Mesenchymal precursor stem cell viability and proliferation in monolayer culture. (a) Bar graph showing the concentration-dependent effect of pentosan polysulfate (PPS) on mesenchymal precursor stem cell (MPC) proliferation. Primary MPCs were cultured in 24-well plates in growth media supplemented with PPS at the indicated concentrations (n = 3). On days 1 (black columns), 3 (white columns) and 6 (hatched columns), the cells were incubated with the tetrazolium salt WST-1 for 2 hours at 37°C to produce a formazan dye. Absorbance at 450 nm for each time point is shown for the indicated concentrations of PPS. Data expressed as mean ± standard error of the mean. A statistically significant increase in proliferation was observed on day 6 at concentrations of PPS in excess of 1 μg/ml (*P < 0.01). (b) Concentration effects of PPS on DNA synthesis in 3-day monolayer cultures of MPCs (n = 3). Data expressed as mean ± standard deviation. Significant elevation in DNA synthesis was observed at PPS concentrations of 1, 2.5 and 5 μg/ml relative to control cultures (P < 0.01). (c) Flow cytometric analysis profiles showing the inhibitory effects of different concentrations of PPS on MPC apoptosis induced by the addition of a combination of 30 ng/ml IL-4 plus 30,000 U/ml IFNγ. Following 5-day culture, cells were harvested by trypsinisation and viabilities assessed by Annexin V staining (n = 3 per PPS concentration). An average 38% reduction in apoptosis was observed when MPCs were cultured at PPS concentrations >1 μg/ml. DPM, Decays Per Minute.