Fig. 2.

In vitro multilineage differentiation potential of PDLSCs cultured in the presence or absence of FBS under hypoxic or normoxic culture conditions. a Alizarin red S (ALZ)- and oil red O (ORO)-staining indicate mineralized deposits and lipid clusters, respectively, in SFM- or FCM-cells under normoxic conditions 4 weeks after culturing in the respective cytodifferentiation medium. In contrast, no positive staining was observed in either SFM- or FCM-cells under hypoxic conditions (3% O₂). Chondrogenic differentiation was achieved in all the PDLSC-pellet cultures after 4 weeks (Safranin O and Alcian blue); particularly, cytodifferentiation was enhanced under hypoxic culture conditions, as indicated by the intense immunoreactivity for type II collagen (Collagen II); scale bars 50 µm. b SFM and FCM cells also achieved osteogenic and adipogenic cytodifferentiation after 2 weeks of normoxic cultivation (Normo 2w), but failed to exhibit cytodifferentiation into either lineage after 2 weeks of hypoxic cultivation (3% O₂ 2w). Notably, switching the culture condition from hypoxia for 2 weeks to normoxia for 2 weeks resulted in the development of ALZ-positive mineralized nodules and ORO-positive lipid droplets in SFM and FCM cultures, respectively (3% O₂ 2w → Normo 2w). c Reverse-transcription polymerase chain reaction analysis revealed that 2-week-hypoxia-cultured PDLSCs that failed to undergo osteogenic (Os) and adipogenic (Ad) lineage differentiation exhibited higher expression of the stemness marker genes Oct3/4, Nanog, and Sox2 (3% O₂); after switching to normoxia, PDLSCs lost, or showed a lower expression of, stemness marker genes during cultivation for differentiation into both lineages (Normo)