Mechanoporation is a potential indicator of tissue strain and subsequent degeneration following experimental traumatic brain injury.

BACKGROUND: An increases in plasma membrane permeability is part of the acute pathology of traumatic brain injury and may be a function of excessive membrane force. This membrane damage, or mechanoporation, allows non-specific flux of ions and other molecules across the plasma membrane, and may ultimately lead to cell death. The relationships among tissue stress and strain, membrane permeability, and subsequent cell degeneration, however, are not fully understood. METHODS: Fluorescent molecules of different sizes were introduced to the cerebrospinal fluid space prior to injury and animals were sacrificed at either 10 min or 24 h after injury. We compared the spatial distribution of plasma membrane damage following controlled cortical impact in the rat to the stress and strain tissue patterns in a 3-D finite element simulation of the injury parameters. FINDINGS: Permeable cells were located primarily in the ipsilateral cortex and hippocampus of injured rats at 10 min post-injury; however by 24 h there was also a significant increase in the number of permeable cells. Analysis of colocalization of permeability marker uptake and Fluorojade staining revealed a subset of permeable cells with signs of degeneration at 24 h, but plasma membrane damage was evident in the vast majority of degenerating cells. The regional and subregional distribution patterns of the maximum principal strain and shear stress estimated by the finite element model were comparable to the cell membrane damage profiles following a compressive impact. INTERPRETATION: These results indicate that acute membrane permeability is prominent following traumatic brain injury in areas that experience high shear or tensile stress and strain due to differential mechanical properties of the cell and tissue organization, and that this mechanoporation may play a role in the initiation of secondary injury, contributing to cell death.

Mesenchymal stromal SB623 cell implantation mitigates nigrostriatal dopaminergic damage in a mouse model of Parkinson's disease.

Regenerative medicine for the treatment of motor features in Parkinson's disease (PD) is a promising therapeutic option. Donor cells can simultaneously address multiple pathological mechanisms while responding to the needs of the host tissue. Previous studies have demonstrated that mesenchymal stromal cells (MSCs) promote recovery using various animal models of PD. SanBio Inc. has developed a novel cell type designated SB623, which are adult bone marrow-derived MSCs transfected with Notch intracellular domain. In this preclinical study, SB623 cells protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal injury when transplanted unilaterally into C57BL/6 mouse striatum 3 days prior to toxin exposure. Specifically, mice with the SB623 cell transplants revealed significantly higher levels of striatal dopamine, tyrosine hydroxylase immunoreactivity and stereological nigral cell counts in the ipsilateral hemisphere vs vehicle-treated mice following MPTP administration. Interestingly, improvement in markers of striatal dopaminergic integrity was also noted in the contralateral hemisphere. These data indicate that MSCs transplantation, specifically SB623 cells, may represent a novel therapeutic option to ameliorate damage related to PD, not only at the level of striatal terminals (i.e. the site of implantation) but also at the level of the nigral cell body. Copyright © 2015 John Wiley & Sons, Ltd.

Comparing the angiogenic potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells.

BACKGROUND: Angiogenesis is a critical part of the endogenous repair process in brain injury and disease, and requires at least two sequential steps. First, angiogenic sprouting of endothelial cells occurs, which entails the initial proliferation of endothelial cells and remodeling of the surrounding extracellular matrix. Second, vessel stabilization is necessary to prevent vascular regression, which relies on vascular smooth muscle recruitment to surround the young vessels. Marrow stromal cells (MSCs) have been shown to promote revascularization after hindlimb ischemia, cardiac ischemia, and stroke. SB623 cells are derived from marrow stromal cells by transfection with a Notch1 intracellular domain (NICD)-expressing plasmid and are known to elicit functional improvement in experimental stroke. These cells are currently used in human clinical testing for treatment of chronic stroke. In the current study, the angiogenic property of SB623 cells was investigated using cell-based assays. METHODS: Angiogenic paracrine factors secreted by SB623 cells and the parental MSCs were identified using the Qantibody Human Angiogenesis Array. To measure the angiogenic activity of conditioned medium from SB623 cells and MSCs, endothelial tube formation in the human umbilical vein endothelial cell (HUVEC) assay and endothelial cell sprouting and branching in the rodent aortic ring assay were quantified. To validate the angiogenic contribution of VEGF in conditioned medium, endothelial cells and aortic rings were treated with SU5416, which inhibits VEGFR2 at low dose. RESULTS: Conditioned medium from SB623 cells promoted survival and proliferation of endothelial cells under serum-deprived conditions and supports HUVEC vascular tube formation. In a rodent aortic ring assay, there was enhanced endothelial sprouting and branching in response to SB623-derived conditioned medium. SU5416 treatment partially reversed the effect of conditioned medium on endothelial cell survival and proliferation while completely abrogate HUVEC tube formation and endothelial cell sprouting and branching in aortic ring assays. CONCLUSIONS: These data indicate that SB623 cell-secreted angiogenic factors promoted several aspects of angiogenesis, which likely contribute to promoting recovery in the injured brain.

Comparing the immunosuppressive potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells.

BACKGROUND: SB623 cells are expanded from marrow stromal cells (MSCs) transfected with a Notch intracellular domain (NICD)-expressing plasmid. In stroke-induced animals, these cells reduce infarct size and promote functional recovery. SB623 cells resemble the parental MSCs with respect to morphology and cell surface markers despite having been in extended culture. MSCs are known to have immunosuppressive properties; whether long-term culture of MSCs impact their immunomodulatory activity has not been addressed. METHODS: To assess the possible senescent properties of SB623 cells, we performed cell cycle related assays and beta-galactosidase staining. To assess the immunomodulatory activity of these expanded NICD-transfected MSCs, we performed co-cultures of SB623 cells or MSCs with either enriched human T cells or monocytes and assessed cytokine production by flow cytometry. In addition, we monitored the immunosuppressive activity of SB623 cells in both allogenic and xenogenic mixed lymphocyte reaction (MLR). RESULTS: Compared to MSCs, we showed that a small number of senescent-like cells appear in each lot of SB623 cells. Nevertheless, we demonstrated that these cells suppress human T cell proliferation in both the allogeneic and xenogeneic mixed lymphocyte reaction (MLR) in a manner comparable to MSCs. IL-10 producing T cells were generated and monocyte-dendritic cell differentiation was dampened by co-culture with SB623 cells. Compared to the parental MSCs, SB623 cells appear to exert a greater inhibitory impact on the maturation of dendritic cells as demonstrated by a greater reduction in the surface expression of the co-stimulatory molecule, CD86. CONCLUSION: The results demonstrated that the immunosuppressive activity of the expanded NICD-transfected MSCs is comparable to the parental MSCs, in spite of the appearance of a small number of senescent-like cells.

Stem cell survival and functional outcome after traumatic brain injury is dependent on transplant timing and location.

PURPOSE: Recent work indicates that transplanted neural stem cells (NSCs) can survive, migrate to the injury site, and facilitate recovery from traumatic brain injury (TBI). The present study manipulated timing and location of NSC transplants following controlled cortical impact injury (CCI) in mice to determine optimal transplant conditions. METHODS: In Experiment 1 (timing), NSCs (E14.5 mouse) were injected into the host striatum, ipsilateral to the injury, at 2, 7, or 14 days. In Experiment 2 (location), NSCs or vehicle were injected into the mouse striatum (7 days post-CCI) either ipsilateral or contralateral to the injury and cognitive and motor abilities were assessed from weeks 1-8 post-transplant. Histological measures of NSC survival, migration, and differentiation were taken at 6 and 8 weeks post-transplant. RESULTS: The results demonstrate that: (1) 2-7 days post-injury is the optimal time-range for delivering NSCs; (2) time of transplantation does not affect short-term phenotypic differentiation; (3) transplant location affects survival, migration, phenotype, and functional efficacy; and (4) NSC-mediated functional recovery is not contingent upon NSC migration or phenotypic differentiation. CONCLUSIONS: These findings provide further support for the idea that mechanisms other than the replacement of damaged neurons or glia, such as NSC-induced increases in protective neurotrophic factors, may be responsible for the functional recovery observed in this model of TBI.

Human mesenchymal stromal cells and their derivative, SB623 cells, rescue neural cells via trophic support following in vitro ischemia.

Cell transplantation is a promising treatment strategy for many neurological disorders, including stroke, which can target multiple therapeutic mechanisms in a sustained fashion. We investigated the ability of human mesenchymal stromal cells (MSCs) and MSC-derived SB623 cells to rescue neural cells via trophic support following an in vitro stroke model. Following oxygen glucose deprivation, cortical neurons or hippocampal slices were cocultured with either MSCs or SB623 cells separated by a semiporous membrane (prohibits cell-cell contact) or with MSC- or SB623 cell-conditioned medium. MSCs, SB623 cells, MSC-conditioned media, and SB623 cell-conditioned media all significantly reduced neural cell damage/death compared to untreated conditions, and the rescue effect of the conditioned media was dose dependent. We identified 11 neurotrophic factors secreted by MSCs and/or SB623 cells. This study emphasizes the importance of trophic support provided by marrow-derived cells, which likely contributes to the efficacy of cell therapy for brain injury.

Extracellular matrix produced by bone marrow stromal cells and by their derivative, SB623 cells, supports neural cell growth.

Several studies have shown the benefits of transplanting bone marrow-derived multipotent mesenchymal stromal cells (MSC) into neurodegenerative lesions of the central nervous system, despite a low engraftment rate and the poor persistence of grafts. It is known that the extracellular matrix (ECM) modulates neuritogenesis and glial growth, but little is known about effects of MSC-derived ECM on neural cells. In this study, we demonstrate in vitro that the ECM produced by MSC can support neural cell attachment and growth. We also compare the neurosupportive properties of MSC to the MSC derivative, SB623 cells, which is being developed as a cell therapy for stroke. Embryonic rat brain cortical cells cultured for 3 weeks on human MSC- and SB623 cell-derived ECM exhibit about a 1.5 and 3 times higher metabolic activity, respectively, compared with the cultures grown on poly-D-lysine (PDL), although the initial neural cell adhesion to cell-derived ECM and PDL is similar. The MSC- and SB623 cell-derived ECM protects neural cells from nutrient and growth factor deprivation. Under the conditions used, only neurons grow on PDL. In contrast, both MSC- and SB623 cell-derived ECMs support the growth of neurons, astrocytes, and oligodendrocytes, as demonstrated by immunostaining. Morphologically, neurons on cell-derived ECM form more complex and extended neurite networks than those cultured on PDL. Together, these data indicate that the beneficial effect of MSC and SB623 cells in neurotransplantation could be explained in part by the neurosupportive properties of the ECM produced by these cells.

Laminin and fibronectin scaffolds enhance neural stem cell transplantation into the injured brain.

Cell transplantation offers the potential to treat central nervous system injuries, largely because multiple mechanisms can be targeted in a sustained fashion. It is crucial that cells are transplanted into an environment that is favourable for extended survival and integration within the host tissue. Given the success of using fetal tissue grafts for traumatic brain injury, it may be beneficial to mimic key aspects of these grafts (e.g. three-dimensionality, cell-cell and cell-matrix support) to deliver cells. Extracellular matrix proteins such as fibronectin and laminin are involved in neural development and may provide adhesive support for donor cells and mediate subsequent cell signalling events. In this study, neural stem cells were transplanted into the traumatically injured mouse brain within a tissue-engineered construct containing either a laminin- or fibronectin-based scaffold. Cells delivered within the scaffolds were more widely distributed in the injured brain compared to cells delivered in media alone. There were no differences in donor cell survival at 1 week post-transplant; however, by 8 weeks post-transplant, cells delivered within the scaffolds showed improved survival compared to those transplanted in media alone. Survival was more enhanced with the laminin-based scaffold compared to the fibronectin-based scaffold. Furthermore, behavioural analyses indicated that mice receiving neural stem cells within the laminin-based scaffold performed significantly better than untreated mice on a spatial learning task, supporting the notion that functional recovery correlates positively with donor cell survival. Together these results suggest that the use of appropriate extracellular matrix-based scaffolds can be exploited to improve cell transplantation therapy.

In vitro neural injury model for optimization of tissue-engineered constructs.

Stem cell transplantation is a promising approach for the treatment of traumatic brain injury, although the therapeutic benefits are limited by a high degree of donor cell death. Tissue engineering is a strategy to improve donor cell survival by providing structural and adhesive support. However, optimization prior to clinical implementation requires expensive and time-consuming in vivo studies. Accordingly, we have developed a three-dimensional (3-D) in vitro model of the injured host-transplant interface that can be used as a test bed for high-throughput evaluation of tissue-engineered strategies. The neuronal-astrocytic cocultures in 3-D were subjected to mechanical loading (inducing cell death and specific astrogliotic alterations) or to treatment with transforming growth factor-beta1 (TGF-beta1), inducing astrogliosis without affecting viability. Neural stem cells (NSCs) were then delivered to the cocultures. A sharp increase in the number of TUNEL(+) donor cells was observed in the injured cocultures compared to that in the TGF-beta1-treated and control cocultures, suggesting that factors related to mechanical injury, but not strictly astrogliosis, were detrimental to donor cell survival. We then utilized the mechanically injured cocultures to evaluate a methylcellulose-laminin (MC-LN) scaffold designed to reduce apoptosis. When NSCs were co-delivered with MC alone or MC-LN to the injured cocultures, the number of caspase(+) donor cells significantly decreased compared to that with vehicle delivery (medium). Collectively, these results demonstrate the utility of an in vitro model as a pre-animal test bed and support further investigation of a tissue-engineering approach for chaperoned NSC delivery targeted to improve donor cell survival in neural transplantation.

Plasma fibronectin is neuroprotective following traumatic brain injury.

Promotion of repair and regeneration following traumatic brain injury remains a challenging clinical problem. While significant efforts have been made to reduce inhibitory extracellular matrix expression following central nervous system injury, much less attention has been given to the role of endogenous reparative matrix proteins, such as fibronectin. Traumatic brain injury leads to increased levels of plasma-derived fibronectin in the brain tissue, though the specific function of this protein following neurotrauma was unknown. In this study, we utilized conditional plasma fibronectin (pFN) knockout mice to examine the role of fibronectin following a traumatic insult. Injured mice deficient in pFN performed significantly worse on both motor and cognitive tasks, had significantly increased lesion volume and apoptotic cell death, and had significantly less phagocytic cells in the injured cortex compared to injured mice with normal pFN levels. Moreover, intravenous injections of fibronectin prior to the injury restored the neural deficits seen in the pFN deficient mice to that of wild type injured mice. These results demonstrate that fibronectin is neuroprotective to the traumatically injured brain and identify a novel target for therapeutic interventions.

Role of plasma fibronectin in the foreign body response to biomaterials.

Host responses to biomaterials control the biological performance of implanted medical devices. Upon implantation, synthetic materials adsorb biomolecules, which trigger an inflammatory cascade comprising coagulation, leukocyte recruitment/adhesion, and foreign body reaction. The foreign body reaction and ensuing fibrous encapsulation severely limit the in vivo performance of numerous biomedical devices. While it is well established that plasma fibrinogen and secreted cytokines modulate leukocyte recruitment and maturation into foreign body giant cells, mediators of chronic inflammation and fibrous encapsulation of implanted biomaterials remain poorly understood. Using plasma fibronectin (pFN) conditional knock-out mice, we demonstrate that pFN modulates the foreign body response to polyethylene terephthalate disks implanted subcutaneously. Fibrous collagenous capsules were two-fold thicker in mice depleted of pFN compared to controls. In contrast, deletion of pFN did not alter acute leukocyte recruitment to the biomaterial, indicating that pFN modulates chronic fibrotic responses. The number of foreign body giant cells associated with the implant was three times higher in the absence of pFN while macrophage numbers were not different, suggesting that pFN regulates the formation of biomaterial-associated foreign body giant cells. Interestingly, cellular FN (cFN) was present in the capsules of both normal and pFN-depleted mice, suggesting that cFN could not compensate for the loss of pFN. These results implicate pFN in the host response to implanted materials and identify a potential target for therapeutic intervention to enhance the biological performance of biomedical devices.

Fibronectin and laminin increase in the mouse brain after controlled cortical impact injury.

The complex environment of the traumatically injured brain exhibits aspects of inhibition and ongoing cell death together with attempts at repair and regeneration. Elucidating these events and exploiting those factors involved in endogenous repair and regeneration may aid in developing more effective treatments for traumatic brain injury. Two extracellular matrix proteins critical to neural development--fibronectin and laminin--may also play a protective or reparative role in the injury response. While both of these proteins have been found to increase following human brain injury,the presence of these proteins has not been studied in a clinically-relevant animal model of blunt head trauma. In this study, we examined the spatiotemporal profile of both fibronectin and laminin in the mouse brain following controlled cortical impact injury. Fibronectin and laminin reactivity was localized to the injury penumbra up to 14 days post-injury and was significantly higher than uninjured controls at 3 days post-injury. Upon examining the spatial relationship of fibronectin and laminin to support cells, we found macrophages/activated microglia prominently present in the fibronectin-rich tissue, consistent with a role for fibronectin in facilitating debris clearing. Furthermore, reactive astrocyte processes were found sheathing laminin positive vasculature, suggesting that laminin may play a role in repairing the blood-brain barrier. These and other hypothesized reparative roles for fibronectin and laminin after traumatic brain injury are discussed.