Its plus

Four weeks after the labelling procedure (day 28), hASCs from passage 6 of all 6 patients were used to evaluate the multilineage differentiation potential after VSOPs-labelling. The adipogenic, chondrogenic and osteogenic differentiation assays were performed as described previously [38 (link)]. According to the protocol of Pittenger [39 (link)] and modified by Nöth et al. [40 (link)], EM-DMEM supplemented with 1 µg/mL insulin and 10 µM dexamethasone, 100 µM indomethacin, as well as 500 µM 1-methyl-3-isobutylxanthine, was used for adipogenic differentiation. For osteogenic differentiation, EM-DMEM with 100 nM dexamethasone, 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid according to Jaiswal et al. was used [41 (link)]. Chondrogenic differentiation was induced with DMEM, plus 1% P/S and 100 nM dexamethasone, 100 µg/mL sodium pyruvate, 50 µg/mL ascorbate-2-phosphate, 40 µg/mL proline, ITS-plus (Sigma-Aldrich), as well as 10 ng/mL TGF-ß3 (LONZA, Basel, Switzerland). Human ASCs cultured in EM-DMEM were used as negative controls.
The differentiation media were replaced every two days for three weeks. The differentiation capacity was documented by histological images and quantified by Real-Time Polymerase Chain Reaction (PCR).

Adipogenic, osteogenic and chondrogenic differentiation were performed to evaluate the multidifferentiation potential and a possible impact of VSOPs labeling. The hASCs were labeled with 1.5 mM VSOPs, the highest concentration used for the experiments. In the literature, an impairment of the differentiation potential of MSCs as a function of IONPs concentration is described [51 (link),52 (link)]. Defined media were used to induce differentiation. For adipogenic differentiation, EM-DMEM supplemented with 10 µM dexamethasone, 1 µg/mL insulin, 100 µM indomethacin and 500 µM 1-methyl-3-isobutylxanthine was used based on a modification [65 (link)] of the protocol of Pittenger et al. [66 (link)]. The osteogenic differentiation medium consists of EM-DMEM with 100 nM dexamethasone, 10 mM ß-glycerophosphate and 50 µg/mL ascorbic acid according to Jaiswal et al. [67 (link)]. Chondrogenic differentiation was induced with DMEM plus 1% P/S and 100 nM dexamethasone, 100 µg/mL sodium pyruvate, 50 µg/mL ascorbate-2-phosphate, 40 µg/mL proline, ITS-plus (Sigma-Aldrich Chemie GmbH) and 10 ng/mL TGF-ß3 (LONZA, Basel, Switzerland).