Sex Dimorphism in the Bone Marrow Niche Contributes to Gender Difference in Mouse Hematopoiesis

Sex dimorphism can be found in hematopoietic stem and progenitor cells, as well as in the development of hematopoietic lineages, resulting in sexual dimorphisms in normal hematopoiesis. Furthermore, males are at higher risk and have a worse prognosis for most hematologic malignancies than females. Under normal conditions, the number of circulating progenitor cells in women is lower than in men. Furthermore, female mice have a higher rate of HSC division than male mice. However, the molecular mechanism underlying normal hematopoiesis sex dimorphism is largely unknown.

We compared male and female hematopoiesis in C57BL/6 mice in the steady state and discovered that male mice have more HSCs than female mice. Female LSK (Lineage-, Sca-1+, and cKit+) cells also had higher apoptosis and lower quiescence than male LSK cells. Additionally, male mice had higher percentage and number of mesenchymal stem cells (MSC) than female mice.

We then conducted two sex/gender mismatch transplantation experiments. First, we injected male or female BM cells into male or female recipients, resulting in four transplantation groups (male to male, male to female, female to male, and female to female). We found that both male and female donor cells engraft better in male recipients than in female recipients, suggesting that male niche provides better hematopoietic engraftment. In the secondary transplantation setting, we mixed equal numbers of male (CD45.1) and female (CD45.2) BM cells and injected the cells into male and female CD45.1/CD45.2 chimeric recipient mice. Male donor cells engraft better than female donor cells in both male and female recipients. Consistently, male recipients have more support for HSC regeneration than female recipients. Altogether, these data suggest that male donor to male recipients produce the best engraftment outcome. This is consistent with the clinic bone marrow transplantation result.

To better understand the underlying molecular mechanism, we performed single-cell RNA sequencing on LSK cells and bone marrow stromal cells from female and male mice (Lineage-, CD45-, Ter119-, and CD71-). Based on gene expression profiling at the single cell level, different populations of HSPCs and stromal cells were clustered. Mesenchymal stem cells (MSC) are the niche cellular component with the most significant sex difference, with male mice having more MSCs than female mice. The LSK cells were then co-cultured with male and female MSCs, and the cobblestone colony formation (CAFC) was assessed over time. The number of CAFC day 35 colonies was significantly higher in male MSC stromal than that in female MSCs. Therefore, male MSCs can thus better support for HSC function, which is consistent with our sex mismatch transplantation result.

We further discovered that male MSC secrete more Cxcl12, which may underlie the increased hematopoietic regeneration. We are currently investigating the mechanism underlying Cxcl12 differential expression in the male/female niche by focusing on the Kdm5c gene. Kdm5c is a gene on the X chromosome that has escaped Xist inactivation. Female cells therefore express twice as much as male cells. It specifically demethylates H3K4me3/me2 and inhibits gene transcription. We hypothesize that a low level of Kdm5c in male MSCs leads to more methylation of H3K4me3, thus increasing Cxcl12 transcription. By using Chip-qPCR, we found that Kdm5c was highly enriched in the Cxcl12 promoter in female MSCs compared to male MSCs, leading to the increased level of demethylated H3K4me3, thus reducing expression of Cxcl12. We next knockdown Kdm5c in female MSCs and found that methylated H3K4me3 was increased in the Cxcl12 promotor, resulting in increased Cxcl12 expression.

In conclusion, sex dimorphism affects normal hematopoietic in a cell intrinsic and extrinsic mechanism. Male MSCs provide a better support for hematopoietic engraftment and regeneration. Differential expression of Kdm5c, sex chromosome-specific gene, may be the underlying mechanism in which a lower level of KDM5C in male MSC leads to higher expression of Cxcl12, which promotes HSC engraftment and maintenance.

No relevant conflicts of interest to declare.