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>Zeigler, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+megakaryocytopoietic+and+thrombopoietic+activity+of+c-mpl+ligand+%28TPO%29+on+purified+murine+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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>
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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>
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<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>
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</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+ cell proliferation. However, this expansion results in reduced MK purity in the culture14. The method presented here, using serum-free media and rhTPO alone, does not allow significant expansion of the progenitor cells, but permits unilineage megakaryocytic proliferation and differentiation and consistent production of MK (90 - 99% CD41+CD42a+ double positive cells) without contamination from other lineages. The culture period for MK formation is 10 to 12 days without the need for supporting stromal or feeder cells. This compares favorably with other methods that require larger culture periods (over 20 days)17,18. The platelet yield from the present protocol is 19 - 42 platelets per MK or up to 420 platelets per input CD34+ cell. Most protocols result in lower platelet yields18,19.
Although yield is high relative to other methods, large sources of CD34 cells are needed to produce sufficient numbers of platelets for therapeutic use. Cord blood MK mature at a lower rate, are generally smaller (of lower ploidy classes), and have reduced platelet production capacity20. Nevertheless, fresh UCB is generally a more accessible resource and this represents a significant advantage for researchers. Other methods that can produce higher yields of MK and platelets with therapeutic aims have also been described13,17.
There are some sources of variability to consider: A) Quality of the cord blood unit. Only cord blood collected within 24 h should be used as a source of CD34+ cells. Cord blood units containing clots should also be discarded. B) Percentage of CD34+ cells in the mononuclear cell fraction (step 1.8): using mononuclear cell fractions with less than 0.3% CD34+ cells may result in low yields of relatively low purity CD34+ cells. These cells are not recommended for MK differentiation. It is essential to let the liquid drain by gravity force only (e.g., steps 1.13, 1.14, 1.16). In case of column blockage, it is recommended to remove the column from the magnet, push the cells gently with the syringe plunger into a new tube, and reload on a new equilibrated column.
A number of conditions affect platelet number and function. Thrombocytopenia refers to a marked decline in platelet numbers that can lead to external and internal bleeding. Auto-immune conditions such as immune thrombocytopenia (ITP) and drug-induced thrombocytopenia (DITP) are well known causes of thrombocytopenia21,22. Other immune diseases such as systemic lupus erythematosus and rheumatoid arthritis can also have detrimental effects on platelets. Non-immune causes of thrombocytopenia include cancer treatment, severe trauma, infections, bone marrow failure, and surgery. Due to the high utilization of platelets by patients undergoing chemotherapy or receiving stem cell transplants, platelet transfusion has steadily increased over the past decades. MK and platelet research will undoubtedly assist in the development of large scale platelet production for clinical applications. Availability of in vitro produced functional platelets would prevent platelet shortages and allow platelet transfusions into refractory patients. MK differentiation and platelet production in vitro are critical tools for the study and understanding of both pathological conditions and the physiological mechanisms that lead to platelet formation.

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

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 as CD41+ cells) are observed early in serum-free CD34+ cell cultures in the presence of rhTPO. On day 7, the percentage of mature MK (CD41+ and CD42a+ cells) is usually 30 - 40% (Figure 1C). The highest levels of CD41+ and CD42a+ double positive cells (90 - 99%) are observed between days 10 and 12 of differentiation (Figure 1C). The variability observed depends mainly on the cord blood source and on the purity of the CD34+/CD45+ cells isolated in step 1.17. The yield of mature MK on day 10 ranges from 5 - 10 per input CD34+ cell. Mature MK (CD41+/CD42b+) observed under fluorescent microscopy are shown in Figure 2A. Cultured MK appear as large, usually multinucleated cells (Figure 2A, arrowheads). MK&#39;s granular content was determined by vWf (green) and CD62p (p-selectin, red) staining (Figure 2B). The ploidy distribution observed in cultured MKs is shown in Figure 2C.
Proplatelets are long, beaded fibers or filaments that extend from the MK body. Proplatelets can be several hundred micrometers long16 and contain branches and distended regions. A characteristic proplatelet-bearing MK is illustrated in Figure 2D. The percentage of proplatelet-bearing MK (pbMK) was 1.3 &#177; 0.17%.
Platelets can be analyzed and counted as described in the protocol. As shown in Figure 3B, most platelets in the cell culture supernatant fall within the analytical gate of peripheral blood platelets and are positive for the platelet marker CD41 (Figure 3B). The platelet yield from this method ranges from 19 to 42 platelets per MK. Platelets produced in culture can be activated by platelet agonists such as ADP as determined by increased binding of the activation-specific PAC1 monoclonal antibody (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56420/56420fig1.jpg" /> 
Figure 1: Flow cytometry plots of CD34+ cell isolation and MK differentiation in culture. (A) Mononuclear cells (gated as shown in the left panel) purified from human cord blood (step 1.8) were stained with anti-CD34-PE antibody to determine the percentage of the CD34+ cells in the sample (1.3%, right panel). (B) After separation, the positive fraction (step 1.17) was stained with anti-CD34-PE and anti-CD45 PerCP antibodies. The enriched CD34+ population is indicated in the figure (98.1%, right panel, upper right quadrant). (C) Phenotypic analysis of MK differentiated in vitro from CD34+ cells for 7 days or 11 days were stained&#160;with anti-CD41 and anti-CD42a antibodies. Mature MK are positive for both CD41+ and CD42a+ (upper right quadrant). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56420/56420fig2.jpg" /> 
Figure 2: MK staining, ploidy and proplatelet formation in vitro. (A) Fluorescent images of day 11 MK stained with anti-CD41-PE and CD42b-APC antibodies. Nuclei were stained with DAPI. Yellow arrowhead indicates multi-nuclear MKs. Scale bar, 30 &#181;m. (B) Fluorescent images of day 14 MK stained with anti vWf (green), anti CD62p (red) and anti-CD42b-APC (magenta) antibodies. Nuclei were stained with DAPI. Scale bar, 15 &#181;m (C) Representative gating strategy showing the ploidy distribution of CD41+ events. The graph (lower panel) shows the observed distribution of ploidy classes (n = 4), error bars, SD. (D) The characteristic morphology of proplatelet-bearing MK is shown. The MK body is indicated by the arrow. The long cytoplasmic processes extending from the MK (proplatelets) are indicated by arrowheads. It may be unclear in some areas/fields of view whether one or two MK are producing these proplatelet extensions. This should be counted as one proplatelet-bearing MK. Images taken with an inverted microscope, 10X objective. Scale Bar, 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56420/56420fig3.jpg" /> 
Figure 3: Abundance of platelets produced in vitro and platelet activation. (A) Human platelets from platelet-rich plasma were used to set the analytical gate using log scale for forward and side scatter (left panel). The right panel shows cells from MK cultures. Platelets produced in vitro are observed in the analytical gate defined for human platelets. Cells and counting beads are indicated in the figure. Plt, platelet (B) Platelets produced in vitro were stained with anti-CD41 antibody. Human platelets from platelet-rich plasma were used to set the analytical gate using log scale for forward and side scatter and to compare the profile within the CD41-FITC gate. C-FITC, FITC isotype control (C) Platelet activation following treatment by ADP to a final concentration of 20 &#181;M. Human platelets from platelet-rich plasma were used as control (upper panels). Platelets produced in culture are shown in the lower panels. Binding of PAC1 antibody indicates platelet activation. C-FITC, FITC isotype control. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56420/56420fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.6K Views.  University of New South Wales. Een zeer zuivere bevolking van megakaryocytes kan worden verkregen van koord bloed afkomstige CD34+ cellen. Een methode voor CD34+ celdifferentiatie isolatie en Megakaryocyt is hier beschreven.

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

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve 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.

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

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 superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

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

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.

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

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

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.

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

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 presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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 ...

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

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

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

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Developmental Biology

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

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

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

Biology

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

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

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.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

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

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

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

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.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Modeling Erythropoiesis Using HSPCs from Cord Blood and Bone Marrow

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

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

Author Spotlight: Advancing Erythropoiesis Research - A Simplified Pipeline for Assessing Hematopoietic Stem Cell Function in Myelodysplastic Syndromes

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.

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 ...

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

Source: Perdomo, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56420">Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells &#8212; isolated from human umbilical cord blood &#8212; differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

Source: Perdomo, J., et al. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (2017)
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Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34⁺ Cells

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