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.