The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.

diseases for which no therapies currently exist including graft-versus-host disease, [1,2] autoimmune diseases, [3] liver diseases, [4] orthopedic injuries, [5] cardiovascular diseases, [6] and cancer. [7] But despite high interest and a large number of clinical trials, only three hMSC-based therapies have been clinically approved yet. This might be largely due to the fact that their medical application still faces many challenges including heterogeneity in therapeutic targets, routes of delivery, range of doses, and manufacturing protocols. [8] With minimal effective doses ranging from 70 to 900 million cells per patient extensive ex vivo expansion and large-scale production are required. [9] Furthermore, the therapeutic efficacy and proliferation capacity of hMSCs decline during aging and passaging, and thus optimal growth conditions in vitro are urgently needed to produce adequate numbers of high-quality cells for clinical use. [10,11] hMSCs have been isolated from a wide range of adult and perinatal tissues and are advantageous to induced pluripotent and embryonic stem cells by not bearing the risk of tumorigenesis and their easy applicability. [12,13] They are a heterogeneous population of tissue-specific stem/progenitor cells that possess the ability to recreate the tissue from which they are isolated. [14] The self-renewal and differentiation potential of hMSCs are influenced by not only their tissue of origin but also donor age, genetics, exposure to environmental stress, and passaging in vitro. [15,16] Since their morphology can easily be mistaken for fibroblasts, the International Society for Cellular Therapy defined minimal criteria for hMSCs in vitro: 1) adherent growth in plastic culture dishes, 2) differentiation capacity into adipocytes, osteocytes, and chondrocytes, and 3) expression of CD73, CD105, and CD90 in at least 95% of the cell population and absence of the hematopoietic markers CD45, CD34, CD11b, or CD14. [17] hMSCs express also other cell surface molecules, for example, CD146, intercellular adhesion molecule (ICAM), CD271, Nestin, and leptin receptor that define them as multipotent tissue-specific stem/progenitor cells. The most frequently used source of hMSCs for clinical trials is bone marrow, from which cells can be isolated using Ficoll density gradient centrifugation. The subsequent ex vivo expansion is facilitated by nutrients, growth factors, and extracellular matrix (ECM) proteins present in the undefined soluble component of Human mesenchymal stromal cells (hMSCs) have enormous potential for the treatment of various inflammatory and degenerative diseases. Their manufacturing for cell-based therapies requires extensive ex vivo expansion and optimal growth conditions. To support cell adhesion, spreading, and growth in serum-free culture conditions, the applied plasticware needs to be functionalized with essential biochemical cues. By employing a recently developed screening tool, a chemically defined functional matrix composed of dextran sulfate and a bone-related extracellular ma...

Aging of the hematopoietic system is characterized by an expansion of hematopoietic stem and progenitor cells (HSPCs) with reduced capacity for engraftment, self-renewal, and lymphoid differentiation, resulting in myeloid-biased hematopoiesis. This process is mediated by both HSPC intrinsic and extrinsic factors, e.g., the stromal environment. A relevant cellular component of the bone marrow (BM) microenvironment are mesenchymal stromal cells (MSCs) which regulate fate and differentiation of HSPCs. The bi-directional communication with HSPCs is mediated either by direct cell-cell contacts or by extracellular vesicles (EVs) which carry bioactive substances such as small RNA, DNA, lipids and proteins. So far, the impact of MSC-derived EVs on human hematopoietic aging is poorly investigated. BM MSCs were isolated from young (n = 3, median age: 22 years) and aged (n = 3, median age: 70 years) donors and the EVs were isolated after culturing the confluent cell layer in serum-free medium for 48 h. CD34+ HSPCs were purified from peripheral blood of healthy donors (n = 3, median age: 65 years) by magnetic sorting. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot detection of EV markers CD63, CD81 and Flotillin-1 revealed no significant differences between young and aged MSC-EVs. Interestingly, young MSCs secreted a significantly higher miRNA concentration than aged cells. However, the amount of distinct miRNAs such as miR-29a and miR-34a was significantly higher in aged MSC-EVs. HSPCs incubated with young EVs showed a significant increase in cell number and a higher viability. The expression of the tumor suppressors PTEN, a known target of mir-29a, and CDKN2A was increased in HSPCs incubated with young EVs. The clonogenic assay demonstrated a decreased colony number of CFU-GM after treatment with young EVs and an increased number of BFU-E/CFU-E after incubation with aged MSC-EVs. Xenogenic transplantation experiments showed no significant differences concerning the engraftment of lymphoid or myeloid cell compartments, but the overall human chimerism 8–16 weeks after transplantation was higher after EV treatment. In conclusion, our data suggest that HSPC characteristics such as cell cycle activity and clonogenicity can be modulated by MSC-derived EVs. Further studies have to elucidate the potential therapeutic relevance of our findings.