Dexamethasone (dex)

The potential of ASC to differentiate into osteoblasts and adipocytes was confirmed in monolayer culture. To induce adipogenic differentiation, cells were cultured in MesenCult™ adipogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) supplemented with MesenCult® MSC basal medium (STEMCELL Technologies, Canada). To induce osteogenic differentiation, we used MesenCult™ osteogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) in the osteogenic medium containing 10⁻⁴ M dexamethasone (STEMCELL Technologies, Canada), 1 M β-glycerophosphate (STEMCELL Technologies, Canada), and 10 mg/ml ascorbic acid (STEMCELL Technologies, Canada). Media from both cultures were replaced every 3 d for 21 d in total. The differentiation potential for adipogenesis and the formation of intracellular lipid droplets were assessed by Oil Red O staining after fixation in 10% formalin. The differentiation potential for osteogenesis was assessed by Alizarin Red S (ARS) staining after fixation in 10% formalin. To induce chondrogenic differentiation, we used a chondrogenic medium (LONZA, Switzerland). For histological analysis, pellets were embedded in paraffin and sectioned. Chondrogenic differentiation was assessed by Masson trichrome staining. Morphology and differentiation potential of ASCs are represented for one individual donor from three independent donors.

To qualify isolated cells from human BM as hMSCs, cells were induced to differentiation into adipogenic, osteogenic and chondrogenic lineages. Briefly, for adipogenic differentiation, 5x10³ hMSCs/cm² were exposed to Complete MesenCult Adipogenic Medium containing MesenCult MSC Basal Medium (Stemcell) and 10% Adipogenic Stimulatory Supplement (Stemcell) for 3 weeks. For osteogenic differentiation, 2x10⁵ cells/cm² incubated with Complete MesenCult Osteogenic Medium including MesenCult MSC Basal Medium, Osteogenic Stimulatory Supplement, β-Glycerophosphate, Dexamethasone, Ascorbic acid (all from Stemcell) for 5 weeks. For chondrogenic differentiation, 7.5x10⁶ cells/cm² were cultured for 3 weeks with Stempro Chondrocyte Differentiation Basal Medium (Gibco) containing 10% Stempro Chondrogenesis Supplement (Gibco). Standard histochemical staining methods were applied. Osteogenic, adipogenic and chondrogenic differentiation were confirmed by Toluidine Blue, Oil Red O, Alcian Blue staining, respectively.

For osteogenic differentiation, the hMSC line was seeded into 24-well plates at a density of 2 × 10⁵ cells/cm². Osteogenic differentiation was stimulated by refreshing the expansion medium with complete MesenCult osteogenic medium including MesenCult MSC basal medium, osteogenic stimulatory supplement, dexamethasone, and ascorbic acid (all from Stemcell). After 5 days, when multilayering had been observed, β-glycerophosphate was added to complete MesenCult osteogenic medium. The chondrogenic pellet was assessed with Toluidine Blue staining after 5 weeks of cultivation.

For further characterizations, osteogenic and adipogenic differentiations of hBM- and
hAD-MSCs were induced using the following protocol. In this stage, 1×10⁴ cells
per well (TPP, Switzerland) were seeded in 6-well plates and treated with conductive
medium for 21 days. The media of wells were changed every three days. At 50% confluency,
the medium was supplemented with 0.5 µM ascorbic acid-2-phosphate (Sigma-Aldrich, USA), 1
µM dexamethasone (Stem Cell Technologies, Canada), and 10 mM β-glycerophosphate
(Sigma-Aldrich, USA) for osteogenic induction. The cells were analyzed for mineralization
using alizarin red (Sigma-Aldrich, USA) staining. To induce adipogenic differentiation,
cells were incubated with complete medium including 50 µg/ml indomethacin (Sigma-Aldrich,
USA), 100 nM dexamethasone (Sigma-Aldrich, USA), insulin (SigmaAldrich, USA), and
3-isobutyl-1-methylxanthine (SigmaAldrich, USA). Finally, the cells were analyzed for
lipid content by oil-red (Sigma-Aldrich, USA) staining.

Cells were plated at density of 5 × 10⁴ cells/well in 6-well plates in LG-DMEM containing 10% FBS, allowed to adhere overnight, and replaced with LG-DMEM containing 10% FBS and 1% PS and supplemented with 0.1 mM dexamethasone (Stemcell Technologies), 10 mM b-glycerol phosphate (Stemcell Technologies), and 100 mM ascorbate-2-phosphate (Stemcell Technologies). The medium was changed every 3 days. After 14–21 days, osteoblast differentiation was determined by alkaline phosphatase and von Kossa stainings.

Sera, obtained from each runner before and after the competition, were mixed in two pools named PRE RUN and POST RUN sera, respectively. In order to study the induction of osteogenic and adipogenic differentiation, respectively, before and after physical exercise, we treated the BM-hMSC cell line (human bone marrow-human mesenchymal stem cells, PromoCell, Heidelberg, Germany) with pooled PRE RUN and POST RUN sera [5 (link)].
Pooled sera were added to the above cell line at 10% final concentration. Cells were plated at a density of 5 × 10⁴ cells per well into 24-well plates and cultured up to 21 days. In particular, osteogenic differentiation was performed with osteogenic medium containing osteogenic stimulatory supplements (15%, Stemcell Technologies Inc., Vancouver, Canada), 10⁻⁸ M dexamethasone, 3.5 mM β-glycerophosphate, and 50 μg/ml ascorbic acid (Stemcell Technologies Inc.). The adipogenic differentiation was performed by using 0.5 mM isobutylmethylxanthine, 200 μM indomethacin, 10⁻⁶ M dexamethasone, and 10 μg/ml insulin in basal medium. Chondrogenic differentiation was performed by culturing hMSCs with mesenchymal stem cell chondrogenic differentiation medium (PromoCell, Heidelberg, Germany). For osteogenic, adipogenic, or chondrogenic differentiation, the medium was changed every 3 days after initial plating.