash the column with 1.5 mL of SB.</li>
	<li>Load the collected flow through (3 mL) back into the column and collect the flow through in the same 15 mL collection tube (negative fraction #1). Wash the LS column 3 times with 3 mL of SB. 
	NOTE: If required, cells in the negative fraction (12 mL) can be stained with anti-CD34 antibody to ascertain capture of CD34+ cells by the LS column.</li>
	<li>Remove column from the magnetic separator and flush cells slowly with a syringe plunger into a new 15 mL tube with 2 mL of SB.</li>
	<li>Place column back on magnetic separator and load the 2 mL of cells back into the column. Collect the flow through and wash with 2 mL SB. This is the negative fraction #2 (4 mL).</li>
	<li>Remove LS column from magnetic separator and add 2 mL of SB. Flush cells steadily and firmly with a syringe plunger to collect the CD34+ cell fraction. Count the cells using trypan blue staining and a hemocytometer or automated cell counter.</li>
	<li>Centrifuge tube with the positive fraction at 400 &#215; g for 15 min at 4 &#176;C. Discard the supernatant. Resuspend cells in serum-free media for CD34+ cells (SFM, see Table of Materials). 
	NOTE: If not required immediately, cells can be frozen in liquid nitrogen at this stage.</li>
</ol>
2. Purity Check of Isolated CD34+ Cells
<ol>
	<li>Stain 2 x 104 cells from the positive fraction with 10 &#181;L anti-CD34-PE antibody and 20 &#181;L anti-CD45-PerCP antibody for 15 min at 4 &#176;C (use a separate tube for isotype controls) (Figure 1B).</li>
	<li>Add 1 mL of SB to wash. Centrifuge at 400 &#215; g for 10 min. Resuspend pellet in 200 &#181;L of SB.</li>
	<li>Perform flow cytometric analysis and gate the live cell population to exclude debris (Figure 1B). Set the gate for PE and PerCP positive populations using the PE and PerCP isotype controls (Figure 1B) and determine the percentage of CD34+/CD45+ cells.</li>
</ol>
3. Megakaryocyte Differentiation
<ol>
	<li>Seed 5 &#215; 105 cells/mL CD34+ cells in 2 mL of SFM supplemented with 50 ng/mL recombinant human thrombopoietin (rhTPO) per well in a 12-well plate. Incubate cells at 37 &#176;C, 5% CO2 in a humidified atmosphere. If cells are confluent before they are required for analysis, harvest the cells and split into multiple wells with fresh media and rhTPO. 
	NOTE: Two or three wells should be prepared specifically to monitor differentiation at different time points (e.g., days 7, 9, and 10).</li>
	<li>Harvest cells from the wells set aside to monitor differentiation without disturbing the cells in the other wells.</li>
	<li>Stain cells with 20 &#181;L anti-GPIIb/CD41-FITC antibody and 10 &#181;L anti-GPIX/CD42a-Alexa Fluor 647 antibody in a final volume of 100 &#181;L. Set up a control tube using the respective isotype control antibodies. Incubate for 15 - 30 min at 4 &#176;C.</li>
	<li>Add 1 mL of SB to wash. Centrifuge at 400 &#215; g for 10 min. Discard the supernatant and resuspend the pellet in 200 - 300 &#181;L of SB. Analyze by flow cytometry.</li>
	<li>For each fluorophore, analyze the isotype controls to set the gating for FITC and Alexa Flour 647 positive populations. Analyze the stained cell samples to determine the percent of CD41+/CD42a+ double positive cells, which represent mature MK (Figure 1C).</li>
	<li>For microscopic visualization of cell surface markers stain cells as described in 3.3.
	<ol>
		<li>Add 1 mL of SB to wash. Centrifuge at 400 &#215; g for 10 min. Resuspend the pellet in 100 &#181;L of SB and spin onto a glass slide at 1,000 x g for 5 min. Fix cells on the slide by dipping in methanol for 30 s. Air dry, add 20 &#181;L of mounting media containing DAPI (see Table of Materials), cover with a coverslip, and visualize using a fluorescent microscope (Figure 2A).</li>
	</ol>
	</li>
	<li>For visualization of intracellular antigens, resuspend cells in PBS and fix with paraformaldehyde (1% final concentration) for 15 min at room temperature. To permeabilize the cells, add triton-X 100 (0.1%) and incubate for 15 min. Wash cells with 2 mL PBS/0.1% triton-X 100. Resuspend in 100 &#181;L of PBS/0.1% triton-X 100, add anti vWf and anti CD62p antibodies (1:200 dilution) and incubate for 30 min at room temperature.
	<ol>
		<li>Wash with 2 mL PBS/0.1% triton-X 100 and centrifuge at 400 &#215; g for 10 min, resuspend in 100 &#181;L of the same buffer, and add anti-mouse IgG-Alexa 594 and anti-rabbit IgG-Alexa 488 (1:100). Incubate for 30 min at room temperature, wash with 2 mL PBS/0.1% triton-X 100, resuspend in 100 &#181;L of the same buffer, and add 20 &#181;L of anti CD42b-APC. Then spin the cells onto glass slides as described in step 3.6.1 and prepare samples for microscopic visualization as described in step 3.6.1.</li>
	</ol>
	</li>
	<li>For ploidy determination, harvest cells at days 12 or 13 of differentiation. Add 1 mL of SB to wash. Centrifuge at 400 &#215; g for 10 min and stain with 20 &#181;L anti-GPIIb/CD41-FITC antibody in a final volume of 100 &#181;L. Incubate at 4 &#176;C for 30 min.
	<ol>
		<li>Wash once with 1 mL of SB and resuspend pellet in 300 &#181;L of hypotonic citrate buffer (1.25 mM sodium citrate, 2.5 mM sodium chloride, 3.5 mM dextrose) containing 20 &#181;g/ml propidium iodide and 0.05% Triton-X 100. Incubate for 15 min at 4 &#176;C protected from light.</li>
		<li>Add RNase to a final concentration of 20 &#181;g/mL and incubate for 30 min at 4 &#176;C protected from light. Determine the intensity of propidium iodide by flow cytometry by collecting 30,000 to 50,000 events of the CD41-FITC+ population (Figure 2C).</li>
	</ol>
	</li>
</ol>
4. Proplatelet Counting, Platelet Enumeration, and Platelet Activation
<ol>
	<li>Harvest cells (from step 3.1) at days 8 or 9 of differentiation and seed at 1 &#215; 104 cells/well in 48-well plates in 200 &#181;L of fresh SFM supplemented with 50 ng/mL rhTPO. Culture for 5 days at 37 &#176;C, 5% CO2. 
	NOTE: For quantitation purposes, seed wells in triplicate. This low density is required for visualization and counting of proplatelet-bearing MK. Proplatelets usually start appearing after 2 days of culture. The peak is between days 4 and 5.</li>
	<li>Count the number of proplatelet-bearing MK in the whole well on an inverted light microscope using 10X or 20X objectives. 
	NOTE: A heated (37 &#176;C) microscope stage is preferable, since keeping the cells at room temperature for extended periods causes shrinkage of the proplatelet extensions. Proplatelets are observed as long extensions from the MK body. Each MK may have several proplatelet protrusions. As proplatelets develop, the body of the MK decreases in size.</li>
	<li>Harvest cells and centrifuge at 400 x g for 10 min at room temperature. Stain cells with 20 &#181;L anti-human CD41-FITC antibody, as described in steps 3.3 and 3.4. Calculate the percentage of proplatelet-bearing MK (pbMK): pbMK (%) = [(Proplatelet-bearing MKs/ well) / (Total CD41+ cells/ well)] x 100</li>
	<li>To count platelets released into the culture medium, gently mix the cells with a Pasteur pipette and collect 100 &#181;L at days 14 or 15 of culture.
	<ol>
		<li>Stain with 20 &#181;L anti-human CD41-FITC antibody for 20 - 30 min at 4 &#176;C. Set up a control tube using the respective isotype control antibody.</li>
		<li>Add 150 &#181;L of SB and 50 &#181;L of counting beads.</li>
		<li>For flow cytometric analysis, set the FSC and SSC to log scale. Use normal human blood platelets from platelet-rich plasma stained with CD41-FITC as described in section 4.4.1 to set the gating for platelets (Figure 3A).</li>
		<li>Analyze the stained platelets with counting beads by flow cytometry. Collect 1,000 events of counting beads using the FSC versus SSC scatter plot (Figure 3A). Calculate the number of platelets based on CD41-FITC positive events (Figure 3B) using the formula: 
		Platelets per &#181;L=[(number of CD41-FITC positive events)/1,000 beads)] x [(number of beads in 50 &#181;L)/sample volume)]</li>
	</ol>
	</li>
	<li>To analyze platelet activation, gently mix the cells with a Pasteur pipette and collect 100 &#181;L at days 14 or 15 of culture. Add 1 mL of Tyrode&#39;s buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0.2 mM Na2HPO4, 12 mM NaHCO3, 5.5 mM D-glucose, pH 6.5) and centrifuge at 200 x g for 5 min to pellet cells.
	<ol>
		<li>Collect the supernatant and centrifuge at 800 x g for 10 min to pellet platelet-sized particles.</li>
		<li>Discard supernatant and resuspend in 100 &#181;L of Tyrode&#39;s buffer. Add 20 &#181;L of PAC1-FITC antibody and adenosine diphosphate (ADP) to a final concentration of 20 &#181;M. Incubate at room temperature for 20 min. Analyze by flow cytometry to determine the percentage of FITC positive events. Use fresh human platelets treated in the same manner as a positive control (Figure 3C).</li>
	</ol>
	</li>
</ol>

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<li>Choi, E., Nichol, J. L., Hokom, M. M., Hornkohl, A. C., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelets+generated+in+vitro+from+proplatelet-displaying+human+megakaryocytes+are+functional%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional.</a> Blood. 85 (2), 402-413 (1995).</li>
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<li>Shim, M. H., Hoover, A., Blake, N., Drachman, J. G., Reems, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profile+of+primary+human+CD34%2BCD38lo+cells+differentiating+along+the+megakaryocyte+lineage%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">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/pubmed?term=((Scalable+generation+of+universal+platelets+from+human+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Stem+cell+reports%22%5BJournal%5D)">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/pubmed?term=((In+vitro+and+in+vivo+megakaryocyte+differentiation+of+fresh+and+ex-vivo+expanded+cord+blood+cells%3A+rapid+and+transient+megakaryocyte+reconstitution%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">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/pubmed?term=((Immune+thrombocytopenia%3A+antiplatelet+autoantibodies+inhibit+proplatelet+formation+by+megakaryocytes+and+impair+platelet+production+in+vitro%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">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/pubmed?term=((Impaired+proplatelet+formation+in+immune+thrombocytopenia%3A+a+novel+mechanism+contributing+to+decreased+platelet+count%5BTitle%5D)+AND+%22Br.+J.+Haematol%22%5BJournal%5D)">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/pubmed?term=((A+novel+strategy+for+generating+platelet-like+fragments+from+megakaryocytic+cell+lines+and+human+progenitor+cells%5BTitle%5D)+AND+%22Blood+Cells+Mol.+Dis%22%5BJournal%5D)">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/pubmed?term=((Platelets+generated+from+human+embryonic+stem+cells+are+functional+in+vitro+and+in+the+microcirculation+of+living+mice%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">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/pubmed?term=((Large-scale+production+of+megakaryocytes+from+human+pluripotent+stem+cells+by+chemically+defined+forward+programming%5BTitle%5D)+AND+%22Nature+Commun%22%5BJournal%5D)">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/pubmed?term=((Ex+vivo+expansion+of+megakaryocyte+progenitor+cells%3A+cord+blood+versus+mobilized+peripheral+blood%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">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/pubmed?term=((A+unique+population+of+CD34%2B+cells+in+cord+blood%5BTitle%5D)+AND+%22Stem+cells%22%5BJournal%5D)">A unique population of CD34+ cells in cord blood.</a> Stem cells. 13 (2), Dayton, Ohio. 158-166 (1995).</li>
<li>Italiano, J. E., Patel-Hett, S., Hartwig, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanics+of+proplatelet+elaboration%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Mechanics of proplatelet elaboration.</a> J Thromb Haemost. 5, Suppl 1. 18-23 (2007).</li>
<li>Matsunaga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+large-scale+generation+of+human+platelets+from+cord+blood+CD34%2B+cells%5BTitle%5D)+AND+%22Stem+cells%22%5BJournal%5D)">Ex vivo large-scale generation of human platelets from cord blood CD34+ cells.</a> Stem cells. 24 (12), Dayton, Ohio. 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/pubmed?term=((Prolonged+continuous+in+vitro+human+platelet+production+using+three-dimensional+scaffolds%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">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/pubmed?term=((Preferential+ex+vivo+expansion+of+megakaryocytes+from+human+cord+blood+CD34%2B-enriched+cells+in+the+presence+of+thrombopoietin+and+limiting+amounts+of+stem+cell+factor+and+Flt-3+ligand%5BTitle%5D)+AND+%22J.+Hematother.+Stem+Cell+Res%22%5BJournal%5D)">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/pubmed?term=((Cord+blood+megakaryocytes+do+not+complete+maturation%2C+as+indicated+by+impaired+establishment+of+endomitosis+and+low+expression+of+G1%2FS+cyclins+upon+thrombopoietin-induced+differentiation%5BTitle%5D)+AND+%22Br.+J.+Haematol%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/23714310">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/pubmed?term=((Idiopathic+thrombocytopenic+purpura%3A+Current+concepts+in+pathophysiology+and+management%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">Idiopathic thrombocytopenic purpura: Current concepts in pathophysiology and management.</a> Thromb Haemost. 99 (1), 4-13 (2008).</li>
</ol>

The authors have nothing to disclose.

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>&lt;strong&gt;Cell Culture Reagents&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;CD34 Isolation Reagents&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Flow Cytometry and Cell Staining Reagents&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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

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% (Figure 1B). MK (defined as CD4...

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.

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

megakaryocyte-differentiation-platelet-formation-from-human-cord

Research

JoVE Journal

Immunology and Infection

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Isolation of Mouse Megakaryocyte Progenitors

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Modeling erythropoiesis provides a valuable chance to comprehend the biological significance of actors in this process and to evaluate novel therapeutic approaches that may alleviate differentiation defects. Here, we describe a simple and reliable method to efficiently differentiate CD34+ hematopoietic stem and progenitor cells ex vivo.

A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

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

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

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

Learn megakaryocyte differentiation and platelet formation from human cord blood-derived CD34+ cells. This method enables researchers to study proplatelet production and analyze platelet activation in vitro.

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

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