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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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+population+of+CD34++cells+in+cord+blood.">A unique population of CD34+ cells in cord blood.</a> Stem cells. 13 (2), 158-166 (1995).</li><li>Italiano, J. E., Patel-Hett, S., Hartwig, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+proplatelet+elaboration.">Mechanics of proplatelet elaboration.</a> J Thromb Haemost. 5, 18-23 (2007).</li><li>Matsunaga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+large-scale+generation+of+human+platelets+from+cord+blood+CD34++cells.">Ex vivo large-scale generation of human platelets from cord blood CD34+ cells.</a> Stem cells. 24 (12), 2877-2887 (2006).</li><li>Sullenbarger, B., Bahng, J. H., Gruner, R., Kotov, N., Lasky, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+continuous+in+vitro+human+platelet+production+using+three-dimensional+scaffolds.">Prolonged continuous in vitro human platelet production using three-dimensional scaffolds.</a> Exp Hematol. 37 (1), 101-110 (2009).</li><li>Proulx, C., Boyer, L., Hurnanen, D. R., Lemieux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+ex+vivo+expansion+of+megakaryocytes+from+human+cord+blood+CD34+-enriched+cells+in+the+presence+of+thrombopoietin+and+limiting+amounts+of+stem+cell+factor+and+Flt-3+ligand.">Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.</a> J. Hematother. Stem Cell Res. 12 (2), 179-188 (2003).</li><li>Bornstein, R., Garcia-Vela, J., Gilsanz, F., Auray, C., Cales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+megakaryocytes+do+not+complete+maturation,+as+indicated+by+impaired+establishment+of+endomitosis+and+low+expression+of+G1/S+cyclins+upon+thrombopoietin-induced+differentiation.">Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.</a> Br. J. Haematol. 114 (2), 458-465 (2001).</li><li>Chong, B. H., Choi, P. Y. 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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>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-013</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM II</td>
			<td>Stem Cell Technologies</td>
			<td>9605</td>
			<td>Serum-free media for CD34+ cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CD34 Isolation Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 MicroBead kit ultrapure</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-453</td>
			<td>This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Alere Technologies</td>
			<td>1114545</td>
			<td>Lymphocyte separation media (density 1.077 g/mL)</td>
		</tr>
		<tr>
			<td>Sterile separation buffer (SB)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td>This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow Cytometry and Cell Staining Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>340669</td>
			<td>Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>PerCP mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>347464</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>PerCP isotype control</td>
			<td>BD Biosciences</td>
			<td>349044</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>FITC Mouse anti-Human CD41a</td>
			<td>BD Biosciences</td>
			<td>340929</td>
			<td>Final antibody concentration 1:5 dilution.</td>
		</tr>
		<tr>
			<td>APC Mouse anti-Human CD42b</td>
			<td>BD Biosciences</td>
			<td>551061</td>
			<td>This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human CD42a</td>
			<td>AbD Serotec</td>
			<td>MCA1227A647T</td>
			<td>Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse Negative Control</td>
			<td>AbD Serotec</td>
			<td>MCA928A647</td>
			<td>Currently distributed by Bio-Rad. Isotype control antibody</td>
		</tr>
		<tr>
			<td>Anti von Willebrand factor rabbit polyclonal</td>
			<td>Abcam</td>
			<td>AB6994</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>V450 mouse anti-humna CD41a</td>
			<td>BD Biosciences</td>
			<td>58425</td>
			<td>1: 20 dilution</td>
		</tr>
		<tr>
			<td>V450 isotype control</td>
			<td>BD Biosciences</td>
			<td>580373</td>
			<td>1:20 dilution</td>
		</tr>
		<tr>
			<td>PAC1-FITC antibody</td>
			<td>BD Biosciences</td>
			<td>340507</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>Anti CD62p mouse monoclonal</td>
			<td>Abcam</td>
			<td>AB6632</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11020</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Ig Isotype Control cocktail-C</td>
			<td>BD Biosciences</td>
			<td>558659</td>
			<td>Isotype control antibody</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C36950</td>
			<td>Counting beads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Smaller and larger columns are also commercially available</td>
		</tr>
		<tr>
			<td>MidiMACS Separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile</td>
			<td>In Vitro technologies</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J3800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIRB inverted microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge</td>
			<td>ThermoScientific</td>
			<td>Cytospin 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser software</td>
			<td>BD Biosciences</td>
			<td>FACS Diva Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell analysis software</td>
			<td>Tree Star</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope software</td>
			<td>Zeiss</td>
			<td>Zen 2 blue edition</td>
			<td></td>
		</tr>
	</tbody>
</table>

megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells

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

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

Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

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

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

Research

JoVE Journal

Immunology and Infection

19.5K Views.  University of New South Wales. 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.

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

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

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...