This work has been supported&#160;by ANR (Agence National de la Recherche) Grant ANR- 17-CE14-0001-1.

Control human samples were obtained from volonteer blood donors who gave written informed consent recruited by the blood transfusion center where the research was performed (Etablissement Fran&#231;ais du Sang-Grand Est). All procedures were registered and approved by the French Ministry of Higher Education and Research and registered under the number AC_2015_2371.The donors gave their approval in the CODHECO number AC- 2008 - 562 consent form, in order for the samples to be used for research purposes. Human studies were performed according to Helsinki declaration.
1. Extraction and selection of CD34+ cells (HP) from LRF
<ol>
	<li>Reagent&#39;s preparation (for one LRF)
	<ol>
		<li>Prepare 25 mL of filtered elution buffer: 21.25 mL of Phosphate Buffered Saline (PBS), 2.5 mL of Acid-Citrate-Dextrose (ACD) and 1.25 mL of decomplemented Fetal Bovine Serum (FBS). Filter on 0.22 &#181;m and place at 37 &#176;C.</li>
		<li>Prepare 500 mL of PBS with 2 mM of ethylenediaminetetraacetic-acid (EDTA).</li>
		<li>Dispose 25 mL of density gradient medium (DGM) (1.077 g/mL) in two 50 mL tubes.</li>
	</ol>
	</li>
	<li>LRF back-flushing modalities (Figure 1) 
	NOTE: This step requires a sterile tube welding machine, allowing sterile connection of thermoplastic tubing.
	<ol>
		<li>First, connect the LRF to an empty 600 mL transfer bag and the LRF to a tubing set (Figure 1A). Under the biosafety cabinet, inject the total volume of filtered and prepared elution buffer corresponding to the number of LRFs handled (xLRF x 25 mL) into the empty bag. Then, using a 30 mL syringe to gently aspirate the entire contents of the bag through the LRF, backflush and transfer the cells into a new 50 mL tube (Figure 1B).</li>
		<li>To the red blood cells sedimentation, dilute the cell suspension by half with Dextran 2% and mix well to aggregate red blood cells. Wait for 30 min at room temperature (RT).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62499/62499fig01.jpg" /> 
Figure 1: LRF back flushing modalities. (A) Representative scheme of (i) the sterile connection of the transfer bag to the LRF and (ii) the tubing set to the LRF. (B) Representative scheme of the connection of the syringes for cell collection. <a href="https://www.jove.com/files/ftp_upload/62499/62499fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>PBMC collection
	<ol>
		<li>Following the red blood cells sedimentation, remove and transfer the supernatant into a 50 mL tube and fill it with PBS-EDTA 2 mM. Gently overlay the supernatant onto the above-prepared DGM. Let the supernatant flow gently without breaking the surface plane of the density gradient. Centrifuge at 400 x g at RT for 30 min in brake off mode.</li>
		<li>Collect the PBMC layer with a disposable transfer pipette. Transfer the cells from each DGM tube into a new sterile 50 mL tube. Fill each tube with PBS-EDTA 2 mM and wash twice in 50 mL of PBS-EDTA 2 mM at 200 x g for 10 min at RT in brake on mode.</li>
		<li>Pipette off and pool the cell pellet with 50 mL PBS-EDTA 2 mM. 
		NOTE: There is a possibility to stop the procedure by maintaining the collected cells under agitation at 4 &#176;C during the night. Then, filter the suspension with a 40 &#181;m cell strainer to remove the aggregates formed.</li>
	</ol>
	</li>
	<li>CD34+ cells selection
	<ol>
		<li>Determine the cell number and centrifuge at 400 x g at room temperature for 10 min with the break on.</li>
		<li>Aspirate the supernatant completely and resuspend in the appropriate volume of PBS-EDTA 2 mM detailed by the manufacturer of the CD34 selection kit (300 &#181;L of PBS-EDTA for 108 cells). Add the FcR blocking Reagent and the CD34 Microbeads in the appropriate concentration (50 &#181;L for 108 cells).</li>
		<li>After 30 min at 4 &#176;C, wash the cell suspension and resuspend in the appropriate volume of PBS-EDTA 2 mM detailed by the manufacturer of the CD34 selection kit (500 &#181;L per 108 cells). 
		&#8203;NOTE: The selection is made on sorting columns for the passage of maximum 2 x 109 cells per column.</li>
		<li>Pass the sample over the wet column of the magnet. Wash twice with 3 mL of PBS-EDTA 2 mM and elute the cells with 5 mL of PBS-EDTA 2 mM. A second run on a new column following the same procedure is necessary to improve the purity of the sample. 
		NOTE: An expected number of 6.1 x 105 cells/LRF (Figure 2A). For higher LRF numbers, scale up reagents and methods accordingly.</li>
	</ol>
	</li>
	<li>Evaluation of CD34+ cell purity
	<ol>
		<li>Add to an aliquot of 100 &#181;L of suspension obtained after the CD34+ selection, 2 &#181;L of human CD34-PE antibody or 2 &#181;L of IgG - PE (control). Mix well and incubate for 15 min at 4 &#176;C.</li>
		<li>Wash cells by adding 2 mL of PBS and centrifuge at 400 x g for 5 min. Aspirate supernatant completely and resuspend in 200 &#181;L of PBS.</li>
		<li>Analyze the purity by flow cytometry as shown in Figure 2A and in the Discussion. 
		&#8203;NOTE: A purity of CD34+ cells above 90% is expected (Figure 2Bii).</li>
		<li>Use the CD34+ cells directly or freeze for further use.</li>
	</ol>
	</li>
	<li>CD34+ cells freezing 
	NOTE: CD34+ cells freezing is done at a density of 106 cells per mL.
	<ol>
		<li>Following the CD34+ cell number determination, prepare the following cryopreservation media: (1) 60% Stemspan + 40% FBS, (2) 40% Stemspan + 40% FBS + 20% Dimethyl Sulfoxide (DMSO) and allow cooling at 4 &#176;C.</li>
		<li>Centrifuge the CD34+ cells at 400 x g at room temperature for 5 min and resuspend the pellet in cold solution 1 and then immediately add to the cold solution 2 (v/v).</li>
		<li>Place the cryotubes immediately into a -80 &#176;C freezer for 24 h and then transfer cryotubes into the liquid nitrogen tank.</li>
	</ol>
	</li>
</ol>
2. Culture and differentiation of CD34+ cells to produce mature proplatelet-bearing megakaryocytes
NOTE: Cell culture protocol (Figure 3A), representative scheme of the cell culture procedure are detailed in this section.
<ol>
	<li>CD34+ cells thawing (if required)
	<ol>
		<li>Prepare the thawing solution: 13 mL of PBS-20% FBS and place at 37 &#176;C for 15 min. Quickly transfer the cryotubes to a 37 &#176;C water bath until only one small ice crystal. Under the biological culture cabinet, pipette the whole content, and slowly transfer in 13 mL of prewarmed thawing solution.</li>
		<li>Determine the cell number and cell viability.</li>
	</ol>
	</li>
	<li>Culture protocol, step 1: from day 0 to day 7 
	NOTE: Usually cultures are made in 24-well plates with 1 mL of medium per well at a density of 40,000 viable cells/mL, corresponding to 20,000 viable cells/cm2. It is crucial to respect this density if any scale up is planned.
	<ol>
		<li>Growth medium preparation : In serum-free hematopoietic cell expansion media (previously heated to 37 &#176;C) add Penicillin-Streptomycin-Glutamine (PSG) 1x, human Low-Density Lipoprotein (hLDL) at 20 &#181;g/mL, cytokine cocktail of megakaryocyte expansion 1x and Stemregenin 1 (SR1) at 1 &#181;M.</li>
		<li>Cell seeding : Centrifuge the thawed CD34+ cells at 400 x g at room temperature for 5 min. Thoroughly remove the supernatant and resuspend the cell pellet in 1 mL of culture media and performa cell numeration and viability to determine the appropriate volume to seed the cells.</li>
		<li>Centrifuge the cells 400 x g at room temperature for 5 min and resuspend the pellet in the appropriate volume of the warm growth medium. Incubate the cells at 37 &#176;C with 5% CO2 for 7 days.</li>
	</ol>
	</li>
	<li>Culture protocol, step 2: from day 7 to day 13 (Figure 3B, representative images at day 13) 
	NOTE: Usually cultures are made in 24-well plates with 1 mL of medium per well at a density of 50,000 viable cells/mL, corresponding to 25,000 viable cells/cm&#178;. It is crucial to respect this density if any scale up is planned.
	<ol>
		<li>Maturation medium preparation: In serum-free hematopoietic cell expansion media (previously heated to 37 &#176;C) add PSG 1x, hLDL at 20 &#181;g/mL, TPO at 50 ng/mL, and SR1 at 1 &#181;M.</li>
		<li>Examine the cells under the microscope. At day 7, cells display a round and homogeneous appearance by filling the wells or the flasks without being too confluent.</li>
		<li>Under a biosafety cabinet, gently transfer the cells in a 15 mL tube. Wash wells with PBS. Then, determine the number of cells and their viability to calculate the appropriate volume to seed the cells.</li>
		<li>Centrifuge the cells at 400 x g at room temperature for 5 min. Remove the supernatant and resuspend the cells in the appropriate volume of warm media calculated in the previous step. Incubate the cells at 37 &#176;C, 5% CO2 for 6 days.</li>
	</ol>
	</li>
	<li>Cultured platelet release at day 13
	<ol>
		<li>Add 0.5 &#181;M of prostaglandine I2 (PGI2) and 0.02 U/mL of apyrase to the culture and perform successive pipetting five times with a 1 mL pipette. 
		&#8203;NOTE: Platelets are now released into the medium.</li>
	</ol>
	</li>
</ol>
3. Flow cytometry analysis (MK phenotyping and cultured platelets count)
NOTE: This protocol can be applied to the phenotyping of the cells on the selected culture days. It also allows the determination of the number of the cultured platelet release (Figure 4A,B).
<ol>
	<li>Preparation for MK analysis
	<ol>
		<li>Label four sets of microcentrifuge tubes as follows: Unlabeled cells as control, cells + 5 &#181;L CD41 - Alexa Fluor 488, Cells + 5 &#181;L CD34 - PECy7, Cells + 5 &#181;L CD41 - Alexa Fluor 488 + 5 &#181;L CD34 - PECy7. Use a minimum of 1.105 cells per tube, do not exceed 1.106 cells per tube. Add to 100 &#181;L of cell suspension per cytometry tube and the different antibodies. Incubate in the dark for 30 min at 4 &#176;C.</li>
		<li>Then, add 2 mL of PBS-EDTA 2 mM per tube and centrifuge at 400 x g for 5 min at room temperature. During centrifugation, prepare a solution of PBS-EDTA 2 mM + 7-Aminoactinomycin-D (7AAD) (1/100), allow for 300 &#181;L solution per tube.</li>
		<li>Remove the supernatant and take up the pellet in 300 &#181;L of PBS-EDTA 2 mM with 7AAD. Run samples through the flow cytometer within 30 min. 
		NOTE: Analysis strategy for flow cytometry is shown in the Figure 3C and in the Discussion.</li>
	</ol>
	</li>
	<li>Tube preparation for cultured platelet analysis
	<ol>
		<li>Label four sets of microcentrifuge tubes as follows: Unlabeled cells as control, Cells + 5 &#181;L CD41 - Alexa Fluor 488, Cells + 20 &#181;L CD42a - PE, Cells + 5 &#181;L CD41 - Alexa Fluor 488 + 20 &#181;L CD42a - PE. Following five successive pipetting in culture well, transfer 300 &#181;L of the suspension in tube for cytometry containing a calibrated number of fluorescent beads.</li>
		<li>Add antibodies and incubate in the dark at RT for 30 min.</li>
		<li>Run the samples through the flow cytometer within 30 min and set the acquisition for the passage of 5,000 beads. 
		NOTE: Analysis strategy for flow cytometry is shown in the Figure 4B and in the Discussion.</li>
	</ol>
	</li>
</ol>

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	<li>Deutsch, V. R., Tomer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+development+and+platelet+production.">Megakaryocyte development and platelet production.</a> British Journal of Haematology. 134 (5), 453-466 (2006).</li><li>Lefrancais, E., 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=The+lung+is+a+site+of+platelet+biogenesis+and+a+reservoir+for+haematopoietic+progenitors.">The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.</a> Nature. 544 (7648), 105-109 (2017).</li><li>de Sauvage, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+megakaryocytopoiesis+and+thrombopoiesis+by+the+c-Mpl+ligand.">Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.</a> Nature. 369 (6481), 533-538 (1994).</li><li>Almomani, M. H., Mangla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> StatPearls. , (2020).</li><li>Strassel, C., Hechler, B., Bull, A., Gachet, C., Lanza, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+mice+lacking+the+GPIb-V-IX+complex+question+the+role+of+this+receptor+in+atherosclerosis.">Studies of mice lacking the GPIb-V-IX complex question the role of this receptor in atherosclerosis.</a> Journal of Thrombosis and Haemostasis. 7 (11), 1935-1938 (2009).</li><li>Delalat, B., 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=Isolation+and+ex+vivo+expansion+of+human+umbilical+cord+blood-derived+CD34++stem+cells+and+their+cotransplantation+with+or+without+mesenchymal+stem+cells.">Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells.</a> Hematology. 14 (3), 125-132 (2009).</li><li>Yin, T., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem+cell+niches+in+bone.">The stem cell niches in bone.</a> The Journal of Clinical Investigation. 116 (5), 1195-1201 (2006).</li><li>Salunkhe, V., Papadopoulos, P., Guti&#233;rrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+megakaryocytes+from+human+peripheral+blood+mononuclear+cells.">Culture of megakaryocytes from human peripheral blood mononuclear cells.</a> Bio-protocol. 5 (21), 1639 (2015).</li><li>Peytour, Y., Villacreces, A., Chevaleyre, J., Ivanovic, Z., Praloran, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discarded+leukoreduction+filters:+a+new+source+of+stem+cells+for+research,+cell+engineering+and+therapy.">Discarded leukoreduction filters: a new source of stem cells for research, cell engineering and therapy.</a> Stem Cell Research. 11 (2), 736-742 (2013).</li><li>Lapostolle, V., 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=Repopulating+hematopoietic+stem+cells+from+steady-state+blood+before+and+after+ex+vivo+culture+are+enriched+in+the+CD34(+)CD133(+)CXCR4(low)+fraction.">Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34(+)CD133(+)CXCR4(low) fraction.</a> Haematologica. 103 (10), 1604-1615 (2018).</li><li>Ivanovic, Z., 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=Whole-blood+leuko-depletion+filters+as+a+source+of+CD+34++progenitors+potentially+usable+in+cell+therapy.">Whole-blood leuko-depletion filters as a source of CD 34+ progenitors potentially usable in cell therapy.</a> Transfusion. 46 (1), 118-125 (2006).</li><li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aryl+hydrocarbon+receptor-dependent+enrichment+of+a+megakaryocytic+precursor+with+a+high+potential+to+produce+proplatelets.">Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets.</a> Blood. 127 (18), 2231-2240 (2016).</li><li>Do Sacramento, V., 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=Functional+properties+of+human+platelets+derived+in+vitro+from+CD34(+)+cells.">Functional properties of human platelets derived in vitro from CD34(+) cells.</a> Scientific Reports. 10 (1), 914 (2020).</li><li>Blin, A., 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=Microfluidic+model+of+the+platelet-generating+organ:+beyond+bone+marrow+biomimetics.">Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.</a> Scientific Reports. 6, 21700 (2016).</li><li>Ito, Y., 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=Turbulence+activates+platelet+biogenesis+to+enable+clinical+scale+ex+vivo+production.">Turbulence activates platelet biogenesis to enable clinical scale ex vivo production.</a> Cell. 174 (3), 636-648 (2018).</li><li>Pallotta, I., Lovett, M., Kaplan, D. L., Balduini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+system+for+the+in+vitro+study+of+megakaryocytes+and+functional+platelet+production+using+silk-based+vascular+tubes.">Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes.</a> Tissue Engineering. Part C, Methods. 17 (12), 1223-1232 (2011).</li></ol>

The authors have nothing to disclose.

This protocol describes a method for producing MK capable of emitting proplatelets from blood-derived HP and to release platelets from the culture medium. HP are obtained from LRF, a by-product of the blood banks, used to remove contaminating leukocytes from cellular blood products and avoid adverse reactions. Although this method is relatively simple, a few points deserve special attention.
Deposition of the cell suspension on the density gradient medium (step 1.3.1) has to be performed gentl...

Blood platelets come from specialized large polyploid cells, the megakaryocytes (MK), that originate from a constant and fine-tuned production process known as megakaryopoiesis (MKP). At the apex of this process are hematopoietic stem cells which, in contact with the bone marrow environment (cytokines, transcription factors, hematopoietic niche), will be able to proliferate and differentiate into hematopoietic progenitors (HP) able to commit toward the megakaryocytic pathway, giving rise to immature MKs1. Under the influence of various cytokines, and in particular thrombopoietin (TPO), which is the major cytokine of MKP; the MK will then undergo two major stages of maturation: endomitosis and the development of demarcation membranes (DMS). This fully mature MK then appears close to a sinusoid vessel in which it can emit cytoplasmic extensions, the proplatelets, which will be released under the blood flow and subsequently remodeled into functional platelets2. The cloning of TPO in 19943 provided a boost in the study of MKP by accelerating the development of in vitro culture techniques allowing HP differentiation and MK maturation.
There are many pathologies affecting blood platelets, both in terms of platelet number (increase or decrease) and function4,5. Being able to recapitulate MKP in vitro from human HP could improve understanding of the molecular and cellular mechanisms underlying this process and ultimately the therapeutic management of patients.
Various sources of human HP are suitable: cord blood, bone marrow, and peripheral blood6,7,8. Harvesting HP from peripheral blood raises less logistical and ethical problems than their recovery from cord blood or the bone marrow. HP can be recovered from leukapheresis or buffy coat, but these sources are expensive and not always available in blood centers. Other protocols, less expensive and easier to perform, allow direct recovery of human peripheral blood mononuclear cells (PBMCs) without the need for prior CD34 driven isolation4,8. However, the purity of megakaryocytes is not satisfactory with this method and a selection of CD34+ cells from PBMC is recommended for optimal differentiation into MK. This led us to implement a HP purification from leukoreduction filters (LRF), routinely used in blood banks to remove white blood cells and thus avoid adverse immunological reactions9. Indeed, since 1998, platelet concentrates have been automatically leukodepleted in France. At the end of this process, LRF are discarded and all the cells retained in the LRF are destroyed. Cells in LRFs are, therefore, readily available at no additional cost. LRFs have a cellular content close to that obtained by leukapheresis or in buffy coats, notably in their composition of CD34+ HP making them a remarkably attractive source10. LRF as a human HP source has already been demonstrated to provide cells with intact functional capacities11. This source has the advantage of being abundant and affordable for laboratory research. In this context, this article describes successively: i) the extraction and selection of CD34+ HP from LRFs; ii) a two-phase optimized culture, which recapitulates the commitment of HP into the megakaryocytic pathway and the maturation of MK capable of emitting proplatelets; iii) a method for efficiently releasing platelets from these MK; and iv) a procedure for phenotyping MK and cultured platelets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >7-AAD</td>
			<td >Biolegend</td>
			<td >558819</td>
			<td ></td>
		</tr>
		<tr >
			<td >ACD</td>
			<td>EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PE&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-081-002</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD34-PECy7</td>
			<td >eBioscience</td>
			<td >25-0349-42</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD41-Alexa Fluor 488</td>
			<td >Biolegend</td>
			<td >303724</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-CD42a-PE</td>
			<td >BD Bioscience</td>
			<td >559919</td>
			<td></td>
		</tr>
		<tr >
			<td >Apyrase</td>
			<td >EFS-Alsace</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >BD Trucount Tubes</td>
			<td >BD Bioscience</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr >
			<td >CD34 MicroBead Kit UltraPure, human&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-100-453</td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td>Heraeus</td>
			<td>Megafuge 1.OR</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Compteur ADAM&#160;</td>
			<td>DiagitalBio</td>
			<td >NA</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Cryotubes</td>
			<td>Dutscher</td>
			<td>55002</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Dextran from leuconostoc spp&#160;</td>
			<td>Sigma</td>
			<td>31392-50g</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >DMSO Hybri-max&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr >
			<td >EDTA 0.5 M&#160;</td>
			<td>Gibco</td>
			<td>15575-039</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf 1,5 mL&#160;</td>
			<td>Dutscher</td>
			<td>616201</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Filtration unit Steriflip PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow Cytometer</td>
			<td >BD Bioscience</td>
			<td >Fortessa</td>
			<td></td>
		</tr>
		<tr >
			<td >Human LDL</td>
			<td>Stemcell technologies</td>
			<td>#02698</td>
			<td></td>
		</tr>
		<tr >
			<td >ILOMEDINE 0,1 mg/1 mL</td>
			<td >Bayer</td>
			<td>MA038EX</td>
			<td></td>
		</tr>
		<tr >
			<td >Inserts</td>
			<td>Fenwal</td>
			<td>R4R1401</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Laminar flow hood&#160;</td>
			<td>Holten</td>
			<td >NA</td>
			<td>Archived product</td>
		</tr>
		<tr >
			<td >LS Columms&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td>Stemcell</td>
			<td>7861</td>
			<td></td>
		</tr>
		<tr >
			<td >Pen Strep Glutamine (100x)</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS (-)</td>
			<td>Life Technologies</td>
			<td>14190-169&#160;</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >PGi2</td>
			<td >Sigma</td>
			<td >P6188</td>
			<td></td>
		</tr>
		<tr >
			<td >Poches de transferts 600ml&#160;</td>
			<td>Macopharma</td>
			<td>VSE4001XA</td>
			<td></td>
		</tr>
		<tr >
			<td >Pre-Separation Filters (30&#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >StemRegenin 1 (SR1)</td>
			<td>Stemcell technologies</td>
			<td>#72344</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan Expansion Supplement (100x)</td>
			<td>Stemcell technologies</td>
			<td>#02696</td>
			<td></td>
		</tr>
		<tr >
			<td >StemSpan-SFEM&#160;</td>
			<td>Stemcell technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr >
			<td >Stericup Durapore 0,22&#181;m PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SCGVU05RE</td>
			<td></td>
		</tr>
		<tr >
			<td >SVF Hyclone&#160;</td>
			<td>Thermos scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringues 30 mL&#160;</td>
			<td>Terumo</td>
			<td>SS*30ESE1</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Syringe filters Millex 0,22&#181;M PVDF</td>
			<td>Merck Millipore Ltd</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr >
			<td >TPO</td>
			<td>Stemcell technologies</td>
			<td>#02822</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubes 50 mL</td>
			<td>Sarstedt</td>
			<td>62.548.004 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubes 15 mL&#160;</td>
			<td>Sarstedt</td>
			<td>62.554.001 PP</td>
			<td>Or equivalent material</td>
		</tr>
		<tr >
			<td >Tubulures</td>
			<td>B Braun</td>
			<td>4055137</td>
			<td>Or equivalent material</td>
		</tr>
	</tbody>
</table>

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.

Extraction and selection of CD34+ cells from LRFs 
Here, the method, derived from Peytour et al.9, describes the extraction and selection of CD34+ cells from discarded LRFs available in blood banks after leukocyte removal. Following the backflush procedure, usually 1.03 x 109 &#177;&#160;2.45 x 108 cells/LRF (Mean&#177;SEM; n = 155) are recovered with a viability of 94.88 &#177; 0.10% (Figure 2A i...

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

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 ...

leukodepletion-filters-derived-cd34-cells-as-cell-source-to-study

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JoVE Journal

Biology

3.8K Views.  Université de Strasbourg, INSERM, EFS Grand Est. 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.

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

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

It is now acknowledged that the three-dimensional environment of cells can play an important role in their behavior, maturation and/or differentiation. This protocol describes a three-dimensional cell culture model designed to study the impact of physical containment and mechanical constraints on megakaryocytes.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D ...

Preparation and In Vitro Differentiation of Mesenchymal Stem Cell-Encapsulated Hydrogels for Bone Formation

Differentiating Human Pluripotent Stem Cells into Pancreatic β-Cells

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

Author Spotlight: Advancements and Challenges in &#946;-Cells Differentiation from Pluripotent Stem Cells

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.