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              <p>Human erythropoiesis is a complex process leading to the production of 2.5 million red blood cells per second. Following commitment of hematopoietic stem cells to erythroid lineage, this process can be divided into two distinct stages: erythroid progenitor differentiation and terminal erythropoiesis. We recently resolved the heterogeneity of erythroid progenitors in human bone marrow into four different subpopulations termed EP1 to EP4. Here, we characterized the growth factor(s) responsiveness of the progenitor populations in terms of proliferation and differentiation. The four progenitor populations exhibited differential dose-dependent responses to SCF and EPO, while their growth and differentiation was IL-3 independent. Using mass spectrometry-based proteomics, we quantified the expression levels of ~5,500 proteins from EP1 to EP4 (Figure 1). Our functional analyses highlighted dynamic changes in cell cycle in these populations with an acceleration of the cell cycle during progenitor differentiation. The cell cycle is controlled at multiple levels; one of them being the G1 to S transition. Using EdU and DNA content to resolve different phases of the cell cycle, we noted a decrease in the percentage of cells in the G1 phase and a concomitant increase in S phase as EPs differentiated from EP1 to EP4. While the proportion of cells in G2/M progressively increased from EP1 to EP3, a significant decrease was noted as EP3 transitioned into EP4. Finally, by measuring the mean fluorescence intensity (MFI) of the S-phase, we found that the S phase speed increased from EP1 to EP4. Mechanistically, E2F4 has recently been identified in fast-cycling cells and in murine erythropoiesis, and we noted a specific 3-4-fold increase in E2F4 protein copy numbers from EP1 to EP4 (Figure 2), along with its binding to cell cycle-related genes at the EP3 stage, suggesting a potential role for this E2F member in the regulation of their proliferation. Finally, we assessed the relative contributions of oxidative phosphorylation and glycolysis required to provide ATP to these fast-cycling cells. Both oxidative phosphorylation and glycolysis provide ATP needed for erythroid progenitors to proliferate and differentiate towards terminal stages. Taken together, our data provide comprehensive insights into normal human erythroid progenitor biology and have implications for understanding ineffective erythropoiesis due to erythroid progenitor defects.</p>
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          <orgName>Université Paris Cité</orgName>
          <orgName type="acronym">UPCité</orgName>
          <date type="start">2020-01-01</date>
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              <addrLine>85 boulevard Saint-Germain75006 Paris</addrLine>
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            <address>
              <addrLine>Stanford University</addrLine>
              <country key="US"/>
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            <ref type="url">https://med.stanford.edu/</ref>
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          <orgName>Stanford University</orgName>
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            <address>
              <addrLine>450 Serra Mall, Stanford, CA 94305-2004</addrLine>
              <country key="US"/>
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            <ref type="url">https://www.stanford.edu/</ref>
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        <org type="institution" xml:id="struct-147527" status="VALID">
          <orgName>Hofstra University [Hempstead]</orgName>
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              <country key="US"/>
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            <ref type="url">http://www.hofstra.edu/home/</ref>
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