3.3. In vitro tri-lineage differentiation and histological analysis
First-passage of cells cultures were subjected to in vitrodifferentiation assays in order to evaluate their mesenchymal multipotent potential. The shape of the cells changed to a spindle-shaped morphology. Cells demonstrated a potential to differentiate to varying degrees as shown by positive osteogenic, adipogenic, and chondrogenic staining.
In osteogenesis differentiation, cells seeded at 5×104cells per well were seeded in 6 well plates in triplicate in culture medium. After 24 hours, confluent cell populations (90% confluency) from SPFP-MSCs were promoted by treating cells cultures with osteogenic induction medium (OM) containing AG at different concentrations (1, 5, and 10 μM) while cells maintained in OM were used as controls. After 3 weeks, cells were stained with Alizarin Red S to confirm the accumulation of calcium (Fig. 3A). Furthermore, their osteogenic potency was confirmed by examining mRNA expression of several osteogenic-specific marker genes including Runt-related transcription factor 2 (Runx2 ) and Osteopontin (OPN ) (Fig. 4A). OM containing AG10 µM profoundly upregulated the mRNA expression of osteogenic markers compared to control groups of cells.
Using these isolated cells from SPFP, we further determined the adipogenic potential of cells with cultures using adipogenic induction medium (AM), these cells became more elongated and flatter. 5×104 cells per well were seeded in 6 well plates in triplicate in expansion medium. After 24 hours, confluent cell populations (90% confluency) from SPFP were promoted by treating expansion medium cultures with AM containing AG at different concentrations (1, 5, and 10 μM). SPFP-MSCs were observed, especially at the sites where lipid droplets became visible and compared with SPFP-MSCs maintained in AM for 3 weeks as control groups. After 3 weeks, lipid droplets were confirmed by Oil Red O staining to visualize accumulated cytoplasmic lipid rich vacuoles (Fig. 3B). According to real-time PCR assay, expression levels of adipogenic-specific genes for Peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2 ) and Lipoprotein lipase (LPL ) (Fig. 4B), the mRNA expression showed significantly lower in induced groups with AM containing AG at all concentrations (1, 5, and 10 μM) than in control groups.
Further analysis made was to evaluate chondrogenic differentiation capacity (pellets) of isolated MSCs from SPFP. Pellets were either introduced to chondrogenic medium (CM) containing AG at different concentrations (1, 5, and 10 μM) or CM with 1% FBS. The cells had grown and formed dense spheroidal pellets (Fig. 3C). After 21 days, the spheroidal pellets were easily processed for staining with H&E (Fig. 3D), Toluidine blue (metachromatic) (Fig. 3E) and Alcian blue (Fig. 3F). The results showed morphology as demonstrated by H&E, the presence of glycosaminoglycans as demonstrated by Toluidine blue and the presence of enrichment of sulphated proteoglycans as illustrated by Alcian blue staining. The mRNA expression levels of chondrogenic-specific genes including SRY-related transcription factor 9 (Sox9 ) and Aggrecan (Fig. 4C) showed significantly higher in the induction pellets containing AG at all concentrations (1, 5, and 10 μM) than in the CM as control group.