Stempro adipogenic differentiation kit

All three MSCs strains (fourth or fifth passage) were introduced in vitro into osteogenic (21 days), chondrogenic (21 days), and adipogenic (18 days) differentiation with respective specific media using StemPro^® Osteogenesis Differentiation Kit, StemPro Chondrogenic Differentiation Kit, and StemPro Adipogenic Differentiation Kit media (Gibco; Invitrogen, Grand Island, NY) and a 12-well cell culture plate (Corning^® Costar^®; Sigma‐Aldrich, St. Louis, MO) (5 × 10³ cells/well). The three types of media were prepared according to manufacturer data sheets.

Primary MSC cultures were cultured in an adipogenic induction medium for eighteen days. After this period, we observed the morphological changes and the formation of intracellular lipid vesicles in the cultured cells.
In a 12-well plate, the cells were seeded at the same density in triplicate (5 × 10³ cells). After 24 hours of culture in basal culture medium, the culture medium was changed to the specific adipogenic culture medium supplemented with growth factors (StemPro® Adipogenic Differentiation Kit; Gibco Invitrogen, Grand Island, NY).
For evaluation, the adipogenic induction medium was removed from the cell cultures, and the cells were stained with oil red (Oil Red O, Sigma Aldrich, St. Louis, MO). For the staining, the wells were washed twice with PBS (Gibco Invitrogen, Grand Island, NY) and fixed with 60% isopropanol (Sigma Aldrich, St. Louis, MO) for five minutes at room temperature. After fixation, the cells were stained with oil red (0.5 mg/ml) for 15 minutes under light at room temperature. For the final wash, 60% isopropanol was used once and distilled water twice.
After the staining of the lipid vesicles, the observation of cellular structures was carried out under inverted microscopy (Olympus CKX31).

For adipogenic differentiation, ADSCs in passage 4 were seeded at 10,000 cells/cm², and allowed to grow to 90% confluence. On Day 5 post-seeding, the adipogenic induction was initiated by replacing basal growth media with adipogenic differentiation media (Stempro adipogenic differentiation kit, Invitrogen) in treatment wells, while control ADSCs was maintained in their growth media. The adipogenic media were individually supplemented with appropriate doses of the Wnt regulators LiCl, BIO, and sFRP4 (as standardised by the MTT cell viability assay) during the period of differentiation. Later, a combination treatment containing both BIO and sFRP4 was also performed to observe the interaction between the Wnt activator and antagonist together on adipogenesis. The media were replenished every 3–4 days. Morphological observations for accumulation of lipid droplets in all treatment conditions were recorded and photographed at regular intervals throughout the adipogenic differentiation period. On Day 7 post-induction, cells were analysed for lipid accumulation by Oil red O staining and were harvested to detect the protein expression of adipogenic markers. 24 well-plates were used for histochemical staining while 6 well-plates were used for protein extraction.

Osteogenic differentiation was performed utilizing a StemPro osteogenic differentiation kit (ThermoFisher, UK) as per the manufacturer’s instructions. OMLP-PC cell lines and cell strains were seeded at a density of 5 × 10³ cells/well. Cells were cultured under normal conditions for 24 h before the medium was removed and replaced with an osteogenic induction medium. The medium was changed every 48 h for 14 days. After 14 days cells were fixed with 4% (v/v) PFA for 20 minutes before staining with 40 mM alizarin red staining solution for 5 minutes. Cell monolayers were extensively washed with ddH₂O. Images were obtained using a brightfield inverted microscope. Data is presented from one of 3 biological repeats.
Adipogenic differentiation was performed utilizing a StemPro adipogenic differentiation kit (ThermoFisher, UK) as per the manufacturer’s instructions. OMLP-PC cell lines and strains were seeded at a density of 3 × 10⁴ cells/well. cultures were re-fed every 48 h for 14 days. After 14 days of culture, cells were washed with PBS and fixed with 4% (w/v) PFA for 20 minutes. Cells were stained with both LipidTox green lipid stain (1:100; Fisher Scientific, UK) and 10 μM Hoechst 33342 in PBS for 30 minutes. Cells were imaged using a Leica SP5 confocal microscope. Data is presented from one of 3 biological repeats.

For the adipogenic differentiation, cells were plated in 24-well plates at 1.5 × 10⁴ cells/cm² in complete medium. Twenty-four hours later, SHEDs were washed in PBS and cultivated in Stem Pro Adipogenic Differentiation Kit medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The medium was refreshed every two days. After 15 days in culture, cells were washed three times in PBS and fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) for 15 minutes. After washing twice in PBS, fixed cells were incubated with 0.16% Oil Red O (Sigma, Saint Louis, MO, USA) solution for 15 minutes. After three rinses in PBS, cells were observed and photographed under a phase-contrast inverted microscope (Axiovert 40C, Carl Zeiss, Oberkochen, Germany). The Oil Red dye was eluted from the cells using isopropanol and the absorbance at 500 nm was determined in the Epoch Microplate Spectrophotometer (BioTek, Vermont, USA). The mRNA for the adipogenic FABP4 gene was analyzed using RT-PCR, as detailed below.

For osteogenic and adipogenic differentiation, CBti MSCs at the end of P4 were seeded at a density of 4,000 cells/cm2 on cell culture coverslips (Thermo Fisher Scientific) and arranged in 24-well plates (Falcon®, Corning, Corning, NY, USA) in the presence of standard growth medium. At 70–80% cell confluence, the medium was replaced with specific differentiation media, then renewed every 3–4 days for 21 days. To induce adipogenic differentiation, cells were evaluated using the StemPro® Adipogenic Differentiation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The presence of intracellular lipid droplets was detected by standard staining with Oil Red O (Diapath, Bergamo, Italy). In parallel, cells were also grown using the StemPro® Osteogenic Differentiation Kit (Thermo Fisher Scientific). The presence of calcium deposits representing osteogenic differentiation were evaluated by von Kossa staining (Sigma-Aldrich). Cells were fixed with 10% formalin for 5 min at room temperature, incubated with 1% silver nitrate solution for 15 min and exposed to ultraviolet light for 2 h. Coverslips were rinsed with distilled water and 5% sodium thiosulfate to remove unreacted silver.