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.remotexs.ntu.edu.sg/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>

<ol>
	<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 gently to avoid mixture (red content). If this step is not carried out carefully, the protocol should stop at this point. Similarly, also in step 1.3.1, the brake has to be on the off mode to avoid mixing the fractions. If not, the HP selection must be suspended. As indicated in the protocol, section 1.4, the procedure can be interrupted following the PBMC collection. In this case, cells can be maintained under agitation overnight at 4 &#176;C. Then, use a 40 &#181;m cell strainer to remove the aggregates formed, which can impact on the subsequent CD34 selection. Of note, interrupting the procedure does not affect the yield and purity of CD34+ cells. At the end of the CD34 selection, the purity must be greater than 75% to seed the cells since, in a previous study, differentiation and maturation of MK were poor below this purity12 (Figure 2).
Special attention must be paid to the thawing of the CD34+ cells, which must be carried out quickly to avoid affecting the viability of the cells. In addition, washing steps must be carried out carefully to leave no trace of serum. The cell seeding density must be respected as it has been rigorously chosen for optimal CD34 commitment to the MKP pathway and MK maturation (Figure 3A).
A cell phenotyping protocol is proposed to follow the differentiation and maturation of MK. This protocol is relatively basic, but it is important to have all the control tubes, both unlabeled and single labeling tubes, available for each day of analysis to ensure reliable settings on the cytometer. To ensure that the culture runs smoothly, it is important to collect information of proliferation along the procedure. At D7, the average proliferation is between 2 to 4 fold13. This proliferation varies little between experiments, since each LRF comprises cells from 8 donors. To smooth out the variations further, it is possible to combine cells obtained in parallel from 4 to 8 LRFs.
It is possible to look at the morphology of the cells by light microscopy but the cells should not be observed every day because they are sensitive to temperature variations. When removing the cells from the incubator, ensure to make slow movements to avoid breaking the proplatelets.
Concerning the platelet release, five times successive pipetting are required. Doing less does not ensure optimal platelet release and doing more is detrimental to their functionality12. The most important aspect in this step is to use precise and regular movements, to generate a regular flow required for the platelet release14,15,16. The method of five successive pipetting is, therefore, simple and easy to perform with satisfactory performance results based on the yields described in the literature. The number of released platelets can be determined as mentioned in the section 3.3 by using the strategy of flow cytometry analysis shown in Figure 4B. The quality of the released platelets has been well documented in Do Sacramento et al. in terms of ultrastructure (morphology, size, granule content) and function (hemostasis), demonstrating that these cultured platelets are very similar to the native ones13.
The protocol described here is particularly suitable for small-volume cultures but is not applicable to large-scale culture. It is therefore an optimal method for the study of platelet biogenesis in order to better understand the molecular and cellular mechanisms governing platelet production by adding small molecules, agonists, or antagonists, for example. In addition, and to further explore the mechanisms that regulate MK commitment, MK maturation, and platelet production, it is now possible to genetically manipulate the CD34+ HP using a CRISPR-Cas9 genome editing method.

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). After the CD34 positive selection, an average of 615.54 x 103 &#177; 12.28 cells/LRF is obtained (n = 155) (Figure 2A ii). If the number of cells is less than 300,000, it must be concluded that the procedure has not been carried out correctly and must be stopped. To evaluate the success of the CD34 selection, the purity of CD34+ cells is assessed by flow cytometry (Figure 2B). Routinely, a purity above 90% (91.88 &#177; 0.79%) is expected (Figure 2B). A purity below 75% could mean that there has been a problem in conducting the protocol and in particular the elution of the columns. Below a 75% purity the cells are not conserved for culture experiments.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62499/62499fig02.jpg" /> 
Figure 2: CD34+ cell number/LRF and CD34 purity analysis. (A) (i) An analysis, by cell counter, of the number of cells and their viability obtained following the procedure including both PBMC collection and CD34 selection is performed ((1.03.109 &#177; 2.45.108 cells/LRF (Mean &#177; SEM; n=155) with a viability of 94.88 &#177; 0.10% (n = 155)). (ii) An analysis, by cell counter, is also performed after CD34 selection (615.54 x 103 &#177; 12.28 cells/LRF (n = 155)). (B) CD34 Purity is analyzed by flow cytometry. (i) Cells were stained with a CD34-PE antibody and identified on their FSC/SSC parameters. (ii) Based on the scatter signal and CD34 expression analysis, the purity is determined. A pre-gate of CD34 positivity was used based on the negative control for the CD34 marker. (iii) As can be seen on the bar graph, a purity of CD34+ cells above 90% (91.88 &#177; 0.79% (n = 17)) is expected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62499/62499fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Differenciation and maturation of MK-bearing proplatelets 
The cell culture procedure described is divided in two steps. The first one, from day (D) 0 to D7, is dedicated to HP proliferation and commitment into the megakaryocytic pathway in response to a combination of cytokines and the addition of a chemical compound SR1. The second one, from D7 to D13, is focused on MK maturation and proplatelet extension following the addition of TPO and SR1 (Figure 3A). As quality control of the culture, cell counting, determination of cell viability, and phenotyping of cells, are perfomed at D7 and D10. These stages have been chosen because they are crucial for HP commitment, D7 and MK maturation, D10, respectively (personal data). At D7 and 10, proliferation is routinely of x4.16 &#177; 0.25 (n = 34) and x2.30 &#177; 0.16 (n = 5), respectively, with a cell viability comprised between 86.38 &#177; 0.73% (n = 34); and 84.80 &#177; 2.67% (n = 5) (Figure 3B). Concerning cell phenotyping, as shown in Figure 3C, at D0, more than 90% of the cells are positive for CD34. Then, CD34+ cells become committed toward the megakaryocytic lineage, as witnessed by the apparition of CD41, a specific and early marker of MKP. Indeed, at D7, 50.20 &#177; 2.90% of the cells are positive for both CD34 and CD41 (Figure 3Bii). Then, MK improves their maturation. At D10, a majority of MK are mature, with less than 15.60 &#177; 4.70% of the cells being negative for CD41, 47.90 &#177; 8.90% being CD34+CD41+ and 36.40 &#177; 12.40% being CD34-CD41+ (Figure 3Biii).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62499/62499fig03.jpg" /> 
Figure 3: Differenciation and maturation of MK-bearing proplatelets. (A) Representative scheme of the cell culture procedure. A two-step method is used: a proliferation step from D0 to D7 (SR1 and cocktail of cytokines) and a maturation step from D7 to D13 (SR1 and TPO). At D13, cultured platelets can be released following five successive pipetting. (B) (i) Proliferation rate at D7 (x4.16 &#177; 0.25 (n = 34)) and cell viability (86.38 &#177; 0.73% (n = 34)). (ii) Proliferation rate at D10 (x2.30 &#177; 0.16 (n = 5)) and cell viability (84.80 &#177; 2.67% (n = 5)). (C) Flow cytometry analysis stategy of the phenotypic evolution of MK in culture. At D0, 95.80 &#177; 0.80% of the cells are CD34 positive (n = 3). At D7, 50.20 &#177; 2.90% of the cells are positive for CD34 and CD41. At D10, less than 15.60 &#177; 4.70% of the cells are negative for CD41. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62499/62499fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cultured platelets release, day 13 
Examination of the wells at D13 shows round MK and proplatelet-bearing MK (Figure 4A). An average of 35% of cultured MK extend proplatelets12. Of note, D13 represents the optimal day for proplatelet extension and platelet release. If the required level of MK capable of emitting proplatelets is not reached, something must have gone wrong along the culture process and the results should not be taken into account.
Although the exact mechanisms promoting platelet release from mature MK are still poorly understood, it is well known that hemodynamic forces are indispensable.To mimic these forces in vitro, the suspension containing proplatelet-bearing MK is aspirated and repelled five times with a P1000 cone and then analyzed by flow cytometry. For this purpose, tubes containing a calibrated number of fluorescent beads are used. First, beads present into the tube are gated on the CD41-Alexa-fluor 488/PErcP-Cy5 window (Figure 4Bi, in red). Then, cultured platelets are visualized in a pre-gate (platelet-like elements), determined on the forward scatter (FSC) and side scatter (SSC) parameters of native platelets (Figure 4Bii). The number of platelets is then determined on their CD41/CD42a positivity (Figure 4Biii). Cell counting is stopped in this protocol at 5,000 beads but another fixed number may be used depending on the supplier&#39;s recommendation. From the data acquired, the number of platelets counted per 5,000 beads is routinely at an average of 24.01 &#177; 92 (n = 15) (Figure 4C). Knowing the volume aspirated by the flow cytometer to count 5,000 beads (to be calculated for each cytometer) and the total volume of culture, it is possible to obtain an approximation of the total number of cultured platelets released.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62499/62499fig04.jpg" /> 
Figure 4: Cultured platelets release at day 13. (A) Representative light microscopy image of MK emitting proplatelets at D13. (B) Strategy to quantify cultured platelet release. (i) Beads are gated on the CD41-Alexa-fluor 488/PErcP-Cy5 window (in red). (ii) Platelet-like elements are visualized in a gate determined on the FSC/SSC parameters of native platelets (grey dots). (iii) Cultured platelets are determined on their CD41/CD42 positivity (purple). (C) Number of platelets counted per 5,000 beads can be obtained, routinely an average of 24,011 &#177; 919 (n = 15). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62499/62499fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The procedure at a glance 
To better summarize the method and understand each step, a poster summarizing the protocol step by step is presented in Figure 5. This summary sheet can be displayed in the culture room and serve as a memo. Of note, the success of the experiments is only guaranteed with the product references indicated in the table provided.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62499/62499fig5v2.jpg" /> 
Figure 5: Isolation of CD34+. Poster summarizing the protocol step by step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62499/62499fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Biology

3.9K 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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

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.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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 ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

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 ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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 ...

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

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