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

Abstract

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.

Analyzing signaling activity and function in hematopoietic cells

“…Long-term single-cell imaging and fate quantification of mammalian HSPCs has been accomplished in vitro, for instance demonstrating the production of blood cells from hemogenic endothelium (Eilken et al, 2009), the lineage instruction of hematopoietic progenitor cells by inflammatory cytokine signaling (Endele et al, 2017;Rieger et al, 2009), the asymmetric cell division of HSCs , or the direct regulation of the core hematopoietic transcription factor PU.1 by TNF and IL-1 in HSCs (Etzrodt et al, 2019;Pietras et al, 2016). This approach also enables the identification and quantification of signaling dynamics in HSPCs (Endele et al, 2014;Wang et al, 2021).…”

“…To enable the required high number of fine-tuned responses with the limited set of existing pathways, these are wired in complex interconnected networks. Their activity is nonlinear, context-dependent, dynamic, and heterogeneous, even between closely related individual cells ( Tay et al, 2010 ; Lane et al, 2017 ; Mitra et al, 2017 ; Wang et al, 2021 ), enabling a much-increased number of combinatoric and dynamic outputs ( Fig. 1 B ).…”

“…Long-term single-cell imaging and fate quantification of mammalian HSPCs has been accomplished in vitro, for instance demonstrating the production of blood cells from hemogenic endothelium ( Eilken et al, 2009 ), the lineage instruction of hematopoietic progenitor cells by inflammatory cytokine signaling ( Endele et al, 2017 ; Rieger et al, 2009 ), the asymmetric cell division of HSCs ( Loeffler et al, 2019 ), or the direct regulation of the core hematopoietic transcription factor PU.1 by TNF and IL-1 in HSCs ( Etzrodt et al, 2019 ; Pietras et al, 2016 ). This approach also enables the identification and quantification of signaling dynamics in HSPCs ( Endele et al, 2014 ; Wang et al, 2021 ). Combining protein analyses for detecting signaling activity with single-cell high-dimensional snapshot endpoint analysis methods like transcriptome sequencing ( Wu et al, 2014 ; Ziegenhain et al, 2017 ) enables the comprehensive detection of molecular regulators and target programs of signaling pathways.…”

Protocol to maintain single functional mouse hematopoietic stem cells in vitro without cell division

“…In Oedekoven et al., 2021 , we showed that single HSCs could have virus delivered to them without undergoing division ( Oedekoven et al., 2021 ). However, there are a wide range of other studies that could be imagined, including monitoring symmetric versus asymmetric partitioning of cellular components using high resolution imaging ( Filippi, 2021 ; Wang et al., 2021 ), single cell immunostaining ( Ema et al., 2007 ), or screening of compounds for improved survival or induction of cell division.…”

Fibroblast growth factor receptor 2 promotes the proliferation, migration, and invasion of ectopic stromal cells via activation of extracellular-signal-regulated kinase signaling pathway in endometriosis

“…Extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 are members of larger family of mitogen-activated protein kinases (MAPK) [ 18 ]. ERKs are cytoplasmic serine and threonine kinases that can be activated rapidly by numerous extracellular and intracellular stimuli, such as viral infection [ 19 ], oxidative stress [ 20 ], cytokines [ 21 ], and the tumor microenvironment [ 22–24 ]. In addition, the ERK signaling pathway is known to be involved in the development of various human diseases, including neurodegenerative diseases [ 25 ], heart diseases [ 26 ], renal fibrosis [ 27 ], and cancer [ 22–24 ].…”