Erythropoietin directly remodels the clonal composition of murine hematopoietic multipotent progenitor cells

The cytokine erythropoietin (EPO) is a potent inducer of erythrocyte development and one of the most prescribed biopharmaceuticals. The action of EPO on erythroid progenitor cells is well established, but its direct action on hematopoietic stem and progenitor cells (HSPCs) is still debated. Here, using cellular barcoding, we traced the differentiation of hundreds of single murine HSPCs, after ex vivo EPO exposure and transplantation, in five different hematopoietic cell lineages, and observed the transient occurrence of high-output myeloid-erythroid-megakaryocyte-biased and myeloid-B-cell-dendritic cell-biased clones. Single-cell RNA sequencing analysis of ex vivo EPO-exposed HSPCs revealed that EPO induced the upregulation of erythroid associated genes in a subset of HSPCs, overlapping with multipotent progenitor (MPP) 1 and MPP2. Transplantation of barcoded EPO-exposed MPP2 confirmed their enrichment in myeloid-erythroid-biased clones. Collectively, our data show that EPO does act directly on MPP independent of the niche and modulates fate by remodeling the clonal composition of the MPP pool.

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The main experimental cohort (4-week timepoint at EPO concentration 1000 ng/ml) was repeated twice with the exact same sorted and analyzed cell populations ( Figure 1 and For each of the experimental cohorts, barcode analysis entailed the analysis of two technical replicates for every sample as in Naik et al 2013, DOI 10.1038/nature12013. More in detail, cell subsets collected through FACS were split into two fractions after cell lysis and processed independently throughout barcode amplification by PCR and sequencing. After sequencing, these technical replicates were used to remove erroneous barcodes based on correlation between them, and the mean of the technical replicates was further used for analysis. These steps are further detailed in the Material and Methods section. The experimental cohorts were started with 4-5 mice per experimental condition (EPO vs control). The final sample sizes indicated in the respective figure or figure supplement legends result from 1) mice dying before readout 2) mice with an engraftment of donor cells of under 5% at read-out 3) filtering during barcode sequencing data analysis. More in detail, mice for which one or more cell subset samples did not pass the filtering steps as detailed in the Material and Methods section, could not be included in the analysis.

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