2.5. Characterization of CXCR4-IL10-MSCs

WT and CXCR4-IL10-MSCs were immunophenotypically characterized by flow cytometry (Fortessa, BD Bioscience, NJ, USA) as described by the Mesenchymal cell kit (Immunostep, Salamanca, Spain). The monoclonal anti-human antibodies included in these studies were the following: CD29, CD44, CD73, CD90, CD105, CD166, CD45, CD19, HLA-DR, CD14, and CD34. Data were analyzed with FlowJo version X (FlowJo LLC, CA, USA).

The osteogenic and adipogenic differentiation ability of WT and CXCR4-IL10-MSCs was determined using the NH-OsteoDiff and NH-AdipoDiff Media (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, according to the manufacturer’s protocols. Alkaline phosphatase deposits were observed after the staining with Fast BCIP/NCP (Sigma Aldrich, St. Louis, MO, USA) while lipid droplets were seen with optic microscopy (Nikon, Düsseldorf, Germany). Gene expression analyses related to the differentiation to bone and adipose tissue was performed after 10 or 21 days of culture, respectively, by qRT-PCR, following RNA extraction with RNeasy^® Plus Mini Kit (QIAGEN) and reverse transcription with RETROscript kit (Thermo Fisher Scientific). qRT-PCR was performed using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific) and mixes of probes and primers specific for BGLAP (Ref: Hs01587814_g1), ALPL (Ref: Hs01029144_m1) and PPARγ (Ref: Hs01115513_m1), all conjugated with FAM (ThermoFisher Scientific), on QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems). Results were normalized for human GAPDH expression (Ref: Hs02786624_g1, conjugated with VIC; Thermo Fisher Scientific) and the expression of control samples according to the 2^-ΔΔCt method.