Sorted GFP+CD34+ HSCs were allowed to expand for a further 72 h in X‐VIVO‐15/FCS/cytokine media at 2.5 × 104 mL−1 before differentiation into DCs according to a modified published protocol.30 In brief, the expanded cells were cultured at a density of 6.25 × 104 mL−1 in RPMI 1640, 10% FCS, 1 mm HEPES, 1 mm sodium pyruvate, 100 U mL−1 penicillin‐streptomycin, 2 mm Glutamax (all Thermo Fisher Scientific) and 50 µm β‐mercaptoethanol (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 100 ng mL−1 human FLT3L, 20 ng mL−1 human SCF, 2.5 ng mL−1 human IL‐4, 2.5 ng mL−1 human GM‐CSF (all cytokines from Peprotech) and 1 µm StemRegenin 1 (STEMCELL Technologies, Vancouver). Media and cytokines were replenished on Day 6 of the differentiation culture and harvested for analysis by flow cytometry after 12 days.
Human scf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human SCF is a recombinant protein that functions as a growth factor for hematopoietic stem cells. It plays a crucial role in the survival, proliferation, and differentiation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Human il 3, by Thermo Fisher Scientific (8 mentions)
Human tpo, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Human flt3 ligand, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Human flt3l, by Thermo Fisher Scientific (3 mentions)
Flt3l, by Thermo Fisher Scientific (3 mentions)
Cd34 microbead kit, by Miltenyi Biotec (3 mentions)
Imdm medium, by Thermo Fisher Scientific (3 mentions)
Human gm csf, by Thermo Fisher Scientific (2 mentions)
Human fgf basic, by Thermo Fisher Scientific (2 mentions)
Methocult media, by STEMCELL (2 mentions)
Human vegf, by Thermo Fisher Scientific (2 mentions)
Human igf1, by Thermo Fisher Scientific (2 mentions)
Human egf, by Thermo Fisher Scientific (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Cd34 apc, by BioLegend (2 mentions)
Cd38 pe, by BD (2 mentions)
Apc cy7 cd49f, by BioLegend (2 mentions)
17 protocols using human scf
1
Differentiation of GFP⁺CD34⁺ HSCs into DCs
2
Culturing Endothelial Progenitor Cells from PBMNCs
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PBMNCs were harvested at a concentration of 1 × 105 cells/ml and resuspended with 30% FBS/PBS 200 µl. The following recombinant human cytokines were then added to the cells: human SCF (#300-07; PeproTech) at a concentration of 66.7 ng/ml; human VEGF (#100-20; PeproTech) at a concentration of 33.3 ng/ml; human IL3 (#200-03; PeproTech) at a concentration of 13.3 ng/ml; human IGF-1 (#100-11; PeproTech) at a concentration of 33.3 ng/ml; human FGF Basic (#100-18B; PeproTech) at a concentration of 33.3 ng/ml; and, human EGF (#100-15; PeproTech) at a concentration of 33.3 ng/ml. The cell mixture was resuspended with complete MethoCult™ media (#04236; STEMCELL Technologies, Inc., Vancouver, British Columbia, Canada) at a final volume of 2 ml, and then cultured in a 37 °C environment for 14 days [11 (link)]. Endothelial progenitor cell (EPC) colony forming cells (EPC-CFCs) were assessed under phase-contrast light microscopy (Eclipse TE300; Nikon Instruments, Tokyo, Japan). A colony was defined as the presence of at least 50 cells [15 (link)]. All experiments were performed in triplicate. The numbers of colonies of PBMNCs cultured in QQ culture media and in standard culture media were compared.
3
Expansion and Xenotransplantation of Human Cord Blood CD34+ Cells
4
Erythroid Differentiation of HUDEP-2 and MEL-745A Cells
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HUDEP-2 cells were maintained in StemSpan SFEM containing 50 ng/mL human SCF (PeproTech), 3 U/mL erythropoietin, 2 mM dexamethasone (Sigma), and 1 mg/mL doxycycline (Sigma). Cells were differentiated for 7 days in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 5% PB plasma (STEMCELL Technologies), 2 U/mL heparin (Sigma), 1% GlutaMAX (Gibco), 330 mg/mL holo-transferrin (Sigma), 100 ng/mL human SCF, 10 mg/mL insulin (Tocris Bioscience), 3 U/mL erythropoietin, and 1 mg/mL doxycycline. MEL-745A cells were cultured in Dulbecco Modified Eagle Medium (DMEM) containing 10% FBS (Gemini Bio), 1% GlutaMAX, and 1% penicillin/streptomycin. For differentiation, cells were cultured for 4 days in culture medium with 2% dimethyl sulfoxide (DMSO) (Fisher).
CD34+ cells were thawed and first cultured in StemSpan H3000 (STEMCELL Technologies) and StemSpan CC100 (STEMCELL Technologies) for 3 days. They were then subjected to CD36+ selection and differentiated as previously described [29 (link)], with cells harvested at D6, D8, and D10 of maturation.
CD34+ cells were thawed and first cultured in StemSpan H3000 (STEMCELL Technologies) and StemSpan CC100 (STEMCELL Technologies) for 3 days. They were then subjected to CD36+ selection and differentiated as previously described [29 (link)], with cells harvested at D6, D8, and D10 of maturation.
5
Isolation and Culture of Primary AML Cells
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Human AML patient material was obtained from patients hospitalized at Amsterdam UMC (location VUmc, Amsterdam, the Netherlands) at time of diagnosis according to HOVON AML protocols. Normal bone marrow samples were obtained from otherwise healthy patients undergoing cardiothoracic surgery. Informed consent was obtained from every used patient, procedures were approved by the ethical committee of Amsterdam UMC, and all experiments were conducted in accordance with the Declaration of Helsinki. Mononuclear cells were isolated from each primary sample using Ficoll-Paque Plus (Amersham Biosciences) separation and cryopreserved in liquid nitrogen. Samples were thawed in IMDM medium (ThermoFisher Scientific) with 20% FCS and incubated with 10 mg/mL DNase I (Roche) and 10 mM magnesium chloride (Sigma Aldrich) for 30 minutes. Samples were cultured in IMDM containing 15% BIT 9500 serum substitute (StemCell Technologies), 50 ng/mL Flt3L (Peprotech), 20 ng/mL IL3 (Peprotech), 100 ng/mL human SCF (Peprotech), and 20 ng/mL G-CSF (Peprotech) in a humidified atmosphere at 37°C and 5% CO2.
6
Generation and Modulation of Dendritic Cells from CD34+ Hematopoietic Precursors
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DC were generated from CD34+ haematopoietic precursors (HPC) obtained from cord blood samples and cultured for 7 d in the presence of human SCF (20 ng/mL, Peprotech), TPO (10 ng/mL, Peprotech), Flt3L (25 ng/mL, Peprotech), GM-CSF (200 U/mL, Amoytop Biotech) and IL-4 (100 U/mL, ImmunoTools), as previously described.55 (link) pDC were generated from CD34+ HPC as previously described.56 (link) In selected experiments, pDC were stimulated with type A CpG ODN at 12 μg/mL (5′-ggGGGACGATCGTCgggggg-3′; Eurogentec). All human samples were collected according to a protocol approved by the Ethics Committee of the University Hospital of Liège.
In selected experiments, DC were treated with human recombinant RANKL (0.1 or 0.5 μg/mL, R&D systems) for 6 d followed by a maturation with LPS (1 μg/mL, Sigma-Aldrich) for another day. OPG (Abcam), an inhibitor of RANKL, was also used at a concentration of 1 μg/mL, renewed once during the 7 d of culture.
In selected experiments, DC were treated with human recombinant RANKL (0.1 or 0.5 μg/mL, R&D systems) for 6 d followed by a maturation with LPS (1 μg/mL, Sigma-Aldrich) for another day. OPG (Abcam), an inhibitor of RANKL, was also used at a concentration of 1 μg/mL, renewed once during the 7 d of culture.
7
Generation of MDSCs from CD34+ Cells
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Human CB was provided from the Catholic Hematopoietic Stem Cell Bank after written informed consent given by normal full-term pregnant women. For MDSCs generation, isolated CD34+ cells (Miltenyi Biotec, Bergisch Gladbach, Germany) were cultured in a 48-well plate (BD Falcon, Bedford, MA) at 1 × 105 cells/ well with 1 ml of IMDM containing 10% FBS (Gibco, Grand Island, NY, United States), 10% penicillin–streptomycin (100 U/ml; Lonza Walkersville, MD, United States), 2 mM L-glutamine (Lonza Walkersville) (10% complete medium), 100 ng/ml human GM-CSF (300–03, PeproTech, Rocky Hill, NJ, United States), 100 ng/ml human G-CSF (300–23, PeproTech), or 100 ng/ml human M-CSF (300–25, PeproTech) and 50 ng/ml human SCF (300–07, PeproTech). After incubation for 7 days, the cells were removed from the 48 well plate and centrifuged at 1,300 rpm for 5 min. After one wash with serum free IMDM, the cells were cultured for 2 weeks and media was changed every 7 days. From weeks 4–6, the cells were cultured at a higher density (5 × 105 cells/well). Media was changed every 7 days throughout 6 weeks of the culture.
8
Cultured Cell Lines and Patient-Derived AML Cells
9
Erythroid Differentiation of G-CSF Mobilized CD34+ Cells
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G-CSF mobilized human peripheral blood CD34+ HSPCs were purchased from STEMCELL Technologies. Cells were thawed on day 0 into StemSpan serum-free expansion medium (09655, STEMCELL Technologies) supplemented with 100 ng/mL Flt3L (Peprotech), 50 ng/mL human stem cell factor (SCF; 300-07, Peprotech), 100 ng/mL TPO (Peprotech), and 1% penicillin/streptomycin (Gibco). Electroporation of RNP complexes was done on days 1 and 2 of the culture. Differentiation of CD34+ CD38 into erythroid progenitors was done in four phases of erythroid differentiation medium (EDM) consist of Iscove’s modified Dulbecco’s medium (IMDM; Gibco) supplemented with 5% human AB serum (H6914, Sigma), 10 μg/mL recombinant human insulin (I2643, Sigma), 2 units/mL heparin (H3393, Sigma), 3 units/mL Epogen (EPO, Amgen), 330 μg/mL holo-transferrin (T4132, Sigma), and 1% penicillin/streptomycin. EDM was further supplemented with 25 ng/mL human SCF and 1 ng/mL human IL-3 (Peprotech) in phase 1 of the culture (days 4–7). IL-3 was withdrawn and human SCF is decreased to 10 ng/mL in phase 2 of the culture (days 7–11). human SCF is further decreased to 2 ng/mL in phase 3 of the culture (days 12–16). human SCF was withdrawn and holo-transferrin is increased to 1 mg/mL (day 17 and beyond). Cells were collected on day 21 for flow cytometry, RNA extraction, and Giemsa stain.
10
Expansion and Xenotransplantation of Human Cord Blood CD34+ Cells
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Human umbilical cord blood-derived CD34+ cells (purchased from Lonza or Stem Cell Technologies) or FACS-purified CD34+CD38-CD90+CD49f+ cells (following staining with APC-CD34 [Biolegend; 560940], PE-CD38 [BD; 347687], FITC-CD90 [Biolegend; 328113], and APC/Cy7-CD49f [Biolegend; 313611]) were cultured in IMDM (Life Technologies) containing 0.1% HSA or 0.1% PVA (Sigma P8136, 363081, or 363146), supplemented with 1% ITSX, 1% P/S/G, 10 mM HEPES. For proliferation assays, 50 cells were seeded per well and supplemented with 10 ng/ml human SCF and 100 ng/ml human TPO (Peprotech). During the cultures, media was refreshed every three days and counted at day-7. For xenograft assays, 2×103 cells were expanded for 7-days before being injected intravenously into sub-lethally-irradiated (1.5 Gy) NOG mice. Human cell chimerism in the PB was calculated at 16-weeks post-transplantation using PE/Cy7-CD45.1 (Biolegend; 110730) and V450-hCD45 antibodies (BD; 560367).
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