Conditions affecting the human neurosphere assay. (A) Dissociated neurosphere cells were plated at 500 cells per well in either 1% N2 or 2% B27 supplement, together with 0–100% neurosphere-conditioned medium. Relative to no conditioned medium (0%), a significant increase in neurosphere formation was observed for 50% conditioned medium only in the presence of N2 supplement. In comparison, 50% conditioned medium significantly reduced neurosphere formation in the presence of B27 supplement. Notably, human neurosphere formation was significantly increased by 2% B27 supplement compared to 1% N2 supplement for all concentrations of conditioned medium, as determined by a one-way ANOVA with Tukey post-test (p < 0.001, n = 2). (B) Graph showing the result of the limiting dilution assay, with cells from dissociated human neurospheres being plated at 10–60 cells per well in 2% B27 supplement. The number of non-responding wells (i.e. without a neurosphere present) was quantified as a fraction of the total number of wells 21 days later. Linear regression was used to produce to the line of best fit. Unless otherwise noted, statistics were determined by a one-way ANOVA with Tukey post-test. n = 2 for all except for the limiting dilution assay, where n = 3. *p < 0.05.