Hgm csf

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25 protocols using hgm csf

Isolation and Differentiation of SARS-CoV-2-Infected Immune Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples of healthy donors using human peripheral lymphocytes separation medium (#LTS1077-1, TBD, Tianjin Haoyang) according to recommended protocol. Isolated PBMCs were then allowed to adhere to cell plates pre-coated with poly-D-lysine. After 2 h of adhesion, non-adherent cells were washed away with pre-warm PBS. Adherent monocytes were cultured in RPMI-1640 (#C11875500BT, Gibco) supplemented with 10% FBS (#10099141C, Gibco) and 1% Penicillin-Streptomycin (#10378016, Gibco) at 37°C with 5% CO₂ atmosphere. Monocytes-derived macrophages (MDMs) were differentiated by providing human granulocyte-macrophage colony-stimulating factor (hGM-CSF, #300-03, 50 ng/mL, Peprotech) for 6 days, while Monocytes-derived dendritic cells (MoDCs) were differentiated by adding hGM-CSF (#300-03, 50 ng/mL, Peprotech) and human interleukin-4 (hIL-4, #200-04, 25 ng/mL, Peprotech) for 6 days. The SARS-CoV-2 virus was provided by Prof. Liu reported previously (8 (link)). For virus infection, MoDCs and MDMs were replaced with fresh RPMI-1640 and then infected with SARS-CoV-2 at a MOI = 0.1 for 2 h. After virus absorption, cells were washed with pre-warmed RPMI-1640 twice and maintained in RPM1640 containing 2% FBS for 24 h before further measurement.

Xiang Q., Feng Z., Diao B., Tu C., Qiao Q., Yang H., Zhang Y., Wang G., Wang H., Wang C., Liu L., Wang C., Liu L., Chen R., Wu Y, & Chen Y. (2021). SARS-CoV-2 Induces Lymphocytopenia by Promoting Inflammation and Decimates Secondary Lymphoid Organs. Frontiers in Immunology, 12, 661052.

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Differentiation of hESCs into Hematopoietic Progenitors

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hESCs were differentiated into hematopoietic progenitors using either an embryoid body culture described previously^{174 (link)} or the STEMdiff Hematopoietic Kit (STEMCELL Technologies) per the manufacturer’s instructions. Hematopoietic progenitors were collected then cultured in flasks coated with 20mg/mL poly-HEMA (Sigma-Aldrich) in α-MEM with 10% fetal bovine serum (FBS), Glutamax, penicillin/streptomycin, and 100ng/mL hGM-CSF (PeproTech) for 8–10d with half medium changes every 4d^{175 (link)}. Cells were collected and spun over 20% Percoll (GE Healthcare). The cells at the interface were collected and cultured in poly-HEMA-coated flasks with StemSpan SFEM (STEMCELL Technologies) supplemented with lipid mixture 1 (Sigma-Aldrich), 100ng/mL hGM-CSF (Peprotech), and 100ng/mL hIL-4 (PeproTech) for 7–9d with half medium changes every 4d. Cells were then collected and cultured in poly-HEMA-coated flasks in StemSpan SFEM with lipid mixture 1 and 400ng/mL A23187 calcium ionophore (Sigma-Aldrich) for 2d. Human PBMCs were used as a control following culture in RPMI supplemented with 10% FBS, 2-mercaptoethanol, penicillin/streptomycin, 100ng/mL hGM-CSF, and 100ng/mL hIL-4 for 7–10d.

Pizzato H.A., Alonso-Guallart P., Woods J., Johannesson B., Connelly J.P., Fehniger T.A., Atkinson J.P., Pruett-Miller S.M., Monsma FJ J.r, & Bhattacharya D. (2023). Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers. bioRxiv.

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Conditional Knockout Mouse Models for AML Research

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Cbl^f/f;Cbl-b^f/f mice were generated as described (Goetz et al. 2016 (link)) and crossed to cre^ERT2 mice from Jackson Laboratories. Lnk^−/− mice were generously provided by Dr. Tony Pawson and Dr. Laura Velazquez (Samuel Lunenfeld Research Institute, Toronto, Canada). BM- or peripheral blood-derived MNCs from deidentified AML patients were obtained from the Stem Cell and Xenograft Core Facility at University of Pennsylvania. TF-1 and HEL cells were purchased from American Type Culture Collection. HEL cells were cultured in RPMI with 10% bovine calf serum. TF-1 cells were maintained in RPMI supplemented with 10% CS and 2 ng/mL hGM-CSF (PeproTech).

Lv K., Jiang J., Donaghy R., Riling C.R., Cheng Y., Chandra V., Rozenova K., An W., Mohapatra B.C., Goetz B.T., Pillai V., Han X., Todd E.A., Jeschke G.R., Langdon W.Y., Kumar S., Hexner E.O., Band H, & Tong W. (2017). CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies. Genes & Development, 31(10), 1007-1023.

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Isolation and Generation of Human PBMCs

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Human PBMCs were isolated from healthy donors using a buffy coat (Gulf Coast Regional Blood Center, Houston, Texas, USA) by Ficoll gradient centrifugation. T-cells were isolated from PBMCs by negative selection using a T-cell isolation kit (#17951, Stemcell Technologies, California, USA). DCs were derived from PBMCs by culturing with 20 ng/mL hGM-CSF (#300-03, PeproTech, USA) and 20 ng/mL hIL-4 (#200-04, PeproTech) for 7 days.

Hong B., Sahu U., Mullarkey M.P., Hong E., Pei G., Yan Y., Otani Y., Banasavadi-Siddegowda Y., Fan H., Zhao Z., Yu J., Caligiuri M.A, & Kaur B. (2023). PKR induces TGF-β and limits oncolytic immune therapy. Journal for Immunotherapy of Cancer, 11(2), e006164.

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iPSC-Derived Microglial Characterization

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iPSCs were first differentiated into hematopoietic progenitor cells following manufacturer’s instructions using a commercially available kit (StemCell Technologies #05310) as described before (Andreone et al., 2020 (link)). HPCs positive for identity markers CD34, CD43, and CD45 were transferred to a plate containing primary human astrocytes and co-cultured using media C adapted from a previous study (Pandya et al., 2017 (link)). Once floating cells in co-culture are predominantly (>80%) mature microglia, cells were plated for 3–4 days prior to experiments. Full characterization of human iPSC-derived microglia and additional details on the differentiation protocol has been published elsewhere (Andreone et al., 2020 (link)). All the experiments using GRN^+/+ and GRN^−/− iMG were performed in IMDM (Gibco) media supplemented with 10% defined FBS (Gibco), 1% Penicillin/Streptomycin (Gibco), 20 ng/mL of hIL3 (Peprotech), 20 ng/mL of hGM-CSF (Peprotech) and 20 ng/mL of hM-CSF (Peprotech) (referred to as “C+++ Media”). Immunostainings of PGRN were conducted using the anti-progranulin goat polyclonal antiserum (R&D Systems # AF2420, 1:250) to verify the absence of immunoreactivity in knockout iMG.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

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Monocyte-Derived Dendritic Cell Protocol

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All human research protocols for this work were reviewed and approved by the IRB of the Mount Sinai School of Medicine. Monocyte-derived DCs were obtained from healthy anonymous human blood donations obtained from New York Blood Center, following a standard protocol described elsewhere [49 (link)]. No information on age, sex or ethnicity from these donors is available. These donations are considered scientific samples and not human subjects according to the IRB guidelines. Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll density gradient centrifugation and positive immunomagnetic purification for CD14 followed by a 5 day incubation with 500 U/ml hGM-CSF (Preprotech, Rocky Hill NJ) and 1000 U/ml hIL-4 (Preprotech, Rocky Hill NJ) All experiments were replicated using cells obtained from different donors.

Thakar J., Hartmann B.M., Marjanovic N., Sealfon S.C, & Kleinstein S.H. (2015). Comparative analysis of anti-viral transcriptomics reveals novel effects of influenza immune antagonism. BMC Immunology, 16, 46.

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Generating Immature Dendritic Cells

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The functional plasticity of the patients’ MO population was assessed by generating immature DC. Fresh monocytes were incubated in X-VIVO 15™ Media with Gentamycin and Phenol Red (Lonza, Cohasset, MN) supplemented with human interleukin 4 (hIL-4; Peprotech, Rocky Hill, NJ) at 500 IU/ml and human granulocyte monocyte colony stimulation factor (hGM-CSF; Peprotech, Rocky Hill, NJ) 1000 IU/ml 1 at 37 °C 5% CO₂ in the dark. On day 3, 50% of the X-VIVO15/10™ was replenished with fresh media along with 50% of the initial cytokine concentration. The cultures were terminated on day 5 [22 ].

Laudanski K., Zawadka M., Polosak J., Modi J., DiMeglio M., Gutsche J., Szeto W.Y, & Puzianowska-Kuznicka M. (2018). Acquired immunological imbalance after surgery with cardiopulmonary bypass due to epigenetic over-activation of PU.1/M-CSF. Journal of Translational Medicine, 16, 143.

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Generating Monocyte-Derived Dendritic Cells

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Blood from healthy donors was obtained from the Blood Transfusion Center (University of Lausanne, Switzerland) under Project P_297. Peripheral blood mononuclear cells were isolated by density gradient centrifugation on Lymphoprep (STEMCELL Technologies). To generate moDCs, CD14⁺ cells were isolated with magnetic beads (Miltenyi) and cultured in RPMI 1640 containing 10% FBS, 100 U ml^–1 penicillin, 100 μg ml^–1 streptomycin, 2 mM glutamine, 50 ng ml^–1 hGM-CSF and 50 ng ml^–1 hIL4 (PeproTech; 200-04), at a density of 10⁶ cells per ml for 7 days.

Ghasemi A., Martinez-Usatorre A., Li L., Hicham M., Guichard A., Marcone R., Fournier N., Torchia B., Martinez Bedoya D., Davanture S., Fernández-Vaquero M., Fan C., Janzen J., Mohammadzadeh Y., Genolet R., Mansouri N., Wenes M., Migliorini D., Heikenwalder M, & De Palma M. (2023). Cytokine-armed dendritic cell progenitors for antigen-agnostic cancer immunotherapy. Nature Cancer, 5(2), 240-261.

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Differentiation of Dendritic Cell Progenitors

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Day-seven cultures were processed to FACS-sort CD34⁺CD115⁺ DCPs (or mock-sorted) using a BD FACSAria II SORP apparatus. Sorted DCPs were cultured in StemSpam SFEMII medium (STEMCELL Technologies) supplemented with 50 units per ml of penicillin (Gibco), 50 μg ml^–1 streptomycin (Gibco), 20 ng ml^–1 hGM-CSF, 100 ng ml^–1 hFLT3L, 20 ng ml^–1 hSCF (all from PeproTech) and 1,000 IU ml^–1 hIFNa2b (InvivoGen) for 7 days. Day-14 cultures contained differentiated cell populations identified as indicated in the Reporting Summary.

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Monocyte Differentiation with HDC

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PBMCs were prepared from healthy blood donor buffy coats by Ficoll-Paque (Lymphoprep, Nycomed, Oslo, Norway) density centrifugation. Monocytes were isolated by adherence and cultured in Iscoves’ medium supplemented with 10% human AB serum, 2 mM l-glutamine, 100 µg/ml penicillin, 100 µg/ml streptomycin, 1 ng/ml interleukin 6 (hIL-6, Sigma-Aldrich) and 10 ng/ml granulocyte macrophage colony-stimulating factor (hGM-CSF, Peprotech, Rocky Hill, USA) in the presence or absence of 100 µM HDC. In control experiments, adherent monocytes were cultured in the absence of cytokines. One-half of the medium was replaced and HDC was again added after 2 days of culture. Cells were examined for expression of HLA-DR (antibody: HLA-DR-APC-Cy7, Clone C243, BD Biosciences) by flow cytometry after 5 days of culture.

Grauers Wiktorin H., Nilsson M.S., Kiffin R., Sander F.E., Lenox B., Rydström A., Hellstrand K, & Martner A. (2018). Histamine targets myeloid-derived suppressor cells and improves the anti-tumor efficacy of PD-1/PD-L1 checkpoint blockade. Cancer Immunology, Immunotherapy, 68(2), 163-174.

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