Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production

Abstract

Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.

Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

“…6 are similar to what has been reported for non-transduced mouse CFU-E [35, 47] but with much higher yields. In the absence of both EPO and SCF, there was significant cell death which is also observed in wild type cell cultures [35]. The observed cytokine-mediated cell survival is an indication of conserved apoptosis regulation in IPE cells.…”

“…The individual and combined effects of EPO and SCF cytokines on IPE cell proliferation shown in Fig. 6 are similar to what has been reported for non-transduced mouse CFU-E [35, 47] but with much higher yields. In the absence of both EPO and SCF, there was significant cell death which is also observed in wild type cell cultures [35].…”

“…After 3.5 weeks of culture, a purified population of IPE cells was isolated by flow cytometry with the surface marker expression profile Mac-1 − Gr-1 − c-Kit (high/+) CD71 + (Additional file 1: Figure S6) which in enriched in CFU-Es [9, 35]. For the other cell isolation experiments, the cultures contained a majority of Mac-1 + and Gr-1 + positive cells after 3.5 weeks (Additional file 1: Figure S7) and erythroid cells were too rare to successfully isolate and culture.…”

Blood stem cell fate regulation by Delta-1–mediated rewiring of IL-6 paracrine signaling

“…For DL1 simulation, starved cells were transferred to plates precoated with DL1 (2.5 mg/mL) to initiate the stimulation period. After the commencement of stimulation, cells were fixed and permeabilized as previously described 16 before being stained with phospho-signal transducer and activator of transcription (pSTAT)3 antibody (Cell Signaling Technology, Danvers, MA) and an AF-647 secondary antibody.…”