Figure 2.
ERK signaling dynamics in human HSCs are dependent on cytokine identity and combinations. (A) Workflow of classifying clusters of different ERK signaling dynamics from pooled (n = 1508) single-cell time series of both HSCs and MPPs. (B) Hierarchical clustering of time series of responders (n = 769) using dynamic time warping and Ward’s linkage method, resulting in 3 major clusters (transient, intermediate, and sustained) that are color coded. Gray bar at the top indicates the timing of cytokine presence, with arrows denoting the timing of stimulation and refreshment. (C) Population average of ERK signaling dynamics across 3 clusters as identified in panel B. Gray band indicates the 95% confidence interval of mean. (D) ERK signaling dynamics in single HSCs upon the stimulation with 12 different cytokine combinations are shown as heatmap (top panel) and time series (middle panel). For heatmaps, cells are organized according to the identified 4 clusters (nonresponder, transient, intermediate, and sustained) and sorted based on cumulative ERK signal (no to high). Heatmap heights are stretched to compensate for different cell numbers analyzed and best illustrate the frequencies of 4 clusters (scaled to 100%). For time series, gray curves represent individual cells, and black curve represents the population average. The 2 dashed lines denote values of 0 (bottom) and mean + 2xSD of the baseline measurements (top). Gray bar at the top indicates the timing of cytokine presence. Distributions of 4 clusters across 12 cytokine combinations (bottom panel) are calculated within individual experiments and shown as mean ± SD from 3 or 4 independent experiments (see supplemental Figure 5B for P values determined by pairwise Student t test with FDR correction). Black solid circles indicate the presence of cytokines. Number of independent experiments (N) performed and single cells (n) analyzed are shown. Two or 3 pooled UCB units were used per experiment. (E) 5F has a “more-than-additive” effect in inducing sustained ERK signaling dynamics. Shown are percentage of cells exhibiting sustained ERK activity upon the stimulation with 5F and the arithmetic sum of the corresponding 5 individual cytokine stimulations from 3 independent experiments (1-tailed, paired Student t test).

ERK signaling dynamics in human HSCs are dependent on cytokine identity and combinations. (A) Workflow of classifying clusters of different ERK signaling dynamics from pooled (n = 1508) single-cell time series of both HSCs and MPPs. (B) Hierarchical clustering of time series of responders (n = 769) using dynamic time warping and Ward’s linkage method, resulting in 3 major clusters (transient, intermediate, and sustained) that are color coded. Gray bar at the top indicates the timing of cytokine presence, with arrows denoting the timing of stimulation and refreshment. (C) Population average of ERK signaling dynamics across 3 clusters as identified in panel B. Gray band indicates the 95% confidence interval of mean. (D) ERK signaling dynamics in single HSCs upon the stimulation with 12 different cytokine combinations are shown as heatmap (top panel) and time series (middle panel). For heatmaps, cells are organized according to the identified 4 clusters (nonresponder, transient, intermediate, and sustained) and sorted based on cumulative ERK signal (no to high). Heatmap heights are stretched to compensate for different cell numbers analyzed and best illustrate the frequencies of 4 clusters (scaled to 100%). For time series, gray curves represent individual cells, and black curve represents the population average. The 2 dashed lines denote values of 0 (bottom) and mean + 2xSD of the baseline measurements (top). Gray bar at the top indicates the timing of cytokine presence. Distributions of 4 clusters across 12 cytokine combinations (bottom panel) are calculated within individual experiments and shown as mean ± SD from 3 or 4 independent experiments (see supplemental Figure 5B for P values determined by pairwise Student t test with FDR correction). Black solid circles indicate the presence of cytokines. Number of independent experiments (N) performed and single cells (n) analyzed are shown. Two or 3 pooled UCB units were used per experiment. (E) 5F has a “more-than-additive” effect in inducing sustained ERK signaling dynamics. Shown are percentage of cells exhibiting sustained ERK activity upon the stimulation with 5F and the arithmetic sum of the corresponding 5 individual cytokine stimulations from 3 independent experiments (1-tailed, paired Student t test).

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