Fig. 4

Effects of prolonged in vitro culture expansion on DPSC ageing characteristics. a Digital images representing the extent of senescence-associated β-galactosidase staining by DPSCs at early PDs (7–8 PDs) and towards the end of their respective proliferative lifespans (A1 at 23 PDs, A3 at 83 PDs and B1 at 34 PDs). At 7–8 PDs, all DPSCs were morphologically similar, with the characteristic MSC morphology and were negative for β-galactosidase staining. At PDs towards the later stages of their respective proliferative lifespans, higher percentages of cells stained positive for β-galactosidase, which were larger, more stellate-like and with prominent stress fibres. Scale bars = 100 μm. b Analysis of cellular surface area throughout the proliferative lifespan of A1, A3 and B1, using ImageJ^® Software (average ± SE, **p < 0.001). Significant increases in each DPSC population’s surface area were identified with proliferative lifespan. Only when high proliferative capacity, A3, reached senescence (85 PDs) were surface areas non-significantly different to senescent A1 populations at 22 PDs (p > 0.05). Senescent B1 surface areas at 35 PDs did not reach the cell area sizes of senescent A1 or A3 at 22 PDs and 85 PDs, respectively. c Gene expression of the MSC markers, CD73, CD90 and CD105, was gradually lost during in vitro expansion. CD105 was lost initially, based on expression levels in A1 (11 PDs) and A3 (24 PDs), whilst CD90 and CD73 expression was subsequently lost towards the end of each population’s respective proliferative lifespans. Whilst the premature loss of CD105 was not as apparent with B1, all three markers were lost towards the end of its proliferative lifespan (24 PDs). NA indicates that no cells were available for analysis at these PDs. β-actin served as the reference housekeeping gene