Cytokine combinations for human blood stem cell expansion induce cell type- and cytokine-specific signaling dynamics.

How hematopoietic stem cells (HSCs) integrate signals from their environment to make fate decisions remains incompletely understood. Current knowledge is based on either averages of heterogeneous populations or snapshot analyses, both missing important information about the dynamics of intracellular signaling activity. By combining fluorescent biosensors with time-lapse imaging and microfluidics, we measured the activity of the extracellular signal-regulated kinase (ERK) pathway over time (i.e. dynamics) in live single human umbilical cord blood HSCs and multipotent progenitor cells (MPPs). In single cells, ERK signaling dynamics were highly heterogeneous and depended on the cytokines, their combinations, and cell types. ERK signaling was activated by SCF and FLT3L in HSCs, but by SCF, IL3 and GCSF in MPPs. Different cytokines and their combinations led to distinct ERK signaling dynamics frequencies, and ERK dynamics in HSCs were more transient than those in MPPs. A combination of 5 cytokines recently shown to maintain HSCs in long-term culture, had a more-than-additive effect in eliciting sustained ERK dynamics in HSCs. ERK signaling dynamics also predicted future cell fates. E.g. CD45RA expression increased more in HSC daughters with intermediate than with transient or sustained ERK signaling. We demonstrate heterogeneous, cytokine- and cell type- specific ERK signaling dynamics, illustrating their relevance in regulating HSPC fates.