Female Mesenchymal Stem Cells Research Articles

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.

Abstract 215: Induced Pluripotent Stem Cell-Derived Cardiomyocyte Proliferation is Enhanced by Co-culture With Female Mesenchymal Stem Cells

Background: Myocardial ischemia remains a leading cause of morbidity and mortality worldwide. Bone marrow-derived adult mesenchymal stem cells (MSCs) improve cardiac structure and function post-MI likely mediated in large part by paracrine factors. There is emerging evidence that female MSCs have greater therapeutic efficacy than male MSCs. Objective: We hypothesized that female MSCs promote cardiomyocyte differentiation, proliferation and contractility of induced pluripotent stem cells (iPSCs) better than male MSCs. Methods & Results: Human (h) iPSCs were co-cultured with male or female murine MSCs during or after differentiation into beating cardiomyocytes. Female MSCs were significantly more potent at stimulating ISL-1 and NKX2-5 expression in hiPSC-derived cardiac precursors and cardiomyocytes. Exosomes from female MSCs had lower levels of miR-17, a miR that directly represses Isl1 . hiPSCs co-cultured with female MSCs, exhibited a 2-fold greater increase in proliferation compared to male MSCs. In addition, ß 2 -adrenergic receptor expression was up regulated 3.5-fold in hiPSCs incubated with female MSCs compared to 2.5-fold for male MSCs. Conclusion: Our findings illustrate that there are significant differences between female and male MSCs in their ability to affect cardiomyogenesis of hiPSCs and suggest that this effect is mediated, at least in part, by reduced levels of miR-17. The observation that female MSCs have greater potential to improve cardiomyocyte proliferation and contractility has important implications for stem cell therapy.

Male and female rat bone marrow-derived mesenchymal stem cells are different in terms of the expression of germ cell specific genes.

Recent studies have shown that mesenchymal stem cells (MSCs), under appropriate conditions, can differentiate into cell types including germ cells (GCs). These studies also show that MSCs without any induction express some GC-specific genes innately. Moreover, one report suggests that female MSCs have a greater tendency to differentiate into female instead of male GCs. Therefore, for the first time, this study attempts to assay and determine the differences between the expression levels of some important GC-specific genes (Stra8, Vasa, Dazl, Stella, Piwil2, Oct4, Fragilis, Rnf17 and c-Kit) in male and female bone marrow (BM)-MSCs of rats. BM sampling of the rate was performed by a newly established method. We cultured rat BM samples, then characterized male and female MSCs according to their adhesion onto the culture dish, their differentiation potential into bone, cartilage and fat cells, and phenotype analysis by flow cytometry. The expression of GC-specific genes and their expression levels were evaluated with reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Our results showed that Dazl and Rnf17 did not express in the cells. The majority of examined genes, except Piwil2, expressed at almost the same levels in male and female MSCs. Piwil2 had higher expression in male MSCs which was probably related to the more prominent role of Piwil2 in the male GC development process. Male BM-MSCs appeared more prone to differentiate into male rather than female GCs. Additional research should be performed to determine the exact role of different genes in the male and female GC development process.

Female stem cells are superior to males in preserving myocardial function following endotoxemia

Mesenchymal stem cells (MSCs) may offer therapeutic benefit in the setting of sepsis and endotoxemia. Previous studies suggest that MSCs from female donors may possess better protective capabilities than their male counterparts. The present study examined whether female MSCs may offer a greater protective advantage in the setting of endotoxemic cardiac dysfunction compared with male MSCs. Adult male Sprague-Dawley rats were injected intraperitoneally with LPS and then treated with intraperitoneal injections of either saline, female MSCs, or male MSCs. Hearts and serum were then collected for analysis of myocardial function, myocardial protein, and myocardial and serum cytokines. Compared with male MSC or vehicle-treated animals, female MSC treatment resulted in greater preservation of myocardial function (P < 0.001). Serum and myocardial levels of all measured cytokines were comparable between rats given MSCs from male or female donors but substantially improved over rats given vehicle (P < 0.05). Reduced myocardial inflammation correlated with reduced levels of phosphorylated p38 MAPK expression in the myocardium of animals injected with MSCs of either sex (P < 0.05). The Bcl-xL/Bax ratio was increased to a greater extent following treatment with female MSCs vs. male MSCs (P < 0.05). Intraperitoneal administration of MSCs is effective in limiting myocardial inflammation and dysfunction in the rat endotoxemia model. Compared with treatment with their male counterparts, MSC treatment from female donors is associated with greater cardiac protection against acute endotoxemic injury.

Raloxifene: its ossification-promoting effect on female mesenchymal stem cells

BackgroundRaloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts’ differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation. MethodsFemale and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 106 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed. ResultsOn histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects. Conclusions10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague–Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females ( p ≤ 0.01), and the bridging of callus was delayed ( p 2weeks = 0.041). μCT revealed a reduced callus size ( p = 0.003), mineralization ( p = 0.003) and polar moment of inertia ( p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae ( p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

Role of Tumor Necrosis Factor Receptor 1 in Sex Differences of Stem Cell Mediated Cardioprotection

Mesenchymal stem cells (MSCs) hold great therapeutic potential for the repair and regeneration of ischemic tissue, possibly through the release of beneficial paracrine factors. Sex differences have been observed in the paracrine function of MSCs. Female stem cells produce lower proinflammatory cytokines and higher levels of growth factors compared with their male counterparts. Ablation of tumor necrosis factor receptor 1 (TNFR1) increases protective growth factor production by male, but not by female, MSCs. We therefore hypothesized the following: (1) that female MSCs would improve myocardial recovery compared with male MSCs after ischemia-reperfusion injury (I/R); and (2) that MSCs isolated from TNFR1 knock out male, but not female, mice, would improve postischemic myocardial recovery compared with their wild type (WT) counterparts. Male adult Sprague-Dawley rat hearts were subjected to I/R by Langendorff isolated heart preparation. The MSCs were harvested from adult mice and cultured under normal conditions. Immediately prior to ischemia, one million MSCs were infused into the coronary circulation. Cardiac functional parameters were recorded continuously. Pretreatment with MSCs from either sex significantly increased postischemic myocardial recovery as evidenced by improved left ventricular developed pressure, contractility, and rate of relaxation. Infusion with female MSCs was associated with a greater degree of myocardial recovery after I/R compared with male MSCs. The TNFR1 deficiency increased the degree of myocardial recovery associated with male MSCs, but not with female MSCs. No additional cardioprotection was observed when TNFR1 was ablated in female MSCs. Sex differences influence the cardioprotective effects of both WT and TNFR1 ablated MSCs.

SEX DIMORPHISMS IN ACTIVATED MESENCHYMAL STEM CELL FUNCTION

The plasticity of bone marrow-derived stem cells (BMSCs) has resulted in positive remodeling and the regeneration of viable tissues. However, BMSC release of growth factors, which limit apoptosis and inflammation, may play an important role in conferring organ protection. Recent studies also indicate that those patients with higher circulating BMSC counts may be more resistant to septic and traumatic insults. There are clear sex differences in response to such insults. Within the population of BMSC, mesenchymal stem cells (MSCs) may have clinical advantages. Therefore, we hypothesize that sex differences in the MSC paracrine response to acute injury exist. Mesenchymal stem cells were obtained from male and female mice. One million MSCs per well (triplicate wells per group) were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide [LPS]) and hydrogen peroxide. Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha production by enzyme-linked immunosorbent assay. Differences were considered significant if P < 0.05. Lipopolysaccharide resulted in significant activation of both male and female MSCs. However, LPS provoked significantly more VEGF production in female MSCs versus male MSCs at all LPS doses. Hypoxia of 1 h and hydrogen pyroxide exposure also caused significantly more VEGF production in female MSCs versus male MSCs. Female MSCs expressed significantly less tumor necrosis factor alpha than male MSCs after acute LPS and hypoxia. This study constitutes the first demonstration that sex differences exist in activated MSC function. Sex differences in progenitor cell function may have important implications in understanding the observed sex differences in the host's response to injury.