Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury.
ABSTRACT: Multipotent human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after experimental traumatic brain injury (TBI). The present study was designed to investigate whether systemic administration of cell-free exosomes generated from hMSCs cultured in 2-dimensional (2D) conventional conditions or in 3-dimensional (3D) collagen scaffolds promote functional recovery and neurovascular remodeling in rats after TBI. Wistar rats were subjected to TBI induced by controlled cortical impact; 24 h later tail vein injection of exosomes derived from hMSCs cultured under 2D or 3D conditions or an equal number of liposomes as a treatment control were performed. The modified Morris water maze, neurological severity score and footfault tests were employed to evaluate cognitive and sensorimotor functional recovery. Animals were sacrificed at 35 days after TBI. Histological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Compared with liposome-treated control, exosome-treatments did not reduce lesion size but significantly improved spatial learning at 33-35 days measured by the Morris water maze test, and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14-35 days post injury (p < 0.05). Exosome treatments significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn mature neurons in the dentate gyrus as well as reduced neuroinflammation. Exosomes derived from hMSCs cultured in 3D scaffolds provided better outcome in spatial learning than exosomes from hMSCs cultured in the 2D condition. In conclusion, hMSC-generated exosomes significantly improve functional recovery in rats after TBI, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing neuroinflammation. Thus, exosomes derived from hMSCs may be a novel cell-free therapy for TBI, and hMSC-scaffold generated exosomes may selectively enhance spatial learning.
Project description:BACKGROUND:Traumatic brain injury (TBI) is one of the major causes of mortality and disability for all ages worldwide. Mesenchymal stem cells (MSCs)-originated exosomes have provided therapeutic effects. However, as an indispensable component of MSCs, whether odontogenic stem cell-generated exosomes could benefit TBI is still unclear. Thus we aimed to explore the potential of stem cells from human exfoliated deciduous teeth-originated exosomes (SHED-Ex) for the management of TBI. METHODS:First, a transwell system was used to co-culture activated BV-2 microglia cells with SHED. The secretion levels of neuroinflammatory factors and nitrite were evaluated by enzyme-linked immunosorbent assay (ELISA) and Griess assay. Furthermore, purified SHED-Ex were co-cultured with activated BV-2. ELISA, Griess assay, flow cytometry, immunofluorescence, and qRT-PCR were performed to test the levels of inflammatory factors as well as the microglia phenotype. Finally, SHED and SHED-Ex were locally injected into TBI rat models. Basso, Beattie, and Bresnahan (BBB) scores were chosen to evaluate the motor functional recovery. Histopathology and immunofluorescence were performed to measure the lesion volume and neuroinflammation. RESULTS:As a result, SHED-Ex could reduce neuroinflammation by shifting microglia polarization. The administration of SHED-Ex improves rat motor functional recovery and reduces cortical lesion compared with the control group 2 weeks post-injury (P?<?0.05). CONCLUSIONS:The current study demonstrates for the first time that SHED-Ex contribute a therapeutic benefit to TBI in rats, at least in part by shifting microglia polarization to reduce neuroinflammation. The use of odontogenic stem cells, and indeed their exosomes, may be expanded for the treatment of TBI or other neurological disorders.
Project description:Transplantation of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Although several hypotheses to account for these therapeutic effects have been suggested, current thinking is that both neuroprotection and angiogenesis are primarily responsible. In this study, we compared the effects of hMSCs and angiopoietin-1 gene-modified hMSCs (Ang-hMSCs) intravenously infused into rats 6 h after permanent middle cerebral artery occlusion. Magnetic resonance imaging and histologic analyses revealed that rats receiving hMSCs or Ang-hMSCs exhibited comparable reduction in gross lesion volume as compared with the control group. Although both cell types indeed improved angiogenesis near the border of the ischemic lesions, neovascularization and regional cerebral blood flow were greater in some border areas in Ang-hMSC group. Both hMSC- and Ang-hMSC-treated rats showed greater improved functional recovery in the treadmill stress test than did control rats, but the Ang-hMSC group was greater. These results indicate the intravenous administration of genetically modified hMSCs to express angiopoietin has a similar effect on reducing lesion volume as hMSCs, but the Ang-hMSC group showed enhanced regions of increased angiogenesis at the lesion border, and modest additional improvement in functional outcome.
Project description:Transplantation of mesenchymal stem cells (MSCs) derived from bone marrow has been shown to improve functional outcome in spinal cord injury (SCI). We transplanted MSCs derived from human bone marrow (hMSCs) to study their potential therapeutic effect in SCI in the rat. In addition to hMSCs, we used gene-modified hMSCs to secrete brain-derived neurotrophic factor (BDNF-hMSCs). After a dorsal transection lesion was induced at T9, cells were microinjected on each side of the transection site. Fluorogold (FG) was injected into the epicenter of the lesion cavity to identify transected corticospinal tract (CST) neurons. At 5 weeks after transplantation, the animals were perfused. Locomotor recovery improvement was observed for the BDNF-hMSC group, but not in the hMSC group. Structurally there was increased sprouting of injured corticospinal tract and serotonergic projections after hMSC and BDNF-hMSC transplantation. Moreover, an increased number of serotonergic fibers was observed in spinal gray matter including the ventral horn at and below the level of the lesion, indicating increased innervation in the terminal regions of a descending projection important for locomotion. Stereological quantification was performed on the brains to determine neuronal density in primary motor (M1) cortex. The number of FG backfilled cells demonstrated an increased cell survival of CST neurons in M1 cortex in both the hMSC and BDNF-hMSC groups at 5 weeks, but the increase for the BDNF-hMSC group was greater. These results indicate that transplantation of hMSCs hypersecreting BDNF results in structural changes in brain and spinal cord, which are associated with improved functional outcome in acute SCI.
Project description:Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young individuals worldwide. There is currently no effective clinical treatment for TBI, but mesenchymal stem cell-derived exosomes have exhibited promising therapeutic effects. In this study, we performed intracerebroventricular microinjection of human adipose mesenchymal stem cell (hADSC)-derived exosomes (hADSC-ex) in a weight-drop-induced TBI rat model. We found that hADSC-ex promoted functional recovery, suppressed neuroinflammation, reduced neuronal apoptosis, and increased neurogenesis in TBI rats. The therapeutic effects of hADSC-ex were comparable to those of hADSC. Sequential in vivo imaging revealed increasing aggregation of DiR-labeled hADSC-ex in the lesion area. Immunofluorescent staining of coronal brain sections and primary mixed neural cell cultures revealed distinct overlap between CM-DiI-labeled hADSC-ex and microglia/macrophages, indicating that hADSC-ex were mainly taken up by microglia/macrophages. In a lipopolysaccharide-induced inflammatory model, hADSC-ex suppressed microglia/macrophage activation by inhibiting nuclear factor ?B and P38 mitogen-activated protein kinase signaling. These data suggest that hADSC-ex specifically enter microglia/macrophages and suppress their activation during brain injury, thereby inhibiting inflammation and facilitating functional recovery. They also offer new insight into the cellular targeting, uptake and migration of hADSC-ex, and provide a theoretical basis for new therapeutic strategies for central nervous system diseases.
Project description:Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.
Project description:Transplanted multipotent mesenchymal stromal cells (MSCs) improve functional recovery in rats after traumatic brain injury (TBI). In this study the authors tested a novel hypothesis that systemic administration of cell-free exosomes generated from MSCs promotes functional recovery and neurovascular remodeling in rats after TBI.Two groups of 8 Wistar rats were subjected to TBI, followed 24 hours later by tail vein injection of 100 ?g protein of exosomes derived from MSCs or an equal volume of vehicle (phosphate-buffered saline). A third group of 8 rats was used as sham-injured, sham-treated controls. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, modified Neurological Severity Score, and foot-fault tests were performed. Animals were killed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation.Compared with the saline-treated group, exosome-treated rats with TBI showed significant improvement in spatial learning at 34-35 days as measured by the modified Morris water maze test (p < 0.05), and sensorimotor functional recovery (i.e., reduced neurological deficits and foot-fault frequency) was observed at 14-35 days postinjury (p < 0.05). Exosome treatment significantly increased the number of newly generated endothelial cells in the lesion boundary zone and dentate gyrus and significantly increased the number of newly formed immature and mature neurons in the dentate gyrus as well as reducing neuroinflammation.The authors demonstrate for the first time that MSC-generated exosomes effectively improve functional recovery, at least in part, by promoting endogenous angiogenesis and neurogenesis and by reducing inflammation in rats after TBI. Thus, MSC-generated exosomes may provide a novel cell-free therapy for TBI and possibly for other neurological diseases.
Project description:Traumatic brain injury is a leading cause of death and disability around the world. So far, drugs are not available to repair brain damage. Human mesenchymal stem cell (hMSC) transplantation therapy is a promising approach, although the inflammatory microenvironment of the injured brain affects the efficacy of transplanted hMSCs. We hypothesize that reducing the inflammation in the cerebral microenvironment by reducing pro-inflammatory chemokines prior to hMSC administration will improve the efficacy of hMSC therapy. In a rat model of lateral fluid percussion injury, combined pioglitazone (PG) and hMSC (combination) treatment showed less anxiety-like behavior and improved sensorimotor responses to a noxious cold stimulus. Significant reduction in brain lesion volume, neurodegeneration, microgliosis and astrogliosis were observed after combination treatment. TBI induced expression of inflammatory chemokine CCL20 and IL1-β were significantly decreased in the combination treatment group. Combination treatment significantly increased brain-derived neurotrophic factor (BDNF) level and subventricular zone (SVZ) neurogenesis. Taken together, reducing proinflammatory cytokine expression in the cerebral tissues after TBI by PG administration and prior to hMSC therapy improves the outcome of the therapy in which BDNF could have a role.
Project description:Intravenous delivery of mesenchymal stem cells (MSCs) prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischaemia models. Placental growth factor (PlGF) is angiogenic to impaired non-neural tissue. To test the hypothesis that PlGF contributes to the therapeutic benefits of MSC delivery in cerebral ischaemia, we compared the efficacy of systemic delivery of human MSCs (hMSCs) and hMSCs transfected with a fibre-mutant F/RGD adenovirus vector with a PlGF gene (PlGF-hMSCs). A permanent middle cerebral artery occlusion (MCAO) was induced by intraluminal vascular occlusion with a microfilament. hMSCs and PlGF-hMSCs were intravenously injected into the rats 3 h after MCAO. Lesion size was assessed at 3 and 6 h, and 1, 3, 4 and 7 days using MR imaging and histology. Functional outcome was assessed using the limb placement test and the treadmill stress test. Both hMSCs and PlGF-hMSCs reduced lesion volume, induced angiogenesis and elicited functional improvement compared with the control sham group, but the effect was greater in the PlGF-hMSC group. Enzyme-linked immunosorbent assay of the infarcted hemisphere revealed an increase in PlGF in both hMSC groups, but a greater increase in the PlGF-hMSC group. These data support the hypothesis that PlGF contributes to neuroprotection and angiogenesis in cerebral ischaemia, and cellular delivery of PlGF to the brain can be achieved by intravenous delivery of hMSCs.
Project description:Exosomes (Exo) secreted from mesenchymal stem cells (hMSCs) are protective against myocardial injury. The purpose of the study was to investigate the role and mechanisms by which exosomes promote cardiomyocyte survival and function following myocardial infarction (MI). hMSCs were cultured under hypoxic and normoxic conditions. Hypoxia-conditioned hMSC-derived exosomes (Hypo-Exo) and normoxic-conditioned hMSC-derived exosomes (Nor-Exo) were collected and intramyocardially injected into rats with MI. The therapeutic effects of Hypo-Exo and Nor-Exo were evaluated after 4 weeks. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of candidate long noncoding RNA urothelial carcinoma associated 1 (lncRNA-UCA1) in Nor-Exo and Hypo-Exo. Intramyocardial injection of lncRNA-UCA1-knockdown-Hypo-Exo in a rat model of MI was then performed and the cardiac function was characterized. The target and downstream of the molecular mechanism lncRNA-UCA1 was disclosed by luciferase reporter assays and western blot. Circulating exosomal lncRNA-UCA1 level in AMI patients and healthy volunteers was assessed. We found that (1) hMSC exosomal (from hypoxic and normoxic conditions) cardioprotection in vitro and in vivo correlated with the presence of encapsulated lncRNA-UCA1 in exosomes; (2) lncRNA-UCA1 targeted miR-873 via sponging, reducing the latter's suppressive effects on its target XIAP, and this translated into AMPK phosphorylation and increased level of the antiapoptotic protein BCL2; and (3) plasma derived from patients with AMI contained exosomes enriched with the lncRNA-UCA1, unlike that from normal subjects. This study demonstrates that Hypo-Exo lncRNA-UCA1 plays a cardioprotective role via the miR-873-5p/XIAP axis and circulating exosomal lncRNA-UCA1 may be a promising novel biomarker for the diagnosis of AMI.
Project description:Microenvironment extracellular matrices (ECMs) influence cell adhesion, proliferation and differentiation. The ECMs of different microenvironments have distinctive compositions and architectures. This investigation addresses effects ECMs deposited by a variety of cell types and decellularized with a cold-EDTA protocol have on multipotent human mesenchymal stromal/stem cell (hMSC) behavior and differentiation. The cold-EDTA protocol removes intact cells from ECM, with minimal ECM damage and contamination. The decellularized ECMs deposited by cultured hMSCs, osteogenic hMSCs, and two smooth muscle cell (SMC) lines were tested for distinctive effects on the behavior and differentiation of early passage ('naïve') hMSC plated and cultured on the decellularized ECMs. Uninduced hMSC decellularized ECM enhanced naïve hMSC proliferation and cell motility while maintaining stemness. Decellularized ECM deposited by osteogenic hMSCs early in the differentiation process stimulated naïve hMSCs osteogenesis and substrate biomineralization in the absence of added dexamethasone, but this osteogenic induction potential was lower in ECMs decellularized later in the osteogenic hMSC differentiation process. Decellularized ECMs deposited by two smooth muscle cell lines induced naïve hMSCs to become smooth muscle cell-like with distinctive phenotypic characteristics of contractile and synthetic smooth muscle cells. This investigation demonstrates a useful approach for obtaining functional cell-deposited ECM and highlights the importance of ECM specificity in influencing stem cell behavior.