DPSC cultures differentiate into neural crest-derived mesenchymal lineages.

(A–D) After 21 days in osteogenic media, differentiated DPSCs were positive for BSP and OPN in a membranous pattern. (F) Cultures in adipogenic media showed lipid droplets-containing cells positive for Oil Red O. (H and J) Cultures in chondrogenic media showed cells in clusters positive for toluidine blue and COL II. (K) Mineralized nodules were observed in cells treated with the chondrogenic media after 21 days. The inset depicts a mineralized nodule in high magnification. (L) RT-PCR confirmed the staining results. For osteogenic differentiation (Osteogenic D21), RT-PCR showed expression of osteoblast- associated genes Runx2, Osx, Opn, Bsp, and Dmp1. For adipogenic differentiation, induced cells (Adipogenic D21) expressed Pparg2 and CEBPa, adipogenic transcription factors, as well as Leptin and Adipsin, markers of adipocytes. For chondrogenic differentiation, treated cells (Chondrogenic D21) expressed chondrocyte-associated genes Sox9 and Col2a1. (A, C, E, G, and I) Confluent undifferentiated cells cultured in stem cell media at day 21 (UD D21) expressed some of differentiation genes, but were negative by staining for all differentiation markers and Oil Red O. Cells were counterstained with hematoxylin. RNA isolated from mouse calvarial bone, adipose tissue, and femur was used as positive control for gene expression. Scale bars indicate 100 µm.