MSC-treated Animals Research Articles

Mesenchymal stem cells induce dynamic immunomodulation of airway and systemic immune cells in vivo but do not improve survival for mice with H1N1 virus-induced acute lung injury.

Introduction: Influenza A virus (IAV)-induced acute lung injury (ALI) is characterized by pronounced proinflammatory activation and respiratory lung dysfunction. In this study, we performed deep immune profiling on airway and circulating immune cells to examine the effect of immunomodulation and therapeutic outcomes of mesenchymal stem cells (MSCs) therapy in mice with IAV-induced ALI. Methods: Animals were inoculated intranasally with H1N1 IAV, followed by intravenous administration of vehicle, or human clinical-grade, bone marrow-derived MSCs 24-h later, and monitored for six days to evaluate the survival. In another set of animals, bronchoalveolar lavage (BAL) fluid and whole blood were collected three days after infection for flow or mass cytometry (CyTOF) immune profiling analysis. Results: Immune cell population and phenotypic shifts in blood were mapped by CyTOF. Increases were observed in granulocytes and myeloid-derived cells in blood from vehicle-treated animals. While MSC treatment accentuated changes in these populations, naïve B, antibody-secreting B cells, and T cells were decreased in MSC-treated animals at day 3. Compared to sham animals, IAV infection induced a significant 5.5-fold increase in BAL total cell counts, including CD4+ and CD8+ T cells, CD19+ B cells, CD11b+Ly6G+neutrophils, and CD11b+Ly6C+monocytes. MSC treatment significantly decreased BAL total cell counts in IAV-infected mice, specifically the number of infiltrating CD4+ T cells and CD11b+Ly6G+neutrophils. In contrast, there were increases in CD8+ T cells, B cells, and monocytes in the alveolar space in MSC-treated animals. Phenotypic immune cell profiling of blood and BAL revealed a significantly higher proportion of the monocyte population with the M2 phenotype (CD206) in MSC-treated animals; however, this failed to confer protective effects in the survival of infected mice or reduce viral titer in the lung. Further investigation revealed that MSCs were susceptible to IAV infection, leading to increased cell death and potentially affecting their efficacy. Conclusion: These findings provided in vivo evidence that MSCs promote the selective recruitment of immune cells to the site of infection during IAV infection, with reductions in proinflammatory phenotypes. However, MSCs offered no survival benefit in IAV-infected animals, possibly due to MSCs' H1N1 IAV susceptibility and subsequent cell death.

Mesenchymal Stem Cell Therapy for Huntington Disease: A Meta-Analysis.

Mesenchymal stem cell (MSC) therapy has been explored in Huntington disease (HD) as a potential therapeutic approach; however, a complete synthesis of these results is lacking. We conducted a meta-analysis to evaluate the effects of MSCs on HD. Eligible studies published before November 2022 were screened from Embase, PubMed, Web of Science, Medline, and Cochrane in accordance with PRISMA guidelines. ClinicalTrial.gov and the World Health Organization International Clinical Trials Registry Platform were also searched for registered clinical trials. The outcomes in rodent studies evaluated included morphological changes (striatal volume and ventricular volume), motor function (rotarod test, wire hang test, grip strength test, limb-clasping test, apomorphine-induced rotation test, and neuromuscular electromyography activity), cognition (Morris water maze test), and body weight. The initial search returned 362 records, of which 15 studies incorporating 346 HD rodents were eligible for meta-analysis. Larger striatal and smaller ventricular volumes were observed in MSC-treated animals compared to controls. MSCs transplanted before the occurrence of motor dysfunction rescued the motor incoordination of HD. Among different MSC sources, bone marrow mesenchymal stem cells were the most investigated cells and were effective in improving motor coordination. MSC therapy improved muscle strength, neuromuscular electromyography activity, cortex-related motor function, and striatum-related motor function, while cognition was not changed. The body weight of male HD rodents increased after MSC transplantation, while that of females was not affected. Meta-analysis showed a positive effect of MSCs on HD rodents overall, as reflected in morphological changes, motor coordination, muscle strength, neuromuscular electromyography activity, cortex-related motor function, and striatum-related motor function, while cognition was not changed by MSC therapy.

Abstract 107: Systemic Allogeneic Mesenchymal Stem Cell Administration Does Not Attenuate Early Post-resuscitation Brain Injury Or Inflammation In A Porcine Model Of Cardiac Arrest

Introduction: Based on emerging evidence that systemic inflammation exacerbates post-resuscitation organ injury, we recently tested the efficacy of allogeneic mesenchymal stem cell (MSC) administration early after return of spontaneous circulation (ROSC) in a porcine model of cardiac arrest (CA). Initial results indicate that MSCs attenuate post-ROSC cardiac dysfunction, but it is unclear if these protective effects extend to the brain. Accordingly, we aimed to determine if systemic allogeneic MSCs reduce early post-ROSC brain injury and inflammation in swine. Methods: Swine (n=33) were subjected to 10 min CA followed by mechanical CPR with defibrillation and epinephrine (EPI; 0.015 mg/kg iv). Animals that achieved ROSC (n=19) were blindly randomized to intra-arterial saline (n=9) or allogeneic bone marrow-derived MSCs (55±2 x 10 6 ; n=10) 30 min post-ROSC. Blood sampling was performed for 4 hours post-ROSC to quantify serum neurofilament light chain (NFL) levels, after which brain tissue was collected for post-mortem quantification of regional neuronal injury (H&E), activated microglia (IBA-1), and inflammatory gene expression (PCR) vs. healthy control swine (n=8). Results: Animals resuscitated from CA exhibited a significant increase in neuronal injury throughout the brain vs. controls that was not affected by MSC treatment ( A ). Similarly, the post-ROSC rise in serum NFL was not different between saline-treated and MSC-treated animals (2.5±0.6 vs. 2.5±0.8 fold-change vs. baseline; p=0.94). A significant increase in activated microglia ( B ) and inflammatory gene expression ( C ) was observed throughout the brain in both CA groups vs. healthy controls, with particularly prominent changes in the striatum but no notable differences between treatment groups. Conclusions: Despite favorable effects on the heart, systemic allogeneic MSC therapy after ROSC does not significantly affect early markers of brain injury and inflammation in a porcine model of CA.

Impact of Adipose-Derived Stem Cells on Aortic Tensile Strength in an Abdominal Aortic Aneurysm Model

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a highly morbid condition with treatment currently limited to operative interventions. Prior literature has demonstrated the ability of mesenchymal stem cells (MSCs) in attenuating AAA formation. We aim to demonstrate the mechanical and structural characteristics of the aorta with MSC treatment. METHODS: 16 Yorkshire pigs underwent retroperitoneal exposure of the aorta. Peri-adventitial elastase and collagenase was applied to induce AAA formation. The aorta was treated with media-soaked (aneurysm) or MSC-soaked (treatment) GelFoam. Aortic diameter was monitored serially at postoperative (POD) 0, 7, 14, and 21. Histopathologic staining was performed for hematoxylin and eosin. Tensile strength was measured using a tensiometer and calculations were made for Young’s modulus and peak maximum strain. RESULTS: All animals survived the surgery and postoperative course. At POD21, mean aortic diameter increased to 1.483 cm +/- 0.31 for the aneurysm group vs 1.11 cm +/- 0.04 for the treatment animals (p < 0.004). The elastic modulus for the aneurysm group was 15.83 mPa +/- 1.61 compared to 22.13 mPa +/- 2.34 for the treatment groups (p = 0.034) (Fig. 1). There was no difference in the peak stress (p = 0.564). Medial thickness in the aneurysm group was reduced compared to the treatment animals (590.5 υm +/- 19.35 vs 646.10 υm +/- 11.97, p = 0.044). CONCLUSION: This is the first study to demonstrate the mechanical effects of stem-cell therapy on a porcine AAA model. Young’s modulus characterizes the capacity of tissue to withstand stress and was preserved in the MSC-treated animals. This mechanical test can be utilized to develop additional cell and drug-based therapies for AAA.

Three-dimensional cultured mesenchymal stem cells enhance repair of ischemic stroke through inhibition of microglia

BackgroundWe show previously that three-dimensional (3D) spheroid cultured mesenchymal stem cells (MSCs) exhibit reduced cell size thus devoid of lung entrapment following intravenous (IV) infusion. In this study, we determined the therapeutic effect of 3D-cultured MSCs on ischemic stroke and investigated the mechanisms involved.MethodsRats underwent middle cerebral artery occlusion (MCAO) and reperfusion. 1 × 106 of 3D- or 2D-cultured MSCs, which were pre-labeled with GFP, were injected through the tail vain three and seven days after MCAO. Two days after infusion, MSC engraftment into the ischemic brain tissues was assessed by histological analysis for GFP-expressing cells, and infarct volume was determined by MRI. Microglia in the lesion were sorted and subjected to gene expressional analysis by RNA-seq.ResultsWe found that infusion of 3D-cultured MSCs significantly reduced the infarct volume of the brain with increased engraftment of the cells into the ischemic tissue, compared to 2D-cultured MSCs. Accordingly, in the brain lesion of 3D MSC-treated animals, there were significantly reduced numbers of amoeboid microglia and decreased levels of proinflammatory cytokines, indicating attenuated activation of the microglia. RNA-seq of microglia derived from the lesions suggested that 3D-cultured MSCs decreased the response of microglia to the ischemic insult. Interestingly, we observed a decreased expression of mincle, a damage-associated molecular patterns (DAMPs) receptor, which induces the production of proinflammatory cytokines, suggestive of a potential mechanism in 3D MSC-mediated enhanced repair to ischemic stroke.ConclusionsOur data indicate that 3D-cultured MSCs exhibit enhanced repair to ischemic stroke, probably through a suppression to ischemia-induced microglial activation.

Human-Induced Neural and Mesenchymal Stem Cell Therapy Combined with a Curcumin Nanoconjugate as a Spinal Cord Injury Treatment.

We currently lack effective treatments for the devastating loss of neural function associated with spinal cord injury (SCI). In this study, we evaluated a combination therapy comprising human neural stem cells derived from induced pluripotent stem cells (iPSC-NSC), human mesenchymal stem cells (MSC), and a pH-responsive polyacetal–curcumin nanoconjugate (PA-C) that allows the sustained release of curcumin. In vitro analysis demonstrated that PA-C treatment protected iPSC-NSC from oxidative damage in vitro, while MSC co-culture prevented lipopolysaccharide-induced activation of nuclear factor-κB (NF-κB) in iPSC-NSC. Then, we evaluated the combination of PA-C delivery into the intrathecal space in a rat model of contusive SCI with stem cell transplantation. While we failed to observe significant improvements in locomotor function (BBB scale) in treated animals, histological analysis revealed that PA-C-treated or PA-C and iPSC-NSC + MSC-treated animals displayed significantly smaller scars, while PA-C and iPSC-NSC + MSC treatment induced the preservation of β-III Tubulin-positive axons. iPSC-NSC + MSC transplantation fostered the preservation of motoneurons and myelinated tracts, while PA-C treatment polarized microglia into an anti-inflammatory phenotype. Overall, the combination of stem cell transplantation and PA-C treatment confers higher neuroprotective effects compared to individual treatments.

Post‐Resuscitation Neuroinflammation in a Porcine Model of Cardiac Arrest: Effect of Systemic Allogeneic Mesenchymal Stem Cell Therapy

Objective Although advances in cardiopulmonary resuscitation (CPR) have increased the likelihood of achieving return of spontaneous circulation (ROSC) after cardiac arrest (CA), >50% of initially resuscitated patients die before hospital discharge. Based on emerging evidence that a systemic inflammatory response exacerbates post-ROSC injury, we recently tested the efficacy of allogeneic mesenchymal stem cell (MSC) administration early after ROSC in a porcine model of CA. Initial results indicate that MSCs attenuate post-ROSC cardiac dysfunction, but it is unclear if these protective effects extend to the brain. Accordingly, the objective of this study was to determine if systemic allogeneic MSCs reduce post-resuscitation neuroinflammation in swine. Methods Swine (n=33) were subjected to 10 min CA followed by mechanical CPR with defibrillation and intravenous epinephrine (EPI; 0.015 mg/kg). Animals that achieved ROSC (n=19) were blindly randomized to intra-arterial saline (n=9) or allogeneic bone marrow-derived MSCs (55±2 x 106; n=10) 30 min post-ROSC. Serial blood sampling was performed for 4 hours post-ROSC to quantify circulating leukocytes and inflammatory cytokines (IL-6, TNF-α, CRP), after which animals underwent diffusion-weighted brain MRI (DWI) to assess cytotoxic edema via apparent diffusion coefficient (ADC) mapping. Five hours post-ROSC, brain tissue samples were collected from the frontal cortex and hippocampus for quantification of inflammatory gene expression via RT-PCR. Gene expression data were normalized to ß-2 microglobulin and compared to results from healthy control swine (n=8). Results Following ROSC, we observed a significant ~3-fold increase in circulating neutrophils and monocytes that was similar between treatment groups (A). Plasma concentrations of inflammatory cytokines also increased post-ROSC and did not differ significantly between groups, although MSCs tended to attenuate the rise in circulating IL-6 (p=0.09; B). A significant ~2-4-fold increase in inflammatory gene expression was observed in the frontal cortex and hippocampus in both CA groups compared with healthy controls, without notable differences between treatment groups (C). DWI showed significantly lower cortical ADC values in saline-treated animals (736±32 x106 mm2/s) vs. normal controls (809±24 x106 mm2/s), consistent with cytotoxic edema. In contrast, cortical ADC values in MSC-treated animals (791±20 x106 mm2/s) did not differ vs. controls (p=0.52). However, between-group differences were not observed in the hippocampus or when ADC thresholds were applied across the entire brain. Conclusions Despite favorable effects on the heart, systemic allogeneic MSC therapy after resuscitation from CA does not significantly affect multiple parameters of neuroinflammation within the first several hours post-ROSC. Although these findings should be confirmed after a longer follow-up duration, the present results suggest that alternative neuroprotective interventions may be necessary to reduce brain inflammation and injury after resuscitation from CA.

Graft infusion of adipose-derived mesenchymal stromal cells to prevent rejection in experimental intestinal transplantation: A feasibility study.

Mesenchymal stromal cells (MSC) have been proposed as a promising complement to standard immunosuppression in solid organ transplantation because of their immunomodulatory properties. The present work addresses the role of adipose-derived MSC (Ad-MSC) in an experimental model of acute rejection in small bowel transplantation (SBT). Heterotopic allogeneic SBT was performed. A single dose of 1.5x106 Ad-MSC was intra-arterially delivered just before graft reperfusion. Animals were divided into CONTROL (CTRL), CONTROL+Ad-MSC (CTRL_MSC), tacrolimus (TAC), and TAC+Ad-MSC (TAC_MSC) groups. Each Ad-MSC groups was subdivided in autologous and allogeneic third-party groups. Rejection rate and severity were similar in MSC-treated and untreated animals. CTRL_MSC animals showed a decrease in macrophages, T-cell (CD4, CD8, and Foxp3 subsets) and B-cell counts in the graft compared with CTRL, this decrease was attenuated in TAC_MSC animals. Pro- and anti-inflammatory cytokines and some chemokines and growth factors increased in CTRL_MSC animals, especially in the allogeneic group, whereas milder changes were seen in the TAC groups. Ad-MSC did not prevent rejection when administered just before reperfusion. However, they showed immunomodulatory effects that could be relevant for a longer-term outcome. Interference between tacrolimus and the MSC effects should be addressed in further studies.

Therapeutic Potential of Mesenchymal Stem Cells in a Pre-Clinical Model of Diabetic Kidney Disease and Obesity.

Diabetic kidney disease (DKD) is a worldwide microvascular complication of type 2 diabetes mellitus (T2DM). From several pathological mechanisms involved in T2DM-DKD, we focused on mitochondria damage induced by hyperglycemia-driven reactive species oxygen (ROS) accumulation and verified whether mesenchymal stem cells (MSCs) anti-oxidative, anti-apoptotic, autophagy modulation, and pro-mitochondria homeostasis therapeutic potential curtailed T2DM-DKD progression. For that purpose, we grew immortalized glomerular mesangial cells (GMCs) in hyper glucose media containing hydrogen peroxide. MSCs prevented these cells from apoptosis-induced cell death, ROS accumulation, and mitochondria membrane potential impairment. Additionally, MSCs recovered GMCs’ biogenesis and mitophagy-related gene expression that were downregulated by stress media. In BTBRob/ob mice, a robust model of T2DM-DKD and obesity, MSC therapy (1 × 106 cells, two doses 4-weeks apart, intra-peritoneal route) led to functional and structural kidney improvement in a time-dependent manner. Therefore, MSC-treated animals exhibited lower levels of urinary albumin-to-creatinine ratio, less mesangial expansion, higher number of podocytes, up-regulation of mitochondria-related survival genes, a decrease in autophagy hyper-activation, and a potential decrease in cleaved-caspase 3 expression. Collectively, these novel findings have important implications for the advancement of cell therapy and provide insights into cellular and molecular mechanisms of MSC-based therapy in T2DM-DKD setting.

Abstract 359: Systemic Administration of Allogeneic Mesenchymal Stem Cells Attenuates Post-Resuscitation Left Ventricular Dysfunction in a Porcine Model of Cardiac Arrest

Introduction: Advances in CPR have increased the likelihood of achieving ROSC after cardiac arrest (CA), but &gt;50% of initially resuscitated patients die before hospital discharge. Evidence that a systemic inflammatory response exacerbates post-ROSC injury supports investigation of anti-inflammatory therapies in this setting. Accordingly, we tested the efficacy of allogeneic mesenchymal stem cell (MSC) administration early after ROSC in a porcine model of CA. Methods: Swine (n=33) were subjected to 10 min CA followed by mechanical CPR with defibrillation and intravenous epinephrine (EPI; 0.015 mg/kg). Animals that achieved ROSC (n=19) were blindly randomized to intraventricular saline (n=9) or allogeneic bone marrow-derived MSCs (55±2 x 10 6 ; n=10) 30 min post-ROSC. Intravenous EPI was given during the post-ROSC period as needed to maintain MAP ≥60 mmHg. Left ventricular (LV) function and plasma cardiac troponin I (cTnI) were assessed for 4 hr post-ROSC, at which time heart tissue was collected to assess myocardial inflammation (RT-PCR). Results: Compared with saline-treated controls, MSC-treated animals exhibited improved post-ROSC LV wall thickening ( A ) and lower cTnI levels, indicative of reduced myocardial injury. By design, both groups had a similar post-ROSC MAP and cardiac output, but the saline group required significantly more EPI ( B ). Although a similar degree of monocyte infiltration (CCR2) and macrophage expansion (CD68) was observed in both groups, MSCs increased expression of the anti-inflammatory marker Arginase1 ( C ) and tended to attenuate the post-ROSC rise in circulating IL-6 ( D ). Conclusion: Early post-ROSC delivery of allogeneic MSCs attenuates LV dysfunction and reduces the need for pharmacologic hemodynamic support after CA in swine. These results indicate that systemic MSC administration may be an effective immunomodulatory strategy to reduce post-resuscitation injury, particularly if protective effects extend to the brain.

Xenogeneic mesenchymal stem cell transplantation for mandibular defect regeneration.

It is commonly accepted that xenogeneic stem cell transplantation for tissue engineering is faced with host immune rejection. Using a rat critical-size mandibular defect model, this study examined whether the immune rejection can be evaded by diminishing T-cell immunity. To examine donor cell survival and host immune reaction, pig bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled with CM-DiI, loaded onto gelatin sponge (5×106 cells/scaffold), and transplanted into 5-mm mandibular defects of immunocompetent and T cell-deficient athymic rats. To examine the effects of xenogeneic BM-MSCs on bone regeneration, athymic rats undergone the same surgeries were terminated at post-operative weeks 1, 3, and 6. Control rats underwent the same jaw surgery without BM-MSC transplantation. The density of CM-DiI-labeled BM-MSCs decreased with time in both strains of rats. Although it was substantially higher in athymic rats than in immunocompetent rats at post-operative day 1, by day 3-7 the density became comparable between the two strains of rats. Apoptosis reflected by cleaved Caspase-3 staining was low in both strains. Stronger infiltration of neutrophils, macrophages, B cells and CD8+ T cells was found in MSC-treated animals. In athymic rats, infiltration of neutrophils and macrophages was strong, but it occurred later than that in immunocompetent rats. While bone volume fraction significantly increased with time (P<.001), no difference was found between MSC-treated and control groups. Even in hosts with deficient T-cell immunity, xenogeneic BM-MSC transplantation into mandibular critical-sized defects still faces challenges from host innate immunity, which compromises their regenerative efficacy.

Iron nanoparticle-labeled murine mesenchymal stromal cells in an osteoarthritic model persists and suggests anti-inflammatory mechanism of action.

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA, but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages, which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™, iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints, and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery, mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1, 2 and 4weeks]). 4 weeks after injection, mice were imaged by MRI, and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS, CD206, F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory, homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively, and iii) Masson’s Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs, although some ferumoxytol label was detected in proximity to MSCs in macrophages, likely due to phagocytosis of apoptotic MSCs, increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time, we show that MSC-treated mice had increased ratios of %CD206+: %F4/80+ (homeostatic macrophages) (p<0.05), and decreased ratios of %iNOS+: %F4/80+ macrophages (p<0.01), supporting our hypothesis that MSCs may modulate synovial inflammation.

The role of Interleukin 1 receptor antagonist in mesenchymal stem cell-based tissue repair and regeneration.

Interleukin (IL)-1 receptor antagonist (IL-1Ra), a naturally occurring antagonist of IL-1α/IL-1β signaling pathways, has been attributed to the immunosuppressive effects of mesenchymal stem cells (MSCs). MSCs, in IL-1Ra-dependent manner, suppressed production of IL-1β in dermal macrophages, induced their polarization in anti-inflammatory M2 phenotype, attenuated antigen-presenting properties of dendritic cells (DCs), and promoted expansion of immunosuppressive T regulatory cells in the skin, which resulted in enhanced repair of the nonhealing wounds. Reduced activation of inflammasome and suppressed production of IL-1β in macrophages were mainly responsible for beneficial effects of MSC-derived IL-1Ra in alleviation of acute lung injury, dry eye syndrome, and corneal injury. Through the production of IL-1Ra, MSCs reduced migration of DCs to the draining lymph nodes and attenuated generation of inflammatory Th1 and Th17 cells that resulted in alleviation of fulminant hepatitis and rheumatoid arthritis. MSCs, in IL-1Ra-dependent manner, reduced liver fibrosis by suppressing production of Type I collagen in hepatic stellate cells. IL-1Ra was, at least partially, responsible for enhanced proliferation of hepatocytes and chondrocytes in MSC-treated animals with partial hepatectomy and osteoarthritis. Despite of these beneficial effects, IL-1Ra-dependent inhibition of IL-1α/IL-1β-signaling significantly increased risk of infections. Therefore, future experimental and clinical studies should delineate potential side effects of MSC-derived IL-1Ra before IL-1Ra-overexpressing MSCs could be used as a potentially new therapeutic agent for the treatment of acute and chronic inflammatory diseases.

Neuroprotection and immunomodulation following intraspinal axotomy of motoneurons by treatment with adult mesenchymal stem cells

BackgroundTreatment of spinal cord injury is dependent on neuronal survival, appropriate synaptic circuit preservation, and inflammatory environment management. In this sense, mesenchymal stem cell (MSC) therapy is a promising tool that can reduce glial reaction and provide trophic factors to lesioned neurons.MethodsLewis adult female rats were submitted to a unilateral ventral funiculus cut at the spinal levels L4, L5, and L6. The animals were divided into the following groups: IA (intramedullary axotomy), IA + DMEM (Dulbecco’s modified Eagle’s medium), IA + FS (fibrin sealant), IA + MSC (106 cells), and IA + FS + MSC (106 cells). Seven days after injury, qPCR (n = 5) was performed to assess gene expression of VEGF, BDNF, iNOS2, arginase-1, TNF-α, IL-1β, IL-6, IL-10, IL-4, IL-13, and TGF-β. The cellular infiltrate at the lesion site was analyzed by hematoxylin-eosin (HE) staining and immunohistochemistry (IH) for Iba1 (microglia and macrophage marker) and arginase-1. Fourteen days after injury, spinal alpha motor neurons (MNs), evidenced by Nissl staining (n = 5), were counted. For the analysis of astrogliosis in spinal lamina IX and synaptic detachment around lesioned motor neurons (GAP-43-positive cells), anti-GFAP and anti-synaptophysin immunohistochemistry (n = 5) was performed, respectively. Twenty-eight days after IA, the gait of the animals was evaluated by the walking track test (CatWalk; n = 7).ResultsThe site of injury displayed strong monocyte infiltration, containing arginase-1-expressing macrophages. The FS-treated group showed upregulation of iNOS2, arginase-1, proinflammatory cytokine (TNF-α and IL-1β), and antiinflammatory cytokine (IL-10, IL-4, and IL-13) expression. Thus, FS enhanced early macrophage recruitment and proinflammatory cytokine expression, which accelerated inflammation. Rats treated with MSCs displayed high BDNF-positive immunolabeling, suggesting local delivery of this neurotrophin to lesioned motoneurons. This BDNF expression may have contributed to the increased neuronal survival and synapse preservation and decreased astrogliosis observed 14 days after injury. At 28 days after lesion, gait recovery was significantly improved in MSC-treated animals compared to that in the other groups.ConclusionsOverall, the present data demonstrate that MSC therapy is neuroprotective and, when associated with a FS, shifts the immune response to a proinflammatory profile.

Effect of Bone Marrow Mesenchymal Stem Cells on Satellite Cell Proliferation and Apoptosis in Immobilization-Induced Muscle Atrophy in Rats.

BackgroundMuscle atrophy due to disuse occurs along with adverse physiological and functional changes, but bone marrow stromal cells (MSCs) may be able to act as muscle satellite cells to restore myofibers. Thus, we investigated whether MSCs could enhance the proliferation of satellite cells and suppress myonuclear apoptosis during immobilization.Material/MethodsWe isolated, purified, amplified, and identified MSCs. Rats (n=48) were randomized into 3 groups: WB group (n=16), IM-PBS group (n=16), and IM-MSC (n=16). Rat hind limbs were immobilized for 14 d, treated with MSCs or phosphate-buffered saline (PBS), and then studied using immunohistochemistry and Western blot analysis to characterize the proteins involved. Apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) method.ResultsWe compared muscle mass, cross-sectional areas, and peak tetanic forces and noted insignificant differences between PBS- and MSC-treated animals, but satellite cell proliferation was significantly greater after MSC treatment (p<0.05). Apoptotic myonuclei were reduced (p<0.05) after MSC treatment as well. Pro-apoptotic Bax was down-regulated and anti-apoptotic Bcl-2 and p-Akt protein were upregulated (p<0.05).ConclusionsMSCs injected during hind limb immobilization can maintain satellite cell activity by suppressing myonuclear apoptosis.

Renovascular hypertension: Effects of mesenchymal stem cells in the contralateral hypertensive kidney in rats

ABSTRACTMesenchymal stem cells (MSC) induced neovascularization and improved renal morphology of the stenotic kidney in 2 kidneys-1 clip (2K-1C) model of renovascular hypertension. The present study evaluated the effects of MSC in the contralateral hypertensive kidney. Three weeks after left renal artery occlusion, MSC were injected into the tail vein of the 2K-1C rats. Renal function and morphology were analyzed in both kidneys. Labeled MSC were found in stenotic and contralateral kidneys. Hypertensive 2K-1C animals presented increased circulating levels of Angiotensin II (Ang II) and renin. MSC prevented the progressive increase of blood pressure and reduced circulating Ang II and renin levels. Stenotic kidney showed reduced renal plasma flow (RPF) and glomerular filtration rate (GFR), whereas the contralateral kidney had a tendency (p > 0.5) of reduction in GFR in spite of unchanged RPF. MSC treatment caused an improvement in GFR with no effect of on RPF in the stenotic kidney. Contralateral kidney showed increased diuresis and natriuresis that were even higher in MSC-treated animals, indicating that cell treatment improved the capacity of the contralateral kidney to excrete sodium. Contralateral kidney expressed higher levels of inflammatory cytokines (IL-6, TNF-α) and signs of fibrosis, which were attenuated by MSC treatment. MSC treatment improved the stenotic kidney function, and it was also beneficial to the contralateral hypertensive kidney because it improved the morphology and preserved its capacity to excrete sodium.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice

Background aimsExisting treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. MethodsInduced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 106 cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. ResultsBlood flow in the MCA-derived MSC-treated animals was higher at each day (P < 0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P < 0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DiscussionMCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival.

BackgroundNumerous studies have described the immunosuppressive capacity of mesenchymal stem cells (MSC) but these studies use mixtures of heterogeneous progenitor cells for in vitro expansion. Recently, multipotent MSC have been prospectively identified in murine bone marrow (BM) on the basis of PDFGRa+ SCA1+ CD45− TER119− (PαS) expression but the immunomodulatory capacity of these MSC is unknown.MethodsWe isolated PαS MSC by high-purity FACS sorting of murine BM and after in vitro expansion we analyzed the in vivo immunomodulatory activity during acute pneumonia. PαS MSC (1 × 106) were applied intratracheally 4 h after acute respiratory Klebsiella pneumoniae induced infection.ResultsPαS MSC treatment resulted in significantly reduced alveolitis and protein leakage in comparison to mock-treated controls. PαS MSC-treated mice exhibited significantly reduced alveolar TNF-α and IL-12p70 expression, while IL-10 expression was unaffected. Dissection of respiratory dendritic cell (DC) subsets by multiparameter flow cytometry revealed significantly reduced lung DC infiltration and significantly reduced CD86 costimulatory expression on lung CD103+ DC in PαS MSC-treated mice. In the post-acute phase of pneumonia, PαS MSC-treated animals exhibited significantly reduced respiratory IL-17+ CD4+ T cells and IFN-γ+ CD4+ T cells. Moreover, PαS MSC treatment significantly improved overall pneumonia survival and did not increase bacterial load.ConclusionIn this study we demonstrated for the first time the feasibility and in vivo immunomodulatory capacity of prospectively defined MSC in pneumonia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0288-1) contains supplementary material, which is available to authorized users.

Chronic spinal cord lesions respond positively to tranplants of mesenchymal stem cells.

Despite substantial advances in surgical care and rehabilitation, the consequences of spinal cord injury (SCI) continue to present major challenges. Here we investigate whether transplantation of mesenchymal stem cells (MSCs) in mice during the chronic stage of SCI has benefits in terms of morphological and functional outcomes. Mice were subjected to laminectomy at the T9 level, followed by a 1 minute spinal cord compression with a vascular clip. Four weeks later, 8 × 105 MSCs obtained from GFP mice were injected into the injury site. After eight weeks the analyses were performed. The spinal cords of MSC-treated animals exhibited better white-matter preservation, greater numbers of fibers, higher levels of trophic factor expression, and better ultrastructural tissue organization. Furthermore, transplanted MSCs were not immunoreactive for neural markers, indicating that these cells mediate functional recovery through a paracrine effect, rather than by transforming into and replacing damaged glia in the spinal cord. MSC-treated mice also showed better functional improvement than control animals. We conclude that MSC-based cell therapy, even when applied during the chronic phase of SCI, leads to changes in a number of structural and functional parameters, all of which indicate improved recovery.

Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.

Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.