1,25D3 effects on proliferation, apoptosis and cumulative population doublings.

A/B Measurement of proliferation capacity and apoptosis induction of hMSC from three different donors after stimulation with 1,25D3 for 24, 48 and 72 h. The proliferation rate was decreased time-dependently after 1,25D3 treatment (gray bars) compared to control cells (black bars), with significant effects after 48 h and 72 h (A). 1,25D3 stimulation showed the strongest reduction of apoptosis rates after 72 h 1,25D3 treatment (gray bars) compared to control cells (black bars, B). The results are shown as mean+SEM of three independent experiments, each normalized to its control and performed in triplicates. Cells from three different donors were used. (*, p<0.05; **, p<0.01;***, p<0.001, student's t-test). C The growth rates of cells were determined by population doublings at each subcultivation. Cumulative population doubling (CPD) was first determined for P2. Population doublings were observed for up to six cell passages. Persistent 1,25D3 supplementation resulted in a reduction of population doublings (gray bar), but the difference was not statistically significant. Independent experiments were performed using hMSC from several human donors. Control: P1, P2, n = 6; P3, P4, n = 5; P5, n = 3; P6, n = 2. 1,25D3: P1, P2, n = 6; P3, P4, n = 4; P5, P6, n = 1. D A representative example of CPDs of cells from one donor. 1,25D3 cultured cells exhibited lower CPDs compared to control hMSC (**, p<0.01, student's t-test). E The time required to reach subconfluence was determined. 1,25D3 treated cells (gray bars) needed more days until they reached subconfluence compared to control hMSC (black bars) (**, p<0.01, student's t-test). Independent experiments were performed using hMSC from several human donors. Control: P1, P2, P3, P4, n = 6; P5, n = 5; P6, n = 3. 1,25D3: P1, P2, P3, n = 6; P4, n = 5; P5, n = 4; P6, n = 1.