Mesenchymal stem cell stimulatory supplement

The isolation and culture of BMSCs from C57BL/6 mice was performed as described previously [16 (link)]. In brief, after anesthesia, the femurs of mice were quickly taken out, and bone marrow cells were flushed form the bone marrow cavities into beaker with MesenCult basal medium supplemented with Mesenchymal Stem Cell Stimulatory Supplement (Stem cell Technologies, Vancouver, BC, Canada). Bone marrow cells were harvested and plated into the dishes and then incubated with the Dulbecco’s Modified Eagle Medium (DMEM, Hyclone, GE Healthcare Life Sciences,USA) supplemented with 10% fetal bovine serum (Gibco, Burlington, ON, Canada), penicillin (100U/ml)/streptomycin (100μg/ml) (Sigma-Aldrich, St. Louis, MO) in a humidified atmosphere of 5% CO2 at 37 °C. Non-adherent cells were removed carefully after 48h and fresh medium was replaced. When primary cultures became almost confluent, the culture was treated with 0.5ml of 0.25% trypsin containing 0.02% ethylene diamine tetraacetic acid (Sigma-Aldrich, St. Louis, MO) for 1 min at room temperature (25 °C). Cultured BMSCs between passages 3-5 were used for the following experiments.

BM samples were obtained from patients diagnosed with MDS and from healthy donors (HD) after obtaining written informed consent, as approved by the institutional procedures of the independent ethics committee and the ‘Comité de Protection des Personnes’-Ile de France (NCT03233074/17.07.2017). HDs were aged matched and had blood counts in the normal range. Detailed characteristics of MDS patients are shown in Table 1.
BM mononuclear cells (BMMNCs) were isolated using Lymphosep (Biowest, Nuaillé, France) gradient separation. 1 × 10⁶ cells were cultured in T25 cell flasks in MesenCult® MSC basal medium (Stem Cell Technologies, Vancouver, BC, Canada) and Mesenchymal Stem Cell Stimulatory Supplement (Stem Cell Technologies, Vancouver, BC, Canada) and in 1% penicillin/streptomycin (Lonza, Basel, Switzerland) at 37 °C in 5% CO₂, with medium replacement twice a week in the beginning of the cultures and every week after date until 80% of confluence is reached.
The resulting CD45-CD73+ CD105+ CD90+ (purity >98%), low-passage BM-stromal cells were used to perform different tests.
Adherent cells were analysed by flow cytometry. The list of antibodies used for BMSC discrimination and characterisation is shown in Table 2.

Bone marrow-derived MSCs were isolated from 6 to 8 week old female C579BL/6J mice (Harlan Laboratories, Udine, Italy), as previously described [37 (link)]. MSCs were expanded and maintained in culture in Mesencult basal medium supplemented with Mesenchymal Stem Cell Stimulatory Supplement (StemCell Technologies, Vancouver, BC, Canada) at 37 °C in a humidified 5% CO₂ incubator. Mature MSCs were defined by a stable CD45⁻ CD34⁻ CD11b⁻ CD9⁺ Sca-1⁺ CD44⁺ phenotype [37 (link)]. MSCs were detached with trypsin, washed 3 times with PBS without Ca²⁺ and Mg²⁺ (Sigma-Aldrich), and counted. Mice induced for EAE were injected in the tail vein with 10⁶ MSCs/mouse diluted in 250 μl PBS (Sigma-Aldrich) at disease onset, i.e., at 12 dpi, or with an equal volume of PBS.