Figure 3. Osteogenic, chondrogenic and adipogenic differentiation of DPPCs.

<br> <p>Osteogenic, chondrogenic and adipogenic differentiation of DPPCs. Tri-lineage differentiation analysis was compared between high proliferative capacity, A3 (30 PDs) and low proliferative capacity DPPCs, A1 (11 PDs) and B1 (15 PDs). (A) Osteogenesis was confirmed for each DPPC population by the detection of alizarin red staining for mineralised calcium nodules. No staining was observed in control DPPC cultures in the absence of osteogenic medium. RT-PCR analysis of osteogenic markers (right panel) indicated increased osteocalcin (OCN) and osteopontin (OPN) expression for DPPCs cultured in osteogenic media. Cells maintained in both osteogenic and control media expressed Runx2. (B) Chondrogenesis was only particularly evident with high proliferative capacity, A3, which exhibited positive Safranin O staining for high proteoglycan content. No Safranin O staining was observed with the low proliferative capacity DPPCs, A1 and B1. (C) Adipogenesis was only particularly evident with high proliferative capacity, A3, which exhibited positive Oil Red O staining for intracellular lipid-rich vacuole accumulation. No Oil Red O staining was observed with the low proliferative capacity DPPCs, A1 and B1; or in control cultures in the absence of adipogenic medium. RT-PCR analysis of adipogenic markers (right panel) indicated increased expression of the early adipogenic marker, PPARγ, in all DPPCs maintained in both adipogenic and control cultures; whilst the late adipogenic marker, lipoprotein lipase (LPL), was only expressed in high proliferative capacity, A3, in adipogenic media. Scale bars = 100 µm. β-actin served as the reference housekeeping gene.