Differentiation of TDSCs into osteogenic lineage was confirmed by matrix mineralization and significant upregulation of bone markers SPP1, RUNX2, and BGLAP. Furthermore, adipogenic differentiation was shown by Oil red-O staining of lipid droplets within cells and by increases in aP2 gene expression, which is a late marker for adipocyte  but not PPAR[gamma], which induced early during adipocyte differentiation .
(c) Graph showing the osteogenic gene (RUNX2, SPP1, and BGLAP) expression compared between osteogenic medium and its respective basal cultures.
Real-time RT-PCR analysis demonstrated elevated expression of BGLAP
mRNA (h) and ALP mRNA (i) in BMP6-treated cells (** P < 0.01, *** P < 0.0001).
After 14 days in hPL-supplemented media, only BH samples cultured under osteogenic conditions showed significant changes in expression; ALPL and BGLAP were both upregulated with a 5-fold change and Runx2 was upregulated by 12-fold over undifferentiated commercial ASCs.
All of the osteogenic transcription factors analyzed (ALPL, RUNX2, and BGLAP) were significantly upregulated in comparison to cells grown in basal media supplemented with FBS.
For osteogenesis, collagen type I alpha 1 (COL1A1; Hs00164004-m1), alkaline phosphatase placental type (ALP; Hs03046558_s1), runt-related transcription factor 2 (RUNX2; Hs00231692_m1), and bone gamma-carboxyglutamate protein (BGLAP; Hs01587814_g1) were used.
In contrast, the gene expressions of RUNX2 and BGLAP genes in cells from the knee were significantly higher than those in cells from the hip (3 donors, Figure 6(c); P < 0.01 (RUNX2), P < 0.01 (BGLAP), Mann-Whitney U test).