Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co‐signaling effects

Abstract

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.

MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells

“…Epo controls growth, survival, and differentiation of erythroid progenitors, either cooperatively with, or independently of KitL. [32][33][34] Despite severe anemia, Epo mRNA expression in kidney tissues of MT1-MMP Ϫ/Ϫ mice was low ( Figure 3A) and the expected compensatory increase in spleen size did not occur (see Figure 2B). …”

The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis

“…Because an elevated Epo level is expected to promote erythropoiesis through increasing the progenitor population, the response to Epo of RHAU fl/fl :vav-iCre mice might be impaired. 27 We therefore examined the different subsets of erythroblasts that correspond to different stages of maturation. ProE is the earliest progenitor that is completely committed to erythropoiesis and undergoes maturation through the following stages: basophilic erythroblast, late basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, and, finally, reticulocyte that is enucleated and released into the blood stream.…”

“…On cell division, the CFSE is equally split between the daughter cells, resulting in decreased fluorescence signals; this allows monitoring of the number of divisions undergone by a cell. 27 ProEs from BM cells were sorted as Ter119 med CD71 hi population and were immediately stained with CSFE, after which time they were grown for 3 days in the presence of Epo. From the histograms ( Figure 7C), the RHAU-ablated ProEs gave a higher CFSE signal compared with the wild-type ProEs.…”

Role Of Stem Cell Factor In The Reactivation Of Human Fetal Hemoglobin

“…The second mechanism suggests that SCF and Epo have simultaneous, joint effects on the same individual cells at a specific stage of erythroid development (proerythroblast stage). In this context, recent studies support a model of erythroid differentiation in which the effects of SCF and Epo on individual erythroid progenitors/precursors are mainly sequential and co-signaling (i.e., signaling by both cytokines on the same cells, resulting or not in crosstalk between pathways) has only a limited contribution to the final erythroid output36.…”