Bone slices

BMCs were isolated from wild-type mice and 200 000 cells per well were added on top of primary osteocyte-enriched fractions or primary osteoblasts cultivated in co-culture medium (α-MEM supplemented with 10 nM 1,25-dihydroxyvitamin D3 (Sigma-Aldrich) and 20 ng/ml M-CSF). For pit assay, bone slices (Immunodiagnostic Systems, Tyne and Wear, UK) were used. Cells were removed from bone slices with 1 M ammonium hydroxide overnight and the resorption pits were visualized using 0.5% (w/v in H₂O) toluidine blue.
For conditioned medium-transfer experiments, conditioned medium from Men1-deficient primary osteocyte-enriched fractions or control primary osteocyte-enriched fractions was diluted 1:1 in co-culture medium.
For neutralizing antibody experiments, CXCL10-neutralizing antibody (20 ng/ml; R&D Systems) or IgG was added to co-cultures of wild-type BMCs with Men1-deficient primary osteocyte-enriched fractions in co-culture medium.
The medium was changed every 3 days. After 9 days, cells were stained for TRAP activity.

A Lymphoprep (STEMCELL Technologies) density gradient was used to isolate PB mononuclear cells (PBMC). CD34⁺ cells were isolated from PBMC using human CD34 MicroBead Kit and autoMACS Pro Separator (Miltenyi Biotec), according to the manufacturer’s instructions. CD34⁺ were pre-stimulated for 24 hours (h) and transduced with 1-hit of LV at MOI 100 overnight, as previously described.^{22 (link),23 (link)} Hematopoietic progenitor cultures were performed plating 2,500 cells in 2.5 mL MethoCult H4434 Classic methylcellulose-based medium (STEMCELL Technologies) and cultured for 12 days.
After transduction, cells were expanded using UM171 compound until day 7, as previously described.^{19 (link)} A fraction of expanded cells was differentiated in vitro towards the myeloid lineage for 1 week and then into osteoclasts for 2 or 3 weeks on plastic wells or bone slices (Immunodiagnostic Systems), as previously described.^{14 (link)}