Human m csf

This study was approved by the Ethics Committee of Shenzhen Third People’s Hospital (approval number: 2019-038). Informed written consents were obtained from participants prior to venous blood collection. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Axis-Shield, AS1114547) and differentiated at 1x10⁶ cells/ml in complete 1640 culture medium supplemented with 30 ng/ml human M-CSF (Gibco, PHC9501) for approximately 7 days.

PBMCs were cultured at 2.5 × 10⁶ cells/mL α-Minimum Essential Media (MEM) supplemented with 10% FBS previously ultracentrifuged, 25 ng/mL of human recombinant RANK Ligand (Gibco, Life Technologies, Rockford, IL, USA), 25 ng/mL of human M-CSF (Gibco, Thermo Fisher Scientific, Rockford, IL, USA), and 10 nM dexamethasone (Sigma-Aldrich, Milano, Italy) (Human OC medium). After 3 days, the culture were washed with α-MEM medium to remove non adherent cells. The remaining cells were mononucleated, expressed TRAP and were considered committed pre-osteoclast cells. For human osteoclastogenesis assays, human OC medium was added and the cultures were continued for additional 6–10 days, at the end of the period they contained large mature multinucleated OCs. The culture period was 14 days for both TRAP staining assay, bone resorption assay and ELISA assay.

PBMCs were cultured at 2.5×10⁶cells/ml α-MEM supplemented with 10% FBS previously ultracentrifuged, 25ng/ml of human recombinant RANK Ligand (Gibco, Life Technologies, USA), 25ng/ml of human M-CSF (Gibco, Life Technologies, USA), and 10nM dexamethasone (Sigma-Aldrich Italy) (Human OC medium). After 2-4 days, the culture were washed with α-MEM medium to remove non adherent cells. The remaining cells were mononucleated, expressed TRAP and were considered committed pre-osteoclast cells. For human osteoclastogenesis assays, OC medium was added and the cultures were continued for additional 6-10 days, at the end of the period they contained large mature multinucleated OCs. The culture period was 16 days for both TRAP staining assay, bone resorption assay and qRT-PCR analysis.

Human PBMC were separated from buffy coats of healthy donors (Hong Kong Red Cross Blood Transfusion Service) using Ficoll-Paque density gradient (GE Healthcare Life Sciences) under Institutional Review Board (IRB) approval (UW 10-124). Monocytes/macrophages were isolated by plastic adherence and were cultured in complete RPMI medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml human M-CSF or GM-CSF (Gibco) for 7 days. Human PBMC-derived dendritic cells were differentiated with 10 ng/ml of IL-4 (Gibco) and 50 ng/ml of GM-CSF for 7 days. The research protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 10-124). For preparation of murine bone marrow-derived macrophages, bone marrow cells were isolated from femurs and tibias and cultured in RPMI medium supplemented with 10% FBS and 10 ng/ml of mouse M-CSF (R&D Systems) for 7 days.

Human PBMCs were seeded (1 × 10⁵ live cells/well) on a 96-well plate, 96-well black plate (Thermo Fisher Scientific), or Osteo Assay Surface Stripwell Microplate (Corning, NY, USA). The cells were then treated with 100 ng/mL glutathione S-transferase (GST)-tagged soluble RANKL (sRANKL) (Oriental Yeast, Tokyo, Japan) and 25 ng/mL human M-CSF (PeproTech, Rocky Hill, NJ, USA) in the presence of 10 µg/mL recombinant human Gal-1C2S protein and allowed to differentiate for 7 days for the cell viability assay and TRAP staining or for 14 days for the actin staining and resorption assay. The medium was replenished every three or four days.
Mouse macrophage RAW264 cells were seeded (1 × 10³ live cells/well) on a 96-well plate, 96-well black plate (Thermo Fisher Scientific), or Osteo Assay Surface Stripwell Microplate (Corning) and cultured for 1 day. The cells were then treated with 250 ng/mL GST-tagged sRANKL, as described previously [23 (link)], in the presence of 10 µg/mL recombinant mGal-1 protein and 10 mM lactose or maltose and allowed to differentiate for 4 or 14 days for the resorption assay. The medium was replenished every three or four days.

Human peripheral blood mononuclear cells (PBMCs) of healthy donors were obtained from the CFAR Gene and Cellular Therapy Core Laboratory at UCLA, without identification information under federal and state regulations. Human monocytes were isolated from healthy donor PBMCs by adherence. Briefly, PBMCs were suspended in serum-free RPMI 1640 media (Corning Cellgro, 10-040-CV) at 10 × 10⁶ cells/ml. In total, 12.5 ml of the cell suspension were added to each 10-cm dish and incubated for an hour in a humidified 37 °C, 5% CO₂ incubator. Medium that contained non-adherent cells was discarded. Dishes were washed twice and adherent monocytes were cultured in C10 media with human M-CSF (10 ng/ml) (Peprotech, 300-25) for 6 days to generate MDMs. At day 6, the resulting MDMs were collected and reseeded in a 6-well plate in C10 medium (1 × 10⁶ cells/ 2 ml/well) for 48 h, in the presence or absence of recombinant human IL-4 (10 ng/ml) (Peprotech, 214-14) and human IL-13 (10 ng/ml) (Peprotech, 214-13) to induce MDM immunosuppressive polarization. In some experiments, MAOIs (phenelzine, 20 μM) were added to the MDM polarization culture 30 min prior to adding recombinant human IL-4 and human IL-13, to block MAO-A activity during MDM polarization. Polarized MDMs were then collected and used for flow cytometry and QPCR analysis or for setting up the 3D human tumor organoid culture experiments.

To generate BMDM, bone marrow cells harvested from femurs and tibias of 6–10-week-old control or EROS knockout mice were grown in complete RPMI medium supplemented with 100 ng/mL of murine M-CSF (PeproTech) for 3 days. At day 3, 10 mL of the same medium was added to the culture and differentiated macrophages were collected at day 7 for mass spectrometry or Western blot analysis. To isolate mouse CD4 lymphocytes, mouse spleens were homogenised by manual disruption and subjected to positive selection using CD4 L3T4 magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. Cells were counted and resuspended at a concentration of 2 × 10⁶ cells per mL in complete RPMI medium for subsequent analysis.
Control or EROS-deficient human iPS were generated by CRISPR Cas 9-targeted deletion of 46 base pairs in exon 5 of the CYBC1 sequence (Yeung et al., 2017 (link)). Human macrophages were obtained from the differentiation of the human control or EROS-deficient iPS line following previously established method (van Wilgenburg et al., 2013 (link); Thomas et al., 2017b (link)). Macrophages were cultured in complete RPMI in the presence of human M-CSF (PeproTech) and were used at day 7 post-differentiation.