CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

Bone marrow-derived mesenchymal stromal cells obstruct AML-targeting CD8+ clonal effector and CAR T-cell function while promoting a senescence-associated phenotype.

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm's tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28-CD57+ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28loCD27loCD57+KLRG1+ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell-cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication. RESULTS: Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes. CONCLUSION: Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment. Video Abstract.

A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xenogeneic/serum-free conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by (i) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell expansion; (ii) donor-specific characteristics, including MSC frequency/quality, that decline with disease state and increasing age; and (iii) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which poses safety and consistency issues. METHODS: To overcome these limitations while enabling robust MSC production with constant high yield and quality, the authors developed a chemically defined biomimetic surface coating called isoMATRIX (denovoMATRIX GmbH, Dresden, Germany) and tested its performance during isolation of MSCs. RESULTS: The isoMATRIX facilitates the isolation of significantly higher numbers of MSCs in xenogeneic (xeno)/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. The high proliferation rates can be maintained up to 5 passages after isolation and cells even benefit from a switch towards a proliferation-specific MSC matrix (myMATRIX MSC) (denovoMATRIX GmbH, Dresden, Germany). CONCLUSION: In sum, isoMATRIX promotes enhanced xeno/serum-free and chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Myelodysplastic Syndromes and Metabolism.

Myelodysplastic syndromes (MDS) are acquired clonal stem cell disorders exhibiting ineffective hematopoiesis, dysplastic cell morphology in the bone marrow, and peripheral cytopenia at early stages; while advanced stages carry a high risk for transformation into acute myeloid leukemia (AML). Genetic alterations are integral to the pathogenesis of MDS. However, it remains unclear how these genetic changes in hematopoietic stem and progenitor cells (HSPCs) occur, and how they confer an expansion advantage to the clones carrying them. Recently, inflammatory processes and changes in cellular metabolism of HSPCs and the surrounding bone marrow microenvironment have been associated with an age-related dysfunction of HSPCs and the emergence of genetic aberrations related to clonal hematopoiesis of indeterminate potential (CHIP). The present review highlights the involvement of metabolic and inflammatory pathways in the regulation of HSPC and niche cell function in MDS in comparison to healthy state and discusses how such pathways may be amenable to therapeutic interventions.

Effects of rigosertib on the osteo-hematopoietic niche in myelodysplastic syndromes.

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3 weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Erythropoietin inhibits osteoblast function in myelodysplastic syndromes via the canonical Wnt pathway.

The effects of erythropoietin on osteoblasts and bone formation are controversial. Since patients with myelodysplastic syndromes often display excessively high erythropoietin levels, we aimed to analyze the effect of erythropoietin on osteoblast function in myelodysplastic syndromes and define the role of Wnt signaling in this process. Expression of osteoblast-specific genes and subsequent osteoblast mineralization was increased in mesenchymal stromal cells from healthy young donors by in vitro erythropoietin treatment. However, erythropoietin failed to increase osteoblast mineralization in old healthy donors and in patients with myelodysplasia, whereas the basal differentiation potential of the latter was already significantly reduced compared to that of age-matched controls (P<0.01). This was accompanied by a significantly reduced expression of genes of the canonical Wnt pathway. Treatment of these cells with erythropoietin further inhibited the canonical Wnt pathway. Exposure of murine cells (C2C12) to erythropoietin also produced a dose-dependent inhibition of TCF/LEF promoter activity (maximum at 500 IU/mL, -2.8-fold; P<0.01). The decreased differentiation capacity of erythropoietin-pretreated mesenchymal stromal cells from patients with myelodysplasia could be restored by activating the Wnt pathway using lithium chloride or parathyroid hormone. Its hematopoiesis-supporting capacity was reduced, while reactivation of the canonical Wnt pathway in mesenchymal stromal cells could reverse this effect. Thus, these data demonstrate that erythropoietin modulates components of the osteo-hematopoietic niche in a context-dependent manner being anabolic in young, but catabolic in mature bone cells. Targeting the Wnt pathway in patients with myelodysplastic syndromes may be an appealing strategy to promote the functional capacity of the osteo-hematopoietic niche.

Functional Interference in the Bone Marrow Microenvironment by Disseminated Breast Cancer Cells.

Skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity. Investigations in other solid tumors have revealed an impairment in hematopoietic function upon bone marrow invasion. However, the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively. Employing advanced co-culture assays, proteomic studies, organotypic models as well as in vivo xenotransplant models, we define the consequences of this interaction on the stromal compartment of bone marrow, affected molecular pathways and subsequent effects on the hematopoietic stem and progenitor cells (HSPCs). The results showed a basic fibroblast growth factor (bFGF)-mediated, synergistic increase in proliferation of breast cancer cells and mesenchymal stromal cells (MSCs) in co-culture. The stromal induction was associated with elevated phosphoinositide-3 kinase (PI3K) signaling in the stroma, which coupled with elevated bFGF levels resulted in increased migration of breast cancer cells towards the MSCs. The perturbed cytokine profile in the stroma led to reduction in the osteogenic differentiation of MSCs via downregulation of platelet-derived growth factor-BB (PDGF-BB). Long term co-cultures of breast cancer cells, HSPCs, MSCs and in vivo studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl) /SzJ (NSG) mice showed a reduced support for HSPCs in the altered niche. The resultant non- conducive phenotype of the niche for HSPC support emphasizes the importance of the affected molecular pathways in the stroma as clinical targets. These findings can be a platform for further development of therapeutic strategies aiming at the blockade of bone marrow support to disseminated breast cancer cells. Stem Cells 2016;34:2224-2235.

Breast cancer cells compete with hematopoietic stem and progenitor cells for intercellular adhesion molecule 1-mediated binding to the bone marrow microenvironment.

Adhesion-based cellular interactions involved in breast cancer metastasis to the bone marrow remain elusive. We identified that breast cancer cells directly compete with hematopoietic stem and progenitor cells (HSPCs) for retention in the bone marrow microenvironment. To this end, we established two models of competitive cell adhesion-simultaneous and sequential-to study a potential competition for homing to the niche and displacement of the endogenous HSPCs upon invasion by tumor cells. In both models, breast cancer cells but not non-tumorigenic cells competitively reduced adhesion of HSPCs to bone marrow-derived mesenchymal stromal cells (MSCs) in a tumor cell number-dependent manner. Higher adhesive force between breast cancer cells and MSCs, as compared with HSPCs, assessed by quantitative atomic force microscopy-based single-cell force spectroscopy could partially account for tumor cell mediated reduction in HSPC adhesion to MSCs. Genetic inactivation and blockade studies revealed that homophilic interactions between intercellular adhesion molecule 1 (ICAM-1) expressed on tumor cells and MSCs, respectively, regulate the competition between tumor cells and HSPCs for binding to MSCs. Moreover, tumor cell-secreted soluble ICAM-1(sICAM-1) also impaired HSPC adhesion via blocking CD18-ICAM-1 binding between HSPCs and MSCs. Xenotransplantation studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice revealed reduction of human HSPCs in the bone marrow via metastatic breast cancer cells. These findings point to a direct competitive interaction between disseminated breast cancer cells and HSPCs within the bone marrow micro environment. This interaction might also have implications on niche-based tumor support. Therefore, targeting this cross talk may represent a novel therapeutic strategy.

Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12.

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFß1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches.

MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells.

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3'UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3'UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche.

Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

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.

Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide.

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients' cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34(+) hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34(+) cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

Discriminant Principal Component Analysis of ToF-SIMS Spectra for Deciphering Compositional Differences of MSC-Secreted Extracellular Matrices.

Identifying characteristic extracellular matrix (ECM) variants is a key challenge in mechanistic biology, bioengineering, and medical diagnostics. The reported study demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted ECM types as induced by exogenous stimulation or emerging pathology. ToF-SIMS spectra of decellularized ECM samples are evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins are determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro are analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with polymers that induce macromolecular crowding and with ascorbic acid are detected from the DPCA of ToF-SIMS spectra. The results are verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome display the potential of the novel methodology to reveal disease-associated alterations of the ECM composition.

Biallelic TET2 mutations confer sensitivity to 5'-azacitidine in acute myeloid leukemia.

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5'-azacitidine (5'-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5'-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5'-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer.

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro.

BACKGROUND: In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34(+) cells and mesenchymal stromal cells with regard to different spatial localizations. DESIGN AND METHODS: Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay. RESULTS: In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner. CONCLUSIONS: We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modulate Hematopoietic Stem and Progenitor Cell Viability and the Expression of Cell Cycle Regulators in an Age-dependent Manner.

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.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.