STAT5-Regulated miRNA193b Controls Hematopoietic Stem and Progenitor Cell Expansion By Fine Tuning Cytokine Signaling

Hematopoietic stem cells (HSCs) regenerate the blood system life-long. Their number must be strictly controlled to avoid either exhaustion or excessive expansion leading to leukemia. Not only proteins but also regulatory RNAs are necessary to maintain HSCs. Hematopoiesis requires the right composition of microRNAs (miRNA), small, non-coding RNAs with the ability to simultaneously target multiple mRNAs for the orchestration of complex gene expression networks.

We identified miR193b to be induced in long-term repopulating (LT)-HSCs by the self-renewal promoting cytokine thrombopoietin via STAT5 signaling. To understand the physiological function of miR193b expression in HSCs, we generated a miR193b knock-out mouse model. These mice showed a selective expansion of the pool of functional LT-HSCs over time that were fully capable of multilineage blood reconstitution. MiR193b-deficient LT-HSCs were less quiescent than wildtype LT-HSCs in homeostasis, and were therefore more susceptible for repeated 5-Fluorouracil-induced stress. Further, they showed a higher expansion in vitro, not by changing the cell cycle length or death rate, but by slowing differentiation. Conversely, ectopic miR193b expression in LT-HSCs restricted their expansion and they were unable to repopulate the blood of lethally irradiated recipients. Long-term videomicroscopy-based tracking of LT-HSCs and their progeny revealed an increased cell death and slowed cell cycle upon miR193b expression. MiR193b-deficient hematopoietic stem and progenitor cells (HSPCs) showed a higher basal level of cytokine receptor signaling (pSTAT5 and pAKT), and an extended signal after cytokine stimulation, determined by phosphoflow cytometry at single cell resolution and in vitro PamGene kinase activity assays. Differential next generation RNA-sequencing of HPSCs with and without miR193b confirmed the regulation of signaling-related gene clusters and revealed potential mRNA targets. We identified the receptor tyrosine kinase ckit as a direct target of the miR193b, suggesting that miR193 modulates cytokine receptor expression and thereby signaling in HSCs.

Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR193b upon self-renewal promoting thrombopoietin – Mpl - STAT5 signaling, that in turn restricts cytokine signaling by targeting cytokine receptor expression. Thereby, we shed light into the essential function of miR193b as a modulator of HSC self-renewal.