Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis

Abstract

Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. Although sex differences have been implicated in differential hematopoietic function in males versus females, the mediators responsible for these effects require further study. Here, we characterized hematopoiesis at a steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared with male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery, we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shape normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.

Figure 1

Female BM lineage(–) cells are transcriptionally more quiescent and less inflammatory. (A) Schematic depicts lineage(–) cell isolation and subsequent RNA sequencing. (B) Gene set enrichment analysis (GSEA) from a list of DEGs to determine statistically significant, concordant differences between two biological states. Fifty well-defined biological pathways are shown. GSEA shown is enriched or depleted in females relative to males. Red color represents statistically significant enrichment; blue color represents enrichment that is trending toward significance. (C) Heatmaps of genes involved in inflammatory response, unfolded protein response, hypoxia, and glycolysis. N = 5 mice/arm. Schematic made using BioRender.com. BM = Bone marrow; DEG = Differentially-expressed gene.

Figure 2

Male mice display accelerated rates of peripheral recovery following sex-matched HST. (A) Schematic depicting transplant conditions. 1e5 lin(–) BM cells from male donors were transplanted into lethally irradiated (10 Gy) male recipients, and the equivalent was performed using female donors and recipients. (B) Complete blood count analysis of circulating white blood cells (WBC) lymphocytes (LYMPH), neutrophils (NE), red blood cells (RBC), and platelets (PLT) in male (blue) and female (purple) mice at indicated time points post–bone marrow transplantation. (C) Cytometric analysis of total BM cellularity, BM LSKs, and BM LT-HSCs at indicated time points post–bone marrow transplantation. N = 10 mice/arm over two replicates. Error bars represent SEM. Repeated measure ANOVA performed on all analyses. (D) Serum concentrations (pg/mL) of CXCL12 and tumor necrosis factor alfa (TNF-α) in male and female mice at days 14 and 42. N = 10 mice/arm. Error bars represent SEM. *p < 0.05. **p < 0.01. ***p < 0.001. Student’s t-test performed at each time point. Schematic made using BioRender.com. ANOVA = Analysis of variance; BM = bone marrow; HST = hematopoietic stem cell transplantation; SEM = standard error of mean.

Figure 3

Male donor cells accelerate peripheral hematopoietic recovery in HST independent of recipient sex. (A) Schematic depicting transplant conditions. 1e5 lin(–) BM cells from male donors were transplanted into lethally irradiated (10 Gy) female recipients, and the equivalent was performed using female donors and male recipients. (B) Complete blood count analysis of circulating white blood cells (WBC), lymphocytes (LYMPH), neutrophils (NE), red blood cells (RBC), and platelets (PLT) in male (red) and female (blue) mice at indicated time points post–bone marrow transplantation. (C) Cytometric analysis of total BM cellularity, BM LSKs, and BM LT-HSCs at indicated time points post–bone marrow transplantation. N = 10 mice/arm over two replicates. Error bars represent SEM. Repeated measure ANOVA performed on all analyses. (D) Serum concentrations (pg/mL) of CXCL12 and tumor necrosis factor alfa (TNF-α) in male and female mice at days 14 and 42. N = 10 mice/arm. Error bars represent SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Student’s t-test performed at each timepoint. Schematic made using BioRender. ANOVA = Analysis of variance; BM = bone marrow; HSC = hematopoietic stem cells; SEM = standard error of mean.