Fig. 2

GBM subpopulations undergo state transitions in a non-hierarchical manner. a Experimental setup of FACS sorting and functional analysis performed on 16 subpopulations (NCH644). See Supplementary Fig. 2E for gating strategy. b Self-renewal test, including sphere formation (mean + /− SEM) and sphere diameter (Box limits indicate the 25th and 75th percentiles and center lines show the medians as determined by R software; whiskers represent the extreme low and high observed values, unless those are above 1.5 times interquartile range (IQR)—thereby whiskers are limited to 1.5 IQR. All outlying data points are represented by dots). Bulk cells were used as control (CTR). No statistical difference observed, except for P11 passages 1 vs. 2 and 1 vs. 3 (*p-value ≤ 0.05, Kruskal–Wallis test). c Proliferation test (mean doubling time + /− SEM). No statistical difference observed. d Multipotency test. Marker expression over time: FACS-sorting day 0 (D0) and re-phenotyping after 20 (D20), 30 (D30), and 70 (D70) days in culture. The order of subpopulations in alluvial plots is based on highest to lowest percentage at each time point. See an alternative representation in Supplementary Fig. 5A for column chart and Supplementary Data 2A for statistics (color code in Fig. 1e). e Markov modeling of state transitions between 16 subpopulations. Arrows represent predicted direct state transitions between subpopulations, thickness of lines corresponds to transition probabilities. See Supplementary Fig. 5C for transition matrix. f Proportions of subpopulations predicted in equilibrium state is similar to initial culture. g Validation of Markov modeling. FACS-sorted admixtures (time 0) were re-phenotyped at the predicted equilibrium time (39 days) showing the accuracy of the mathematical model. See Supplementary Data 1A for statistics. h Predicted time to reach equilibrium for each subpopulation