Recombinant human csf 1

Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France) or newly diagnosed CMML patients whose samples were collected with informed consent following the authorization provided by the ethical committee Ile-de-France 1 (DC-2014-2091), separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood CD14⁺ monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Paris, France)^{41 (link)}. Monocytes used for nucleofection experiments were sorted with Monocyte isolation kit II (Miltenyi Biotech). After sorting, monocytes were cultured overnight at 10⁶/mL in RPMI 1640 Glutamax medium (ThermoFisher Scientific) supplemented with 10% heat inactivated fetal bovine serum (FBS, Lonza, Amboise, France), 1% penicillin/streptomycin, and 2 mM L-Glutamine (ThermoFisher Scientific). Cells were then seeded at 0.5 × 10⁶/mL and differentiated into macrophages in the same medium containing recombinant human CSF-1 (100 ng/mL Peprotech, Neuilly-Sur-Seine, France) during 1 to 3 days. Cells were detached using PBS-EDTA and centrifuged at 300×g into dry pellets for further RNA or protein extraction.

For in vitro OC differentiation, either primary BM cells or splenocytes were enriched for MPs initially by culturing on non-adherent plastic plates in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 50 U·mL^–1 penicillin/streptomycin (P/S, Gibco) and 50 ng·mL^–1 recombinant human CSF1 (Peprotech, Rocky Hill, NJ). After 3 days of culture, the myeloid enriched non-adherent fraction was mechanically isolated and replated on adherent tissue culture plates in DMEM containing 10% FBS, 50 U·mL^–1 P/S, 50 ng·mL^–1 CSF1, and 100 ng·mL^–1 recombinant human RANKL (Peprotech) and harvested at the indicated time points. Where indicated, primary BM-derived myeloid cells (BMDM) undergoing in vitro OC differentiation were treated with 250 nmol·L^–1 JQ1 or equivalent volume of DMSO (Vehicle control) in the culture media. For extraction and culture of Pu.1 KO cells derived from Pu.1^fl/nullCSF1rTAMCre + mice, mice were treated in vivo with 5 daily intraperitoneal (IP) injections of 1 mg tamoxifen/mouse/day. One day following the final injection, BMs were flushed from femurs and tibias and plated on non-adherent plates as described above with the addition of 1 μmol·L^–1 4-OHT in the media. When BMDMs were replated on adherent plates for in vitro osteoclast differentiation, 4-OHT was removed from the media.

Human recombinant CSF-1 (Peprotech) was coupled with Alexa Fluor 488 using the Alexa fluor 488 Microscale Protein Labeling Kit (ThermoFisher Scientific) according to the supplier instructions.

For phagocytosis assays, non-GMP hMDMs were differentiated from cryopreserved primary CD14+ monocytes using serum-containing Iscove's Modified Eagle's Medium essentially as described using human recombinant CSF-1 (100 ng/ml, Peprotech).²¹ (link) hCAMs and hAAMs were polarised from hMDMs by overnight stimulation with LPS/hIFNγ (50/20 ng/ml), and hIL-4/IL-13 (20 ng/ml each) respectively. For clinical-grade hMDMs, we utilised a serum-free GMP-compliant process as described.²² (link) Peripheral blood mononuclear cells (PBMCs) were centrifuged and collected from healthy volunteer buffy coats using Ficoll-paque 1.077 (GE Healthcare). CD14+ cells were isolated from PBMCs using CliniMACS CD14 MicroBeads (Miltenyi Biotec) on LS separation columns (Miltenyi Biotec). CD14+ cells were cultured (37°C, 5% CO₂) for 7 days in TexMACS GMP media (Miltenyi Biotec), supplemented with GMP-grade human recombinant CSF-1 (100 ng/ml, R&D Bio-Techne) with an additional feed at day 3 to generate hMDMs. Clinical-grade hAAMs were generated from hMDMs using human recombinant cytokines (R&D Bio-Techne) as above. Successful hMDM differentiation was confirmed using flow cytometry to demonstrate a minimum 5-fold increase in mean fluorescence intensity (MFI) on 25F9 and CD206 compared to initial CD14+ cells.