Marrow Multipotent Mesenchymal Stromal Cells Research Articles

Bone marrow mesenchymal stromal cells support regeneration of intestinal damage in a colitis mouse model, independent of their CXCR4 expression.

Inflammatory bowel disease (IBD) is characterized by a chronically dysregulated immune response in the gastrointestinal tract. Bone marrow multipotent mesenchymal stromal cells have an important immunomodulatory function and support regeneration of inflamed tissue by secretion of soluble factors as well as through direct local differentiation. CXCR4 is the receptor for CXCL12 (SDF-1, stromal-derived factor-1) and has been shown to be the main chemokine receptor, required for homing of MSCs. Increased expression of CXCL12 by inflamed intestinal tissue causes constitutive inflammation by attracting lymphocytes but can also be used to direct MSCs to sites of injury/inflammation. Trypsin is typically used to dissociate MSCs into single-cell suspensions but has also been shown to digest surface CXCR4. Here, we assessed the regenerative effects of CXCR4high and CXCR4low MSCs in an immune-deficient mouse model of DSS-induced colitis. We found that transplantation of MSCs resulted in clinical improvement and histological recovery of intestinal epithelium. In contrary to our expectations, the levels of CXCR4 on transplanted MSCs did not affect their regenerative supporting potential, indicating that paracrine effects of MSCs may be largely responsible for their regenerative/protective effects.

Acute Myeloid Leukemia Causes Serious and Partially Irreversible Changes in Secretomes of Bone Marrow Multipotent Mesenchymal Stromal Cells.

In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.

Angioprotective and Follicle-Stimulating Effects of Administration of Bone Marrow Mesenchymal Stromal Cells in Chronic Inflammation of Internal Reproductive Organs.

Induced inflammation of reproductive organs in female Wistar rats was associated with an increase in the diameters of arteries and veins and number of blood vessels in the ovary medulla in combination with an increase in the number of lymphatic vessels; these changes were accompanied by reduction of the ovarian reserve and number of yellow bodies. Intravenous and submucosal injection of bone marrow multipotent mesenchymal stromal cells (BМ-ММSC) led to further increase in the diameters of arteries and veins and number of primordial and primary follicles. The injection of conditioned medium of BМ-ММSC cultures generally produced the same effects, which could demonstrate the secretory mechanisms of their influence on local angiogenesis and folliculogenesis.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions.We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes.The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied.MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA.Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun.Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression.The work is supported by the RFBR, project 19-015-00280. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Multipotent Mesenchymal Stromal Cells as Autologous Therapy for Osteonecrosis: Effects of Age and Underlying Causes.

Bone marrow (BM) is a reliable source of multipotent mesenchymal stromal cells (MSCs), which have been successfully used for treating osteonecrosis. Considering the functional advantages of BM-MSCs as bone and cartilage reparatory cells and supporting angiogenesis, several donor-related factors are also essential to consider when autologous BM-MSCs are used for such regenerative therapies. Aging is one of several factors contributing to the donor-related variability and found to be associated with a reduction of BM-MSC numbers. However, even within the same age group, other factors affecting MSC quantity and function remain incompletely understood. For patients with osteonecrosis, several underlying factors have been linked to the decrease of the proliferation of BM-MSCs as well as the impairment of their differentiation, migration, angiogenesis-support and immunoregulatory functions. This review discusses the quality and quantity of BM-MSCs in relation to the etiological conditions of osteonecrosis such as sickle cell disease, Gaucher disease, alcohol, corticosteroids, Systemic Lupus Erythematosus, diabetes, chronic renal disease and chemotherapy. A clear understanding of the regenerative potential of BM-MSCs is essential to optimize the cellular therapy of osteonecrosis and other bone damage conditions.

Neuropeptide Y is involved in the regulation of quiescence of hematopoietic stem cells

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G0 phase and an increase in the gene expression levels of FOXO3, DICER1, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence.

Changes in the Circulatory and Lymphatic Systems of Internal Genitals in Female Rats after Intravenous and Lymphotropic Administration of Multipotent Mesenchymal Stem Cells and Products Secreted by These Cells.

We studied the effects of intravenous and lymphotropic administration of bone marrow multipotent mesenchymal stromal cells and products secreted by these cells into conditioned medium on the blood and lymph circulation in the uterus and ovaries, as well as on folliculogenesis in female Wistar rats. It was shown that stromal cells and conditioned media of these cells administered via both routes lead to an increase in the number and diameter of blood vessels in the uterine wall and in the cortical layer of the ovaries. Neither mesenchymal stromal cells, not conditioned media affected the ovarian follicular apparatus.

The Influence of an Electret-Generated Electric Field Based on a Tantalum Oxide Anode on Differentiation Properties of Bone Marrow Stromal Cells from Patients with Osteoarthritis

The ability of the bone marrow multipotent mesenchymal stromal cells (MMSCs) of a patient with osteoarthritis to differentiate into the osteogenic and chondrogenic lineages under the influence of an electret-generated electric field created on the surface of anodic tantalum oxide was investigated. It has been shown that the synthesis of protein-differentiation markers in MMSCs is intensified in the presence of samples with anodic oxide in the electret state. The effect of electrets with uniform charge distribution over the surface was compared with linear charge distribution, MMSCs osteogenic differentiation was accompanied with enhanced expression of osteocalcin and type I collagen. These features were exhibited much stronger in cells exposed to electret specimens with a charge distribution on the surface close to linear.

Effect of Extracellular Vesicles Formed by Multipotent Mesenchymal Stromal Cells on Irradiated Animals.

C57Bl/6J mice were exposed to γ-radiation in a dose of 7.5 Gy. A week later, the experimental group received intravenous injection of extracellular vesicles isolated from the culture medium of human bone marrow multipotent mesenchymal stromal cells. Changes in the physiological parameters of animals were assessed by laser correlation spectroscopy, histological examination, cytometry, and by differential leukocyte count. In 3 and 6 weeks, the parameters of the experimental group occupied an intermediate position between the intact and irradiated groups or did not differ significantly from the parameters of the intact group. The rate and efficiency of recovery varied at different levels of organization. Reduction of damage caused by irradiation in a sublethal dose at different levels of organization of experimental animals was shown.

Tributyltin induces a transcriptional response without a brite adipocyte signature in adipocyte models

Tributyltin (TBT), a peroxisome proliferator-activated receptor γ (PPARγ)/retinoid X receptor (RXR) ligand and founding member of the environmental obesogen chemical class, induces adipocyte differentiation and suppresses bone formation. A growing number of environmental PPARγ ligands are being identified. However, the potential for environmental PPARγ ligands to induce adverse metabolic effects has been questioned because PPARγ is a therapeutic target in treatment of type II diabetes. We evaluated the molecular consequences of TBT exposure during bone marrow multipotent mesenchymal stromal cell (BM-MSC) differentiation in comparison to rosiglitazone, a therapeutic PPARγ ligand, and LG100268, a synthetic RXR ligand. Mouse primary BM-MSCs (female, C57BL/6J) undergoing bone differentiation were exposed to maximally efficacious and human relevant concentrations of rosiglitazone (100nM), LG100268 (100nM) or TBT (80nM) for 4 days. Gene expression was assessed using microarrays, and in silico functional annotation was performed using pathway enrichment analysis approaches. Pathways related to osteogenesis were downregulated by all three ligands, while pathways related to adipogenesis were upregulated by rosiglitazone and TBT. However, pathways related to mitochondrial biogenesis and brown-in-white (brite) adipocyte differentiation were more significantly upregulated in rosiglitazone-treated than TBT-treated cells. The lack of induction of genes involved in adipocyte energy dissipation by TBT was confirmed by an independent gene expression analysis in BM-MSCs undergoing adipocyte differentiation and by analysis of a publically available 3T3 L1 data set. Furthermore, rosiglitazone, but not TBT, induced mitochondrial biogenesis and respiration. This study is the first to show that an environmental PPARγ ligand has a limited capacity to induce health-promoting activities of PPARγ.

PO-286 Evaluating the influence of mesenchymal stromal cells derivatives on early stage of breast cancer

IntroductionTumour microenvironment plays a decisive role in cancer development and metastasis, and affects the therapeutic effectiveness of anticancer drugs. Mesenchymal stromal cells (MSCs) are an important elements of tumour stroma. MSCs can both stimulate and inhibit tumour progression, depending on the components of microenvironment, genesis and stage of cell differentiation. Special attention focused on the paracrine effect of products secreted by MSCs. The aim of this study was to characterise the influence of derivatives from human bone marrow MSCs on proliferation, survival, receptor profile of MCF-7 in 2D and 3D cell cultures in vitro.Material and methodsThe monolayer MCF-7 cell culture was cultured in standard conditions in DMEM nutrient medium (Sigma, USA), with 2 mM l-glutamine (Sigma, USA), 40 mg/mL Gentamicin (Biopharma, Ukraine). The initial density of MCF-7 cells was 2 × 104 cells/cm2. Human bone marrow multipotent mesenchymal stromal cells (MSCs) were used. MCF-7 cells were incubated in full nutrient conditioned media from MSCs in the ratio 1:1. For the initial generation of spheroids the DMEM nutrient medium with 2% carboxymethyl cellulose (Bio-Rad, USA) was used. Plates with spheroids were being incubated on an orbital shaker (PSU-10i, Biosan, Latvia) at 80 rpm for 3–5 hours. The spheroid culture was maintained for 7 days. Cell viability was evaluated by MTT assay. The Stemi2000 software Axio Vision Red 4.7(Zeiss, Germany) was used for processing the images. The volume of aggregates was calculated by Bjerkvig formula after 7 days of cultivation. Markers were detected by applying IHC method with primary monoclonal antibodies Ck (clone AE1/AE3, IS053, Dako, USA), vim (Clone V9, IS630, Dako, USA), EpCAM (Sigma, HPA026761, USA).Results and discussionsIncubation of tumour cells with c-medium from MSCs leaded to decreasing of cell viability by 45% and 60% after 48 and 96 hours, respectively, compared with the control samples. MSCs derivatives reduced the volume of tumour spheroids by 45% and 60% on 2nd and 4th day of cultivation respectively compared with the control. Expression of epithelial markers was increased under influence of MSCs derivatives in both 2D and 3D cell cultures, but expression of vimentin was only in 3D cell culture.ConclusionMSCs derivatives inhibited proliferative activity, reduced migration ability and increased differentiation properties of breast cancer cells in 2D and 3D cell cultures of MCF-7.

The Effect of Multipotent Mesenchymal Stromal Cell Derivatives on the Properties of Breast Cancer Cells in vitro and in vivo

The effects of multipotent mesenchymal stromal cell (MMSC) derivatives on breast cancer cells have been studied in vitro and in vivo. A cytostatic effect of substances produced by human bone marrow MMSCs on the tumor cell population and the inhibition of tumor cell migration into the suspension fraction in vitro were demonstrated. These phenomena were accompanied by upregulation of tumor-associated marker expression: cytokeratin and EpCAM expression was upregulated in 2D and 3D cell cultures and vimentin expression was upregulated in 3D cultures. A single injection of mouse bone marrow MMSC lysate into the experimental animals suppressed tumor growth in thigh muscles. Moreover, this treatment contributed to the preservation of muscle tissue integrity and the normalization of biochemical parameters of the blood in animals that received grafts of Ehrlich adenocarcinoma tumor cells.

Effect of Bone Marrow Multipotent Mesenchymal Stromal Cells on Blood and Lymphatic Vasculature in Uterine Wall of Wistar Rats during Experimental Chronic Metroendometritis.

The study examined the effect of bone marrow multipotent mesenchymal stromal cells and their products secreted into the conditioned medium on uterine microcirculatory bed in Wistar rats during chronic inflammation. The parameters of blood and lymphatic microcirculation in the uterus changed in various directions in relation to the administration routes of biomedical cell product, which is important when the cell therapy is employed in the treatment of inflammatory-degenerative alterations in the lesser pelvis organs.

Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice.

The retinoid X receptors (RXR), peroxisome proliferator activated receptor gamma (PPARγ), and liver X receptors (LXR) all have been shown to regulate bone homeostasis. Tributyltin (TBT) is an environmental contaminant that is a dual RXRα/β and PPARγ agonist. TBT induces RXR, PPARγ, and LXR-mediated gene transcription and suppresses osteoblast differentiation in vitro. Bone marrow multipotent mesenchymal stromal cells derived from female C57BL/6J mice were more sensitive to suppression of osteogenesis by TBT than those derived from male mice. In vivo, oral gavage of 12 week old female, C57Bl/6J mice with 10 mg/kg TBT for 10 weeks resulted in femurs with a smaller cross-sectional area and thinner cortex. Surprisingly, TBT induced significant increases in trabecular thickness, number, and bone volume fraction. TBT treatment did not change the Rankl:Opg RNA ratio in whole bone, and histological analyses showed that osteoclasts in the trabecular space were minimally reduced. In contrast, expression of cardiotrophin-1, an osteoblastogenic cytokine secreted by osteoclasts, increased. In primary bone marrow macrophage cultures, TBT marginally inhibited the number of osteoclasts that differentiated, in spite of significantly suppressing expression of osteoclast markers Nfatc1, Acp5, and Ctsk and resorptive activity. TBT induced expression of RXR- and LXR-dependent genes in whole bone and in vitro osteoclast cultures. However, only an RXR antagonist, but not an LXR antagonist, significantly inhibited TBTs ability to suppress osteoclast differentiation. These results suggest that TBT has distinct effects on cortical versus trabecular bone, likely resulting from independent effects on osteoblast and osteoclast differentiation that are mediated through RXR.

Some Peculiarities of Local Distribution of Multipotent Mesenchymal Stromal Cells after Their Injection into Intact Muscle Tissue in Experiment.

Changes in the muscular tissue after subcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and additionally stained with cell membrane dye Vybrant CM-Dil in the projection of ligated femoral vein were studied by light microscopy with luminescence. Stromal cells injected through the skin can appear not only in the damaged tissue where acceleration of regeneration processes is required, but also in intact structures located in superficial or deeper layers. In intact muscular tissue, stromal cells spreading in the perivascular tissue initiate inflammation and migration of macrophages, activate and even trigger sclerotic processes due to differentiation into connective tissue cells (fibroblasts) and stimulation of proliferation and collagen synthesis by host fibroblasts. Injected multipotent mesenchymal stromal cells are gradually phagocytized by macrophages.

Prospect of Using Cell Product for the Therapy of Skin Defects in Diabetes Mellitus.

The effectiveness of autologous cell product in the therapy of skin burn wounds was studied in C57B1/6 male mice against the background of streptozotocin-induced diabetes mellitus. In animals with and without modeled diabetes mellitus, significant decrease in skin defect area was observed after single administration of the cell product (bone marrow multipotent mesenchymal stromal cells, fibroblasts or media conditioned by these cells).

Effects of Bone Marrow Multipotent Mesenchymal Stromal Cells and Their Secretion Products on the Cellular Composition of the Thymus and Spleen of Female Wistar Rats with Experimental Chronic Inflammation of the Internal Genitals.

The effects of bone marrow multipotent mesenchymal stromal cells and their secretion products on the subpopulation composition of thymic and splenic lymphocytes were studied in female Wistar rats with experimental chronic inflammatory process in the internal genitals. Stromal cells and medium conditioned by these cells in different administration routes (intravenous or lymphotropic injection) produces different modulating effect on blood leukocyte count and on subpopulation composition of the splenic and thymic lymphocytes. The most manifest anti-inflammatory effect was observed after lymphotropic injection of multipotent mesenchymal stromal cells creating a high concentration and long persistence of the factors produced by these cells in the focus of inflammation.

Correction to: Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The author name G. A. Chastikina should read G. A. Chastikin.

Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The peculiarities of tissue sclerosis after injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained with Vybrant CM-Dil cell membrane dye were studied by light microscopy with luminescence. The surgical intervention consisting in ligation of the great vein was followed by tissue sclerotic transformation caused by direct damage and chronic inflammation caused by the presence of slowly resorbed ligature. Injection of stromal cells after this intervention led to formation of more extensive scar. This can attest to the possibility of stromal cells differentiation into connective tissue cells, fibroblasts, and stimulation of proliferation and collagen synthesis by host fibroblasts. A decrease in the volume of dense fibrous connective tissue due to scar reorganization at latter terms cannot not excluded.