Oil red staining

As formerly mentioned, the third passage cells were collected for adipogenic differentiation and cultured in MesenCult medium along with 10% adipogenic stimulatory supplements (both from STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) with a density of 2×10³ cells in a milliliter of medium within the two-chamber cultivation slides according to the manufacturer's instructions. The cells were cultured for 3-4 weeks. Half of the media were changed only when the ambient color turned yellow. When the cells exhibited appropriate morphological changes, they were analyzed by using Oil Red staining (Sigma). In order to perform the staining, the cells were incubated in 4% formalin containing 1% calcium chloride for one hour. The cells were then stained with Oil Red O solution for 10-15 minutes, and in ethanol 70% for one minute; and then, washed with distilled water.

Adipogenic, chondrogenic, and osteogenic differentiation of SHED and ASC were carried out as previously described at the third passage [19 (link)]. The cultures were initiated at a density of 1000 cells/cm² in six-well plates and grown until confluence and then subjected to differentiation into adipogenic, chondrogenic, and osteogenic lineages. Briefly, the adipogenic lineage was initiated by inducing the cells with 10% FBS, 200 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine (IMBX), 10 μg/mL insulin, and 1 μM dexamethasone (all from Sigma-Aldrich). Lipid droplets were visualized using oil red staining (Sigma-Aldrich). For chondrogenic differentiation, cells were cultured in medium supplemented with ITS+1 (Sigma-Aldrich), 50 μM L-ascorbic acid 2-phosphate, 55 μM sodium pyruvate (Invitrogen), 25 μM L-proline (Sigma-Aldrich), and 10 ng/mL transformation growth factor-β (TGF-β; Sigma-Aldrich). Assessment of proteoglycan accumulation was visualized by Alcian Blue staining (Sigma-Aldrich). Osteogenic differentiation was stimulated in a 3-week culture period in medium supplemented with 10% FBS, 10–7 M dexamethasone, 10 mM glycerol phosphate (Fluka, Buchs, Switzerland), and 100 μM L-ascorbic acid 2-phosphate. The assessment of calcium accumulation was visualized using von Kossa staining (Sigma-Aldrich).

Cells at P3 were seeded in a 24-well tissue culture plate with a density of 3.7 × 10⁴ cells in each well and supplemented with complete DMEM/F12 Media, 10% FBS, and 1X antibiotic/antifungal. The stromal media was removed from each well every 2–3 days till it reached 100% confluency and adipogenic induction media was added in HG-DMEM (Lonza, Switzerland) for 21 days while there were Ad-MSCs cultured in growth complete DMEM/F12 medium deprived of differentiation factors serve as a control. After this specified period, the cells were washed with PBS and fixed by incubation in 10% buffered formalin for 10 min at room temperature (RT). The cell monolayer was stained with a working solution of 0.5% Oil Red staining (Sigma-Aldrich) which appeared as lipid vacuoles under a microscope.

Adipogenic, osteogenic, and chondrogenic differentiation experiments were performed following the instructions of human mesenchymal stem cell functional identification kit (R&D systems, Inc., Wiesbaden, Germany).
For adipogenic differentiation, MSCs were seeded into a 24-well plate at the density of 3.7 × 10⁴ cells/well, and maintained in culture medium until 100% confluency. Cells were then exposed to adipogenic differentiation medium for 3 weeks. Lipid droplets of the resultant differentiated cells were detected using Oil red staining (Sigma-Aldrich, St. Louis, MO, USA).
For osteogenic differentiation, 4.2 × 10³ cells were seeded per well. When cells reached 50–70% confluency, the medium was replaced with osteogenic differentiation medium and kept for 3 weeks. To assess osteogenic differentiation, Alizarin Red S staining (Sigma-Aldrich) was performed for the calcium-rich extracellular matrix.
For chondrogenic differentiation, 2.5 × 10⁵ cells resuspended in chondrogenic differentiation medium were centrifuged for 5 min at 200×g in a 15-mL conical tube (Corning). After 3 weeks, a chondrogenic pellet was harvest and fixed in 4% paraformaldehyde (PFA). Cryosection was performed and sections were stained with Alcian Blue (Sigma-Aldrich).

We performed adipogenic, osteogenic and chondrogenic differentiation using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems Inc., MN, USA). The induction processes for the three lineages were performed according to the manufacturer’s instructions. Noninduced control WJ-MSCs were fed with complete growth medium (10 % FBS LG-DMEM) on the same schedule of each investigated lineage. Regarding adipogenic differentiation, after about 7 days lipid vacuoles started to appear in the induced cells. The detection of the resultant differentiated cells was carried out using Oil Red staining (Sigma-Aldrich, USA). For the osteogenic lineage, cells changed from spindle shaped to cuboidal shaped during differentiation, and differentiation was confirmed by Alizarin Red-S staining (Sigma-Aldrich, USA) for the calcium-rich extracellular matrix. Finally, regarding chondrogenic induction, cells changed from spindle shaped to cuboidal shaped during differentiation, and differentiation was confirmed by Alcian 8GX blue staining (Sigma-Aldrich, USA) for sulfated proteoglycan.

Adipogenic differentiation was induced using adipogenic differentiation medium (regular growth medium supplemented with 50 mg/ml ascorbic 2-phosphate (Sigma-Aldrich), 10 nM dexamethasone (Sigma-Aldrich) and 10 mM ß-glycerol phosphate (Sigma-Aldrich) at P1 and P2. After 3 weeks, the adipogenesis was assessed using Real-time PCR (see below) and Oil Red staining (Sigma Aldrich™). Images were captured using a digital camera system (Olympus DP72) connected to an inverted optical microscope (Olympus CKX41-Olympus Optical CO).