Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy.

Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.

Early Intravenous Infusion of Mesenchymal Stromal Cells Exerts a Tissue Source Age-Dependent Beneficial Effect on Neurovascular Integrity and Neurobehavioral Recovery After Traumatic Cervical Spinal Cord Injury.

Localized vascular disruption after traumatic spinal cord injury (SCI) triggers a cascade of secondary events, including inflammation, gliosis, and scarring, that can further impact recovery. In addition to immunomodulatory and neurotrophic properties, mesenchymal stromal cells (MSCs) possess pericytic characteristics. These features make MSCs an ideal candidate for acute cell therapy targeting vascular disruption, which could reduce the severity of secondary injury, enhance tissue preservation and repair, and ultimately promote functional recovery. A moderately severe cervical clip compression/contusion injury was induced at C7-T1 in adult female rats, followed by an intravenous tail vein infusion 1 hour post-SCI of (a) term-birth human umbilical cord perivascular cells (HUCPVCs); (b) first-trimester human umbilical cord perivascular cells (FTM HUCPVCs); (c) adult bone marrow mesenchymal stem cells; or (d) vehicle control. Weekly behavioral testing was performed. Rats were sacrificed at 24 hours or 10 weeks post-SCI and immunohistochemistry and ultrasound imaging were performed. Both term and FTM HUCPVC-infused rats displayed improved (p < .05) grip strength compared with vehicle controls. However, only FTM HUCPVC-infusion led to significant weight gain. All cell infusion treatments resulted in reduced glial scarring (p < .05). Cell infusion also led to increased axonal, myelin, and vascular densities (p < .05). Although post-traumatic cavity volume was reduced with cell infusion, this did not reach significance. Taken together, we demonstrate selective long-term functional recovery alongside histological improvements with HUCPVC infusion in a clinically relevant model of cervical SCI. Our findings highlight the potential of these cells for acute therapeutic intervention after SCI.

Splenic involvement in umbilical cord matrix-derived mesenchymal stromal cell-mediated effects following traumatic spinal cord injury.

BACKGROUND: The spleen plays an important role in erythrocyte turnover, adaptive immunity, antibody production, and the mobilization of monocytes/macrophages (Mφ) following tissue injury. In response to trauma, the spleen initiates production of inflammatory cytokines, which in turn recruit immune cells to the inflamed tissue, exacerbating damage. Our previous work has shown that intravenous mesenchymal stromal cell (MSC) infusion has potent immunomodulatory effects following spinal cord injury (SCI), associated with the transplanted cells homing to and persisting within the spleen. Therefore, this work aimed to characterize the relationship between the splenic inflammatory response and SCI pathophysiology, emphasizing splenic involvement in MSC-mediated effects. METHODS: Using a rodent model of cervical clip-compression SCI, secondary tissue damage and functional recovery were compared between splenectomised rodents and those with a sham procedure. Subsequently, 2.5 million MSCs from the term human umbilical cord matrix cells (HUCMCs) were infused via tail vein at 1-h post-SCI and the effects were assessed in the presence or absence of a spleen. RESULTS: Splenectomy alone had no effect on lesion volume, hemorrhage, or inflammation. There was also no significant difference between the groups in functional recovery and those in lesion morphometry. Yet, while the infusion of HUCMCs reduced spinal cord hemorrhage and increased systemic levels of IL-10 in the presence of a spleen, these effects were lost with splenectomy. Further, HUCMC infusion was shown to alter the expression levels of splenic cytokines, suggesting that the spleen is an important target and site of MSC effects. CONCLUSIONS: Our results provide a link between MSC function and splenic inflammation, a finding that can help tailor the cells/transplantation approach to enhance therapeutic efficacy.

Early Intravenous Delivery of Human Brain Stromal Cells Modulates Systemic Inflammation and Leads to Vasoprotection in Traumatic Spinal Cord Injury.

UNLABELLED: : Spinal cord injury (SCI) is a life-threatening condition with multifaceted complications and limited treatment options. In SCI, the initial physical trauma is closely followed by a series of secondary events, including inflammation and blood spinal cord barrier (BSCB) disruption, which further exacerbate injury. This secondary pathology is partially mediated by the systemic immune response to trauma, in which cytokine production leads to the recruitment/activation of inflammatory cells. Because early intravenous delivery of mesenchymal stromal cells (MSCs) has been shown to mitigate inflammation in various models of neurologic disease, this study aimed to assess these effects in a rat model of SCI (C7-T1, 35-gram clip compression) using human brain-derived stromal cells. Quantitative polymerase chain reaction for a human-specific DNA sequence was used to assess cell biodistribution/clearance and confirmed that only a small proportion (approximately 0.001%-0.002%) of cells are delivered to the spinal cord, with the majority residing in the lung, liver, and spleen. Intriguingly, although cell populations drastically declined in all aforementioned organs, there remained a persistent population in the spleen at 7 days. Furthermore, the cell infusion significantly increased splenic and circulating levels of interleukin-10-a potent anti-inflammatory cytokine. Through this suppression of the systemic inflammatory response, the cells also reduced acute spinal cord BSCB permeability, hemorrhage, and lesion volume. These early effects further translated into enhanced functional recovery and tissue sparing 10 weeks after SCI. This work demonstrates an exciting therapeutic approach whereby a minimally invasive cell-transplantation procedure can effectively reduce secondary damage after SCI through systemic immunomodulation. SIGNIFICANCE: Central nervous system pericytes (perivascular stromal cells) have recently gained significant attention within the scientific community. In addition to being recognized as major players in neurotrauma, pericytes have been discovered to share a common origin and potentially function with traditionally defined mesenchymal stromal cells (MSCs). Although there have been several in vitro comparisons, the in vivo therapeutic application of human brain-derived stromal cells has not been previously evaluated. This study demonstrates that these cells not only display a MSC phenotype in vitro but also have similar in vivo immunomodulatory effects after spinal cord injury that are more potent than those of non-central nervous system tissue-derived cells. Therefore, these cells are of great interest for therapeutic use in spinal cord injury.

Very high resolution ultrasound imaging for real-time quantitative visualization of vascular disruption after spinal cord injury.

Spinal cord injury (SCI) is characterized by vascular disruption with intramedullary hemorrhage, alterations in blood-spinal cord barrier integrity, and perilesional ischemia. A safe and easily applied imaging technique to quantify evolving intraspinal vascular changes after SCI is lacking. We evaluated the utility of very high resolution ultrasound (VHRUS) imaging to assess SCI-induced vascular disruption in a clinically relevant rodent model. The spinal cords of Wistar rats were lesioned at the 11th thoracic vertebra (Th11) by a 35 g 1-minute clip compression. Three-dimensional quantification of intraspinal hemorrhage using VHRUS (at an acute 90-min and subacute 24-h time point post-SCI) was compared with lesional hemoglobin and extravasated Evans blue dye measured spectrophotometrically. The anatomy of hemorrhage was comparatively assessed using VHRUS and histology. Time-lapse videos demonstrated the evolution of parenchymal hemorrhage. VHRUS accurately depicted the structural (gray and white matter) and vascular anatomy of the spinal cord (after laminectomy) and was safely repeated in the same animal. After SCI, a hyperechoic signal extended from the lesion epicenter. Significant correlations were found between VHRUS signal and hemorrhage in the acute (r=0.88, p<0.0001) and subacute (r=0.85, p<0.0001) phases and extravasated Evans blue (a measure of vascular disruption) in the subacute phase (r=0.94, p<0.0001). Time-lapse videos demonstrated that the expanding parenchymal hemorrhage is preceded by new perilesional hemorrhagic foci. VHRUS enables real-time quantitative live anatomical imaging of acute and subacute vascular disruption after SCI in rats. This technique has important scientific and clinical translational applications.

Adult-derived pluripotent stem cells.

The global incidence of spinal cord injury (SCI) is 15-40 cases per million people, with the socioeconomic and healthcare costs amounting to nearly $10 billion per annum in the USA alone. Despite substantial advances in medical care and surgical technology, many patients with SCI still experience significant long-term neurologic disability. Cellular transplantation offers a promising therapy to address the multifactorial nature of SCI in both the subacute and chronic phase of the injury to promote central nervous system repair and regeneration and to augment existing therapies. Adult-derived stem cells are the least ethically challenging stem cells but, until recently, a major hurdle has been inducing pluripotency to generate the required neural lineages. Improved generation and transfection techniques, combined with positive experimental outcomes in SCI models, suggest that adult-derived induced pluripotent stem cells could be a genuine alternative to embryonic stem cells for clinical treatments. For translation from bench to bedside, the efficacy of induced pluripotent stem cell-derived neural stem and progenitor cells in suitable SCI models needs to be validated further and backed up with rigorous early-stage clinical trials.

Mesenchymal cells in the treatment of spinal cord injury: current & future perspectives.

Nearly 1.5 million people in the US live with a spinal cord injury (SCI). The cost to the healthcare system is estimated at over $10 billion annually likely because over 65% of SCIs occur at the cervical level. Despite medical advances, many SCI patients still experience substantial neurological disability and high dependency with severe loss of motor, sensory and autonomic function. The consensus from a combination of in vivo studies and in vivo models is that mesenchymal or stromal cells, and possibly even neural progenitors, regardless of derivation act through the provision of trophic support and inflammatory modulation. Indeed, they have been found to secrete a wide spectrum of diffusible factors with known roles in both. As such, mesenchymal cells, obtainable from multiple tissues, are ideally suited to addressing many pathophysiological consequences of SCI. Advances in understanding the latter, structural and functional magnetic resonance imaging, image-guided microneurosurgical techniques and transplantable cell biology have enabled the clinical use of cell-based therapies. Of the twenty most recent cell therapy clinical trials for SCI, seven involve adult bone marrow mesenchymal cells and six others umbilical cord cells. This reflects the growing recognition of the clinical potential of perinatal cells. However, a limited understanding of how best to exploit the capabilities of these cells impedes a full-scale clinical deployment. This mini-review focuses on recent developments that are likely to facilitate the targeted application of these cells to treat specific secondary pathophysiological processes.

Cellular treatments for spinal cord injury: the time is right for clinical trials.

More than 1 million people in the United States live with a spinal cord injury (SCI). Despite medical advances, many patients with SCIs still experience substantial neurological disability, with loss of motor, sensory, and autonomic function. Cell therapy is ideally suited to address the multifactorial nature of the secondary events following SCI. Remarkable advances in our understanding of the pathophysiology of SCI, structural and functional magnetic resonance imaging, image-guided micro-neurosurgical techniques, and transplantable cell biology have enabled the use of cell-based regenerative techniques in the clinic. It is important to note that there are more than a dozen recently completed, ongoing, or recruiting cell therapy clinical trials for SCI that reflect the views of many key stakeholders. The field of regenerative neuroscience has reached a stage in which the clinical trials are scientifically and ethically justified. Although experimental models and analysis methods and techniques continue to evolve, no model will completely replicate the human condition. It is recognized that more work with cervical models of contusive/compressive SCI are required in parallel with clinical trials. It is also important that the clinical translation of advances made through well-established and validated experimental approaches in animal models move forward to meet the compelling needs of individuals with SCI and to advance the field of regenerative neuroscience. However, it is imperative that such efforts at translation be done in the most rigorous and informed fashion to determine safety and possible efficacy, and to provide key information to clinicians and basic scientists, which will allow improvements in regenerative techniques and the validation and refinement of existing preclinical animal models and research approaches. The field of regenerative neuroscience should not be stalled at the animal model stage, but instead the clinical trials need to be focused, safe, and ethical, backed up by a robust, translationally relevant preclinical research strategy.

Loss of photoreceptor potential from retinal progenitor cell cultures, despite improvements in survival.

Retinal degeneration (RD) results from photoreceptor apoptosis. Cell transplantation, one potential therapeutic approach, requires expandable stem cells that can form mature photoreceptors when differentiated. Freshly dissociated primary retinal cells from postnatal day 2-6 (PN2-6) mouse retina can give rise, post-transplantation, to photoreceptors in adult recipients. Unfortunately, incorporation rates are low; moreover, photoreceptor potential is lost if the same PN2-6 cells are cultured prior to transplantation. We investigated the identity of the cells forming photoreceptors post-transplantation, using FACS sorted primary postnatal day (PN) 3-5 Rho-eGFP retinal cells. Higher integration rates were achieved for cells that were expressing Rho-eGFP at PN3-5, indicating that post-mitotic photoreceptor precursors already expressing rhodopsin form the majority of integrating rods. We then investigated improvement of cell culture protocols for retinal progenitor cells (RPCs) derived from PN3-5 retinal cells in vitro. We succeeded in improving RPC survival and growth rates 25-fold, by modifying retinal dissociation, replacing N2 supplement with B27 supplement minus retinoic acid (B27-RA) and coating flasks with fibronectin. However, levels of rhodopsin and similar photoreceptor-specific markers still diminished rapidly during growth in vitro, and did not re-appear after in vitro differentiation. Similarly, transplanted RPCs, whether proliferating or differentiated, did not form photoreceptors in vivo. Cultured RPCs upregulate genes such as Sox2 and nestin, markers of more primitive neural stem cells. Use of these cells for RD treatment will require identification of triggers that favour terminal photoreceptor differentiation and survival in vitro prior to transplantation.

Stem cells are resistant to TRAIL receptor-mediated apoptosis.

New therapeutic approaches aim to eradicate tumours by expression of tumouricidal proteins in the tumour stroma. One such anti-neoplastic protein is tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) because it induces apoptosis in cancerous cells, but not in non-transformed cells. Stem cells can migrate to, survive and proliferate in tumours. We examined the suitability of bone marrow-derived adult mesenchymal stem cells (bmMSC), foetal-MSC and umbilical cord matrix stem cells (Wharton's Jelly MSCs) as TRAIL-delivery vehicles. Although all MSC types expressed DR4 and/or DR5, none of them were sensitive to TRAIL-induced apoptosis. Selective activation of DR4 or DR5 with agonistic antibodies or DR5-selective TRAIL-mutant (D269H/E195R) revealed that the TRAIL receptors are inactive in MSCs. In fMSC DR5 was not fully inactivated, its activity however was minimal in comparison to the colon carcinoma cell, Colo205. The intracellular components of the TRAIL-apoptotic pathway, such as pro-caspase-8 and -9 were also expressed at very low; almost undetectable levels in all three MSC types. In conclusion, the MSC species examined are resistant to TRAIL and thus can be suitable tools for TRAIL delivery to tumours.

Stem cell therapies for perinatal brain injuries.

This chapter reviews four groups of paediatric brain injury. The pathophysiology of these injuries is discussed to establish which cells are damaged and therefore which cells represent targets for cell replacement. Next, we review potential sources of cellular replacements, including embryonic stem cells, fetal and neonatal neural stem cells and a variety of mesenchymal stem cells. The advantages and disadvantages of each source are discussed. We review published studies to illustrate where stem cell therapies have been evaluated for therapeutic gain and discuss the hurdles that will need to be overcome to achieve therapeutic benefit. Overall, we conclude that children with paediatric brain injuries or inherited genetic disorders that affect the brain are worthy candidates for stem cell therapeutics.