Project description:Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.
Project description:Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche. Prior studies showed that signaling from members of the transforming growth factor (TGF) superfamily in mesenchymal stromal cells is required for normal niche development. Here, we assessed the impact of TGF family signaling on niche maintenance and stress responses by deleting Smad4 in mesenchymal stromal cells at birth, thereby abrogating canonical TGF signaling. No alteration in the number or spatial organization of CXCL12-abundant reticular (CAR) cells, osteoblasts, or adipocytes was observed in Osx-Cre, Smad4fl/fl mice, and expression of key niche factors was normal. Basal hematopoiesis and stress erythropoiesis responses to acute hemolytic anemia were normal. TGF-? potently inhibits stromal CXCL12 expression in vitro; however, G-CSF induced decreases in bone marrow CXCL12 expression and subsequent hematopoietic stem/progenitor cell mobilization were normal in Osx-Cre, Tgfbr2fl/fl mice, in which all TGF-? signaling in mesenchymal stromal is lost. Finally, although a prior study showed that TGF-? enhances recovery from myeloablative therapy, hematopoietic recovery following single or multiple doses of 5-flurauracil were normal in Osx-Cre, Tgfbr2fl/fl mice. Collectively, these data suggest that TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.
Project description:Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.
Project description:Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFR?), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.
Project description:Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials, their variable and heterogeneous supporting activity pose major challenges. While three-dimensional (3D) MSC cultures are emerging as alternative approaches, key changes in cellular characteristics during 3D-spheroid formation remain unclear. Here, we show that MSCs in 3D spheroids undergo further progression towards the epithelial-mesenchymal transition (EMT), driven by upregulation of EMT-promoting microRNAs and suppression of EMT-inhibitory miRNAs. The shift of EMT in MSCs is associated with widespread histone modifications mimicking the epigenetic reprogramming towards enhanced chromatin dynamics and stem cell-like properties, but without changes in their surface phenotype. Notably, these molecular shifts towards EMT in 3D MSCs caused enhanced stem cell niche activity, resulting in higher stimulation of hematopoietic progenitor self-renewal and cancer stem cell metastasis. Moreover, miRNA-mediated induction of EMT in 2D MSCs were sufficient to mimic the enhanced niche activity of 3D spheroid MSCs. Thus, the molecular hierarchy in the EMT gradient among phenotypically indistinguishable MSCs revealed the previously unrecognized functional parameters in MSCs, and the EMT-enhanced "naïve" mesenchymal state represents an 'activated mesenchymal niche' in 3D spheroid MSCs.
Project description:Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu ?-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu ?-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu ?-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.
Project description:Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo-expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.
Project description:The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.
Project description:: Mesenchymal stromal cells (MSCs) are crucial elements in the bone marrow (BM) niche where they provide physical support and secrete soluble factors to control and maintain hematopoietic stem progenitor cells (HSPCs). Given their role in the BM niche and HSPC support, MSCs have been employed in the clinical setting to expand ex-vivo HSPCs, as well as to facilitate HSPC engraftment in vivo. Specific alterations in the mesenchymal compartment have been described in hematological malignancies, as well as in rare genetic disorders, diseases that are amenable to allogeneic hematopoietic stem cell transplantation (HSCT), and ex-vivo HSPC-gene therapy (HSC-GT). Dissecting the in vivo function of human MSCs and studying their biological and functional properties in these diseases is a critical requirement to optimize transplantation outcomes. In this review, the role of MSCs in the orchestration of the BM niche will be revised, and alterations in the mesenchymal compartment in specific disorders will be discussed, focusing on the need to correct and restore a proper microenvironment to ameliorate transplantation procedures, and more in general disease outcomes.
Project description:Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased G(s) signaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of G(s) signaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.