Mesencult basal medium

The mice were sacrificed by cervical dislocation. The bone marrow was obtained from the tibias and femurs and the cells were seeded using MesenCult basal medium, supplemented with 20% Mesenchymal Mouse Stimulatory Supplement and 1% Pen-Strept complete medium (Life Technologies, Monza, Italy). The cells were grown at 37 °C in a humidified atmosphere at 5% CO₂, trypsinized at confluence and reseeded at 2 × 10⁴ cells/cm² (passage 1, p1). All experiments were performed at passage 2 (p2). The cellular density seeded onto the disks was 10⁵/cm². The cell count was performed at the undifferentiated state and after differentiation at 12, 24, 48 h and 8 days. The RT-PCR was performed at 0, 3 and 8 days. The viable and dead cells were evaluated with Trypan blue exclusion. All experimental protocols on mice were conducted in compliance with the Italian DL 26/2014 act, the implementation of the European Directive 2010/63 on the protection of animals used for scientific purposes. All experimental protocols were approved by the Institutional Ethics Committee for Animal Use of the University of Pisa.

Myeloma BMSCs were isolated from remaining bone marrow samples of myeloma patients for routine diagnostic. Cells in the bone marrow sample were obtained by Ficoll density gradient centrifugation (AXIS-SHIELD). The cells were then plated in the tissue culture flasks at a concentration of 10⁶ cells/mL in Mesencult basal medium supplemented with MSC stimulatory supplements (both from Life technology). After 24 h incubation at 37°C in a 5% CO² humidified atmosphere, non-adherent cells were removed, and the adherent fraction was cultured in fresh medium. Cells used for future experiment were no more than 10 passages. Normal BMSCs cell line HS5 was obtained from ATCC, and cultured in Mesencult basal medium supplemented with MSC stimulatory supplements.