Multipotent Mesenchymal Stromal Cells Administration Research Articles

T Cell and Cytokine Dynamics in the Blood of Patients after Hematopoietic Stem Cell Transplantation and Multipotent Mesenchymal Stromal Cell Administration

Multipotent mesenchymal stromal cells (MSCs) are currently under intensive investigation for the treatment and prevention of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), owing to their substantial immunomodulatory properties. The responses of recipients to MSC infusion following allo-HSCT are not yet well understood. T cells are central to the adaptive immune system, protecting the organism from infection and malignant cells. Memory T cells with different phenotypes, gene expression profiles, and functional properties are critical for immune processes regulation. The aim of this study was to study the dynamics of memory T cell subpopulations and cytokines in the blood of allo-HSCT recipients after MSC administration. In clinical trial NCT01941394, patients after allo-HSCT were randomized into 2 groups, one receiving standard GVHD prophylaxis and the other also receiving MSC infusion on the day of leukocyte recovery to 1000 cells/μL (engraftment, day E0). Blood samples of patients from both groups were analyzed on days E0, E+3, and E+30. T cell subpopulations were studied by flow cytometry, and cytokine concentrations were evaluated by the Bio-Plex Pro Human Cytokine Panel. Administration of MSCs to patients on day E0 did not affect the overall dynamics of restoration of absolute numbers and proportions of T and B lymphocytes after 3 and 30 days. At 3 days after MSC injection, only the numbers of CD8+ effector cells (CD8+TE, CD8+TM, and CD8+EM) were found to increase significantly. A significant increase in the number of CD4+ cells after 30 days compared to day E0 was observed only in patients who received MSCs, indicating faster recovery of the CD4+ cell population following MSC injection. An increase in CD8+ cell number by day E+30 was significant regardless of MSC administration. To characterize the immune status of patients following allo-HSCT in more detail, changes in the cytokine concentration in the peripheral blood of patients on days E0, E+3, and E+30 after MSC administration were investigated. On day E+30, significant increases in the numbers of CD4+CM and activated CD4+CD25+ cells were observed. The concentrations of proinflammatory and anti-inflammatory cytokines IL-6, IL-8, IL-17, TNF-α, and IFN-γ were increased significantly in patients injected with MSCs. Analysis of growth factor levels showed that in the group of patients who received MSCs, the concentrations of G-CSF, GM-CSF, PDGFbb, FGFb, and IL-5 increased by day E+30. Among the cytokines involved in regulation of the immune response, concentrations of IL-9, eotaxin, IP-10, MCP-1, and MIP-1a were increased after 30 days irrespective of MSC administration. The administration of MSCs exerts a positive effect on the restoration of T cell subpopulations and immune system recovery in patients after allo-HSCT.

The neuroprotective effect of mesenchymal stem cells in colistin-induced neurotoxicity

Colistin is an effective antibiotic against multidrug-resistant gram-negative bacterial infections; however, neurotoxic effects are fundamental dose-limiting factors for this treatment. Stem cell therapy is a promising method for treating neuronal diseases. Multipotent mesenchymal stromal cells (MSC) represent a promising source for regenerative medicine. Identification of neuroprotective agents that can be co-administered with colistin has the potential to allow the clinical application of this essential drug. This study was conducted to assess the potential protective effects of MSC, against colistin-induced neurotoxicity, and the possible mechanisms underlying any effect. Fourty adult female albino rats were randomly classified into 4 equal groups; the control group, the MSC-treated group (A single dose of 1 × 106/mL MSCs through the tail vein), the colistin-treated group (36 mg/kg/day colistin was given for 7 days) and the colistin and MSC treated group (36 mg/kg/day colistin was administered for 7 days, and 1 × 106/mL MSCs). Colistin administration significantly increased GFAP, NGF, Beclin-1, IL-6 and TNF-α immunreactivity intensity. MSC administration in colistin-treated rats partially restored each of these markers. Histopathological changes in brain tissues were also alleviated by MSC co-treatment. Our study reveals a critical role of inflammation, autophagy and apoptosis in colistin-induced neurotoxicity and showed that they were markedly ameliorated by MSC co-administration. Therefore, MSC could represent a promising agent for prevention of colistin-induced neurotoxicity.

Dynamics of Cytokines Concentration in the Blood Plasma of Patients after Multipotent Mesenchymal Stromal Cells Administration for Graft Versus Host Disease Prevention

IntroductionDespite the large number of clinical studies on the use of multipotent mesenchymal stromal cells (MSCs) for the treatment and prevention of graft-versus-host disease (GVHD), the mechanism of their action in the organism is not well understood. The known data refer either to clinical effects or obtained in vitro. Due to the immunomodulatory effect of MSCs in the body, subpopulations of T cells and the concentration of cytokines involved in the immune response can change. The role of T cells and certain cytokines (TNF alpha, IL6, IL8, etc.) in the pathophysiology of GVHD is described. The aim of this investigation was to study the composition of T cells subpopulations and the concentration of cytokines in the peripheral blood of patients who received MSCs for GVHD prophylaxis.MethodsThe study included 21 patients who received hematopoietic stem cells donor's derived MSCs for the prevention of GVHD as part of the ClinicalTrials.gov Identifier NCT01941394 trial. The control group included 16 patients who did not receive MSCs. After signing informed consent, blood samples were taken from all patients during routine examinations on the day of restoration of the number of leukocytes to 1000 in μl (day 0), after 3 and after 30 days. MSCs were injected on day 0. None of the patients developed GVHD during this time. To analyze plasma cytokines and chemokines, the Bio-Plex Pro Human Cytokine Panel kit, 27-Plex (BioRad) was used, to determine the concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, bFGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-bb, RANTES, TNF-α, VEGF according to manufacturer's recommendations. Forward and side scattering parameters determined the peripheral blood lymphocytes population and then CD4+ or CD8+ lymphocytes were gated. For each of this population the composition of memory cells subset were determined by flow cytometry.ResultsSignificant differences were found between the 2 groups only on day 30 in the concentration of IL8 (17.0±3.2 pg/ml in the control group versus 32.8±4.1 pg/ml in the MSC group, p<0.0001). It has been shown that 30 days after MSCs, the number of CD4+ T cells in the peripheral blood of patients significantly increases compared to the group without MSCs (CD4 CM 38.9±9.0 vs 22.4±7.8, CD4 TM 97.0±32.5 vs 91.0±43.2, CD4 TE 5.0±1.9 vs 1.4±1.0, CD4 EM 34.6±20.1 vs 19.9±7.9, CD4CD25+ 27.6±6.6 vs 12.9±5.0). These cells produce IL8, which play an important role in immune cell homeostasis by activating antimicrobial neutrophils. Without the introduction of MSCs, the concentration of this protective against GVHD cytokine practically did not change within a month, whereas after the introduction of MSCs, it gradually increased almost 2 times. However, the dynamics of changes in the levels of the studied cytokines differed greatly between the 2 groups (Table). The concentration of IP10, which is involved in the development of GVHD, increased significantly faster and stronger in the group without MSCs. An increase in the concentration of other investigated cytokines associated with the activation of macrophages (MCP-1, MIP-1a, MIP-1b) did not depend on the MSCs administration. At the same time, in the MSC group, the concentration of the growth factor PDGF-bb necessary for the HSC proliferation increased significantly more actively. The IL9 concentration on day 0 was comparable to the level in healthy donors, and then gradually increased in both groups and after 30 days it was significantly higher than on day 0 in the MSC administration group. The concentration of G-CSF changed in a similar way.ConclusionChanges in the dynamics of T cells subpopulations and the concentration of cytokines in the blood after MSCs administration contribute to a faster recovery of patients after allogeneic bone marrow transplantation.The work were supported by the Russian Foundation for Basic Research, Project No. 19-29-04023. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multipotent Mesenchymal Stromal Cells for Poor Graft Function after Allogeneic Hematopoietic Cell Transplantation:a Multicenter Prospective Study

Selleslag: Celyad: Other: Clinical trial research (no honoraria recieved); Novartis: Consultancy, Honoraria, Speakers Bureau.

Immediate results of intranasal administrations of multipotent mesenchymal stromal cells (MMSC) to Parkinson's disease (PD) patients

Introduction PD is a steadily progressive neurodegenerative disease. MMSC administration is considered one of the most promising therapies to modify the course of the disease. Objective Assessment of the immediate results of intranasal transplantation of autologous MMSC on motor symptoms of patients with PD. Materials and methods Four patients with PD were subjected to cell transplantation (all males; age = 42,7 ± 8,3 SD). Neurological examination was carried out in the morning after a 12 or (24) - hour break in taking anti-Parkinsonian drugs («OFF-state»), and one hour after the drugs were taken («ON-state»). The intensity of motor symptoms was assessed using the third part of the Unified Parkinson's disease Rating Scale (UPDRS) of the International Society for Movement Disorders. Initial assessment was performed 1 day before the transplantation, and the progress was measured one week after the procedure. Non-parametric methods of statistical analysis were used. The study was approved by local ethics committee. All subjects signed informed consent form before participating in the study. Results An improvement was detected in all the patients. Before the transplantation median levels of UPDRS in «OFF»-state was 33.4 [28.7; 39.2], and in «ON»-28.9 [21.2; 34.9]. After the procedure - «Off»-period was 31.1 [26.4; 36.5], «ON»-26.2 [19.4; 31.4]. Conclusion Transplantation of MMSC is a promising therapeutic intervention in treatment of PD that could influence the symptoms, and potentially the pathogenesis, of the disease. However, further examination is needed to estimate the consequences of this complex and insufficiently explored method.

Effect of Priming of Multipotent Mesenchymal Stromal Cells with Interferon γ on Their Immunomodulating Properties.

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate subpopulational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4+ and CD8+ expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naïve cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naïve state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.

The properties of multipotent mesenchymal stromal cells samples vary depending on their ability to induce HLA-DR expression on T-cells co-cultured with them

Interactions between lymphocytes and multipotent mesenchymal stromal cells (MSCs) in vitro sometimes increase HLA-DR expression on T-cells. On lymphocytes derived from one donor the elevation of HLA-DR was observed after co-cultivation with part of MSCs samples (group A), on the others the HLA-DR expression level did not change (group B). Study of T-cell subpopulations after interactions with MSCs could explain ineffectiveness of some MSCs, as an immunomodulating agent in clinical applications. The aim of the study was to discriminate variations in T-cell subpopulations, co-cultured with MSCs from groups A and B. MSCs were isolated from bone marrow of 13 donors for allogeneic hematopoietic cells and cultured by a standard method. MSCs were seeded 100000 cells per flask, and then 1000000 allogeneic lymphocytes from single donor were added to all MSCs cultures. Lymphocytes and MSCs were immunophenotyped separately during 4 days of co-cultivation. The expression of HLA-DR, activation markers CD25, CD38, CD69, HLA-DR and PD-1 and the distribution of naïve and effector T-cells were studied by flow cytometry. Expression of HLA-DR on lymphocytes after 4 days of cultivation without MSCs did not change compared to 1st day. HLA-DR expression on lymphocytes co-cultured with MSCs from group A was 3 times greater than from group B. In lymphocytes co-cultured with MSCs there were higher number of naïve cells compared to control, p < 0.001. Group B showed lower number of EM and TM cells. Differences between groups were more pronounced when lymphocytes were activated. In group B proportion of HLA-DR CD4+ and CD8+ cells was significantly lower, compared to group A and control samples. At the same time the number of CM and PD-1+ CD4+ cells was lower in group A, but number of TE was increased. These data indicate that MSCs from group A became more immunogenic after interaction with lymphocytes and could not show immunomodulating properties in the same way as MSCs from group B. Data obtained can explain why administration of MSCs is not always successful. Preliminary study of MSCs prior to their administration may be used to predict their efficiency in the future.

Administration of multipotent mesenchymal stromal cells restores liver regeneration and improves liver function in obese mice with hepatic steatosis after partial hepatectomy

BackgroundThe liver has the remarkable capacity to regenerate in order to compensate for lost or damaged hepatic tissue. However, pre-existing pathological abnormalities, such as hepatic steatosis (HS), inhibits the endogenous regenerative process, becoming an obstacle for liver surgery and living donor transplantation.Recent evidence indicates that multipotent mesenchymal stromal cells (MSCs) administration can improve hepatic function and increase the potential for liver regeneration in patients with liver damage. Since HS is the most common form of chronic hepatic illness, in this study we evaluated the role of MSCs in liver regeneration in an animal model of severe HS with impaired liver regeneration.MethodsC57BL/6 mice were fed with a regular diet (normal mice) or with a high-fat diet (obese mice) to induce HS. After 30 weeks of diet exposure, 70% hepatectomy (Hpx) was performed and normal and obese mice were divided into two groups that received 5 × 105 MSCs or vehicle via the tail vein immediately after Hpx.ResultsWe confirmed a significant inhibition of hepatic regeneration when liver steatosis was present, while the hepatic regenerative response was promoted by infusion of MSCs. Specifically, MSC administration improved the hepatocyte proliferative response, PCNA-labeling index, DNA synthesis, liver function, and also reduced the number of apoptotic hepatocytes.These effects may be associated to the paracrine secretion of trophic factors by MSCs and the hepatic upregulation of key cytokines and growth factors relevant for cell proliferation, which ultimately improves the survival rate of the mice.ConclusionsMSCs represent a promising therapeutic strategy to improve liver regeneration in patients with HS as well as for increasing the number of donor organs available for transplantation.

Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS). Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs) in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs) suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS-) induced maturation of dendritic cells (DCs) in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG) E2 in this process. In vivo, early administration of murine and human ASCs (hASCs) ameliorated myelin oligodendrocyte protein- (MOG35-55-) induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+) DCs in draining lymph nodes (DLNs). In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function.

Intravitreal administration of multipotent mesenchymal stromal cells triggers a cytoprotective microenvironment in the retina of diabetic mice.

BackgroundDiabetic retinopathy is a common complication of diabetes and the leading cause of irreversible vision loss in the Western world. The reduction in color/contrast sensitivity due to the loss of neural cells in the ganglion cell layer of the retina is an early event in the onset of diabetic retinopathy. Multipotent mesenchymal stromal cells (MSCs) are an attractive tool for the treatment of neurodegenerative diseases, since they could differentiate into neuronal cells, produce high levels of neurotrophic factors and reduce oxidative stress. Our aim was to determine whether the intravitreal administration of adipose-derived MSCs was able to prevent the loss of retinal ganglion cells in diabetic mice.MethodsDiabetes was induced in C57BL6 mice by the administration of streptozotocin. When retinal pro-damage mechanisms were present, animals received a single intravitreal dose of 2 × 105 adipose-derived MSCs or the vehicle. Four and 12 weeks later we evaluated: (a) retinal ganglion cell number (immunofluorescence); (b) neurotrophic factor levels (real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA)); (c) retinal apoptotic rate (TUNEL); (d) retinal levels of reactive oxygen species and oxidative damage (ELISA); (e) electrical response of the retina (electroretinography); (f) pro-angiogenic and anti-angiogenic factor levels (RT-qPCR and ELISA); and (g) retinal blood vessels (angiography). Furthermore, 1, 4, 8 and 12 weeks post-MSC administration, the presence of donor cells in the retina and their differentiation into neural and perivascular-like cells were assessed (immunofluorescence and flow cytometry).ResultsMSC administration completely prevented retinal ganglion cell loss. Donor cells remained in the vitreous cavity and did not differentiate into neural or perivascular-like cells. Nevertheless, they increased the intraocular levels of several potent neurotrophic factors (nerve growth factor, basic fibroblast growth factor and glial cell line-derived neurotrophic factor) and reduced the oxidative damage in the retina. Additionally, MSC administration has a neutral effect on the electrical response of the retina and did not result in a pathological neovascularization.ConclusionsIntravitreal administration of adipose-derived MSCs triggers an effective cytoprotective microenvironment in the retina of diabetic mice. Thus, MSCs represent an interesting tool in order to prevent diabetic retinopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0299-y) contains supplementary material, which is available to authorized users.

A31: Patterns of mesenchymal stem cell distribution in a model of melanoma B16F10

Background Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) are multipotent progenitor cells used to function as cellular delivery vehicles for antitumor agents (Miletic et al., 2007; Gunnarsson et al., 2010; van Eekelen et al., 2010; Studeny et al., 2002). The use of mesenchymal stem cells for the delivery of anticancer drugs based on the hypothesis that stem cells migrate predominantly into tumor tissue. However, the property of MSCs migrate to the tumor is not clear demonstrated and requires further experimental confirmation. The aim of our study is to identify how tumor growth affects on MSCs distribution after systemic administration on a model melanoma B16F10. Materials and methods There were two groups of mice in this study: healthy + intravenously transplantation MSCs (n = 5), B16F10 + intravenously transplantation MSCs (3 days later tumor inoculation) (n = 5). Preparation of MSCs for transplantation: the isolation of MSCs from bone marrow of male C57BL/6 mice was performed using the methods described in work of Esposito M. (Bazan et al., 2004). To investigate their capacity for mesodermal differentiation adipogenic and osteogenic differentiation was carried out. To phenotype cell-surface antigens, MSCs were stained with FITC, PE or APC-conjugated antibodies specific for the following mice antigens CD45-FITC, CD90-FITC, CD34-PE, CD73- PE, and CD105-APC. Stained cells were analysed using FACS Calibur flow cytometer (Becton Dickinson, USA). One hour, 3, 7 and 14 days after cell transplantation (3, 7, 10, 17 days after tumor inoculation, respectively), the animals were sacrified by cervical dislocation. The blood, tumor, lymph nodes, sentinel lymph nodes (regional lymph nodes), lung, liver, spleen, bone marrow, brain, heart from each animal were immediately excised and processed for qPCR analysis. Fur further validatuion of PCR measurements, fluorescence microscopy of a section of bone marrow of mice bearing melanoma B16F10 was performed to detect Hoechst 33342 labeled transplanted cells. Results MSCs isolated from bone marrow possess ability to differentiate into osteocytes and adipocytes, plastic adherence and expression of cluster of differentiation (CD) markers such as CD105, CD73, and CD90 in >95% of the culture with absent expression of markers including CD34 and CD45. In order to study the effect of the melanoma B16F10 growth on the distribution of transplanted MSCs, we have shown the similarities and differences in the distribution of MSCs in the body of healthy animals and animal bearing tumor. 50–65% of Y-positive MSCs detected in lungs as healthy mice, as mice bearing melanoma B16F10 after one hour of MSCs administrated. We found Hoechst labeled MSCs in capillaries of interalveolar septa by fluorescent microscopy. In other examined organs, Y-positive cells were not found. Tumor tissue contained the maximum number of transplanted MSCs 3 days after cell transplantation. In 3 days after MSCs administration heart and brain of mice bearing melanoma B16F10 contained significantly greater number of Y-positive cells compared to those of healthy mice. In 7 and 14 days after transplantation, bone marrow of mice bearing tumor contained more than 2000 Y-positive cells/100,000, while in the bone marrow of healthy animals, onlysingle cellswere detected. Conclusion Melanoma B16F10 affects on pattern of mesenchymal stem cell distribution, which depends on stage of tumor growth.

Mechanisms of improving the neuroprotective effects of multipotent stromal cells after Co-culturing with neurons

We explored a possibility to maximally alleviate a negative impact of stroke by increasing the therapeutic potential of multipotent mesenchymal stromal cells (MMSC). To do that, we studied the intercellular communication between MMSC and neural cells under their co-cultivation in vitro. Co-cultivation of MMSC and neurons from rat cerebral cortex resulted in a cell contents transfer between cells. Evidence of intercellular exchange was obtained using cytoplasmic fluorescent probes. Thus, we showed that cytosol transfer occurs preferentially from neurons toward MMSC. On the other hand, we observed a reverse intercellular transport of mitochondria (from MMSC toward neural cells). Intravenous administration of MMSC in the post-ischemic period reduced the pathological consequences of stroke, which resulted in a decrease of cerebral lesion and a partial recovery of the neurological status. At the same time, MMSC co-cultured with neurons demonstrated more profound neuroprotective efficiency than the unprimed MMSC. The production of one of the most important neurotrophic factors, BDNF, was slightly increased in MMSC after co-cultivation, and the factor was redistributed in the cytoplasm. We conclude that intercellular communications between neural and stem cells improve the protective capacity of MMSC after stroke. This could be a possible approach to increasing the therapeutic efficiency of cell therapy of cerebral ischemia.

Effect of the treatment of focal brain ablation in rat with bone marrow mesenchymal stromal cells on sensorimotor recovery and cytokine production

Increased efficacy of the multipotent mesenchymal stromal cells (MSCs) for the treatment of CNS injuries has been shown when they are administrated within a collagen scaffold, an environment in three dimensions (3D), when compared to the cultivation over a plastic surface (2D). We evaluated the MSC therapeutic effect in the 2D and 3D conditions using the model of focal cortical ablation. Male Wistar rats were submitted to the ablation by aspiration. Intravenous injection (IV) of MSC cultured in 2D, and the intralesional administration (IL) of MSC cultured in 2D or 3D were tested. Administrations were made 24h after ablation. Unskilled and skilled forelimb movements were evaluated by sensorimotor tests. The level of cytokines was measured two days after ablation in the 2D IV groups. Only the MSC 3D IL promoted recovery of the skilled movements. MSC 2D IV promoted recovery of the unskilled movements in all tests, and the MSC 3D IL promoted it only in the adhesive test. MSC 2D IL was unable to promote any recovery. DAPI-stained MSC was found in the perilesional parenchyma at the third post-ablation day after 2D and 3D IL. A significant reduction in the levels of cytokines by the MSC 2D IV was observed in the plasma. Our study strengthens the evidences of the MSC as a prospective therapeutic approach for the CNS injuries.

Multipotent mesenchymal stromal cells decrease transforming growth factor β1 expression in microglia/macrophages and down-regulate plasminogen activator inhibitor 1 expression in astrocytes after stroke

Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor β1 (TGFβ1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFβ1 after stroke, we therefore tested whether MSCs regulate TGFβ1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFβ1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFβ1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFβ1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFβ1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFβ1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes.

Multipotent Mesenchymal Stromal Cells for the Prophylaxis of Acute Graft-versus-Host Disease—A Phase II Study

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9–1.3 × 106/kg when the blood counts indicated recovery. aGVHD of stage II–IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, (P = 0.002). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study.

Multipotent Mesenchymal Stromal Cells as Graft-Versus-Host Disease Prophylaxis: a Prospective Randomised Clinical Trial

AbstractAbstract 1256Acute and chronic graft-versus-host disease (GvHD) develops in more than 50% patients after hemopoietic stem cell transplantation (HSCT) and remains one of main causes of mortality. That's why it is still very important to find new methods of GvHD prophylaxis and treatment. There is a wellknown algorithm of GvHD treatment with glucocorticoids as first line and there are many other options in the treatment of steroid-resistant GvHD, including multipotent mesenchymal stromal cells (MMSC). The efficacy of MMSC in the treatment of steroid-resistant GvHD was reported to be 71–94% (Le Blanc et al., Lancet, 2008; Kebriaei et al., Biol Blood Marrow Transplant, 2009). There are few data on using MMSC as GvHD prophylaxis. So we designed a trial based on a random patient allocation in two groups: receiving standard GvHD prophylaxis (cyclosporine A + methotrexate, cyclosporine A + methotrexate + prednisolon, or cyclosporine A +methotrexate+ mycophenolate mofetil) and receiving MMSC in addition to the standard GvHD prophylaxis. The goal of the study was to investigate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. MMSC were derived from the bone marrow of hemopoietic stem cells donors and cultured in alpha-MEM with 4% human platelet lysate derived from peripheral blood of the same donors. The bone marrow sample was aspirated at the day of HSCT. The trial was approved by local ethic committee and started in October, 2008. Currently 22 patients with stem cell transplantation from related donors are randomised. 10 patients received standard GvHD prophylaxis and 12 received MMSC in addition. 5 patients received fresh cultivated MMSCs while others – MMSCs after freezing and thawing procedures, carried out in standard conditions. Patients characteristics are presented in the Table 1.Table 1Group characteristicsFirst groupSecond groupstandard GvHDprophylaxisstandard GvHDprophylaxis +MMSCMen/women4/62/10Median age, years (range)30.5 (19-59)40.5 (20-63)Diagnosis, nAML/MDS610ALL21CML21Disease stage, nCR912Non-CR10Conditioning regimen, nRIC25MAC87Death at +100 days, n, %1 (10%)0aGvHD (2-4 grade), n, %5 (50%)0cGvHD (lim+ext), n, %4/9 (44,4%)212 (16,7%)Observation time, months3-18.52.5-20Alive, n, %8 (80%)11 (91,6%)MMSCs were administered at the time of blood counts recovery. The dose of MMSC was 0.9–1.25×106/kg. The median day of administration was + 30 day after HSCT (25-54 dd). Most of the patients had fever and chills after MMSC administration. There were no other complications.The clear difference in acute GvHD development was observed in two groups. In standard prophylaxis group acute GvHD II-IV developed in 50% patients, in MMSC group no acute GvHD II-IV occurred (p=0,009). Two patients in the second group had acute GvHD I developed before MMSC administration. The regression of symptoms was observed after MMSC injection. Chronic GvHD was diagnosed in 40% patients in the first group and in 16.7% patients in the second group. There was no difference in disease recurrence rate and graft rejection rate. More infectious complications occurred in patients from MMSC prophylaxis group, mostly of viral origin. There were no lethal infections in this group. The overall mortality was similar in both groups (20% compared to 8.4% in MMSC group).The results of our small study clearly demonstrate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. We intend to continue the study and we incorporated the MMSC as treatment option in steroid-resistant GvHD. Disclosures:No relevant conflicts of interest to declare.

Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.

Endovenous Administration of Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells Prevents Renal Failure in Diabetic Mice

Twenty-five to 40% of diabetic patients develop diabetic nephropathy, a clinical syndrome that comprises renal failure and increased risk of cardiovascular disease. It represents the major cause of chronic kidney disease and is associated with premature morbimortality of diabetic patients. Multipotent mesenchymal stromal cells (MSC) contribute to the regeneration of several organs, including acutely injured kidney. We sought to evaluate if MSC protect kidney function and structure when endovenously administered to mice with severe diabetes. A month after nonimmunologic diabetes induction by streptozotocin injection, C57BL/6 mice presented hyperglycemia, glycosuria, hypoinsulinemia, massive beta-pancreatic islet destruction, low albuminuria, but not renal histopathologic changes (DM mice). At this stage, one group of animals received the vehicle (untreated) and other group received 2 doses of 0.5 x 10(6) MSC/each (MSC-treated). Untreated DM mice gradually increased urinary albumin excretion and 4 months after diabetes onset, they reached values 15 times higher than normal animals. In contrast, MSC-treated DM mice maintained basal levels of albuminuria. Untreated DM mice had marked glomerular and tubular histopathologic changes (sclerosis, mesangial expansion, tubular dilatation, proteins cylinders, podocytes lost). However, MSC-treated mice showed only slight tubular dilatation. Observed renoprotection was not associated with an improvement in endocrine pancreas function in this animal model, because MSC-treated DM mice remained hyperglycemic and hypoinsulinemic, and maintained few remnant beta-pancreatic islets throughout the study period. To study MSC biodistribution, cells were isolated from isogenic mice that constitutively express GFP (MSC(GFP)) and endovenously administered to DM mice. Although at very low levels, donor cells were found in kidney of DM mice 3 month after transplantation. Presented preclinical results support MSC administration as a cell therapy strategy to prevent chronic renal diseases secondary to diabetes.

Systemic Administration of Multipotent Mesenchymal Stromal Cells Reverts Hyperglycemia and Prevents Nephropathy in Type 1 Diabetic Mice

Multipotent mesenchymal stromal cells (MSCs), often labeled mesenchymal stem cells, contribute to tissue regeneration in injured bone and cartilage, as well as in the infarcted heart, brain, and kidney. We hypothesize that MSCs might also contribute to pancreas and kidney regeneration in diabetic individuals. Therefore, in streptozotocin (STZ)-induced type 1 diabetes C57BL/6 mice, we tested whether a single intravenous dose of MSCs led to recovery of pancreatic and renal function and structure. When hyperglycemia, glycosuria, massive beta-pancreatic islets destruction, and mild albuminuria were evident (but still without renal histopathologic changes), mice were randomly separated in 2 groups: 1 received 0.5 × 10 6 MSCs that have been ex vivo expanded (and characterized according to their mesenchymal differentiation potential), and the other group received the vehicle. Within a week, only MSC-treated diabetic mice exhibited significant reduction in their blood glucose levels, reaching nearly euglycemic values a month later. Reversion of hyperglycemia and glycosuria remained for 2 months at least. An increase in morphologically normal beta-pancreatic islets was observed only in MSC-treated diabetic mice. Furthermore, in those animals albuminuria was reduced and glomeruli were histologically normal. On the other side, untreated diabetic mice presented glomerular hyalinosis and mesangial expansion. Thus, MSC administration resulted in beta-pancreatic islets regeneration and prevented renal damage in diabetic animals. Our preclinical results suggest bone marrow-derived MSC transplantation as a cell therapy strategy to treat type 1 diabetes and prevent diabetic nephropathy, its main complication.

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation.

The major obstacle to successful bone marrow transplantation (BMT) is graft versus host disease (GVHD). Multipotent mesenchymal stromal cells (MSC) have shown immunosupressive effects. The aim of this study was to investigate the effect of MSC on the prevention of GVHD in a rat BMT model. We prepared rat GVHD model using allogeneic BMT+4x108 donor splenocyte infusion (DSI). Allogeneic BMT were performed from Spraque Dawley (SD) rats to female Wistar rats by applying 1x108 mononucleer cells (MNC) via tail vein. The recipient animals were conditioned with a myeloablative regimen consisted of 950 cGy total body irradiation prior to BMT. GVHD prophylactic regimen was performed 3 mg/kg/day cyclosporine-A (CsA)+0.25 mg/kg methotrexate (Mtx) IP +1., +3., +6. days. The rats were divided into three control group (CG) and four study group (SG) (n=7). CG-I; only allogeneic BMT+DSI (GVHD model), CG-II; only myeloablative regimen, CG-III; only inspection. GVHD model was performed in all SGs and then SG-I receiving only GVHD prophylactic regimen, SG-II receiving only MSC, SG-III receiving GVHD prophylactic regimen+MSC, SG-IV receiving MSC after GVHD findings developed. Gross (diarrhea, lose weighting, skin findings), histopathological, and immunophenotypical (CD4, CD8, CD25 and plasma IL-2 levels) examinations of GVHD following allogeneic BMT, and the survival analysis of all groups were performed. The survival following allogeneic BMT was significant longer (p<0.05) in SG-I, SG-II and SG-IV than in CG-I (only GVHD model). However, the survival of SG-III (both GVHD prophylactic regimen and MSC administration) was not significant longer than in CG-I (only GVHD model). Gross and histopathological findings of GVHD was significant lower (p<0.05) in SG-I and SG-II than in CG-I (only GVHD model). The expression of CD25 was showed significant increase (p<0.001) in all SGs and CG-I (only GVHD model) than in CG-III (healthy control). Plasma IL-2 levels were showed significant increase in SG-I (p=0.032), SG-II (p=0.018) and SG-IV (p=0.032) than in CG-I (healthy control). This increase was very prominent (p<0.001) in CG-I (only GVHD model) than in CG-III (healthy control). The ratio of CD4/CD8 expression was showed significant lower in SG-I (P=0.008) than in CG-III (healthy control). In contrast, this expression was showed significant increase in SG-II and SG-IV (p=0.014 for both) than in CG-III (healthy control). Finally, clinical use of MSC in both prophylactic and after GVHD developed was as effective as GVHD prophylactic regimen in preventing GVHD. However, MSC combined with GVHD prophylactic regimen was caused strong immunosuppression, and therefore this combination may results in early mortality. This study may form the basis for use of MSC in the prophylaxis and the treatment of GVHD. Our results are encouraging in the use of MSC in GVHD further to clinical trials.