Anti-Inflammatory Effects of Encapsulated Human Mesenchymal Stromal/Stem Cells and a Method to Scale-Up Cell Encapsulation.

Mesenchymal stem/stromal cells (MSC) promote recovery in a wide range of animal models of injury and disease. They can act in vivo by differentiating and integrating into tissues, secreting factors that promote cell growth and control inflammation, and interacting directly with host effector cells. We focus here on MSC secreted factors by encapsulating the cells in alginate microspheres, which restrict cells from migrating out while allowing diffusion of factors including cytokines across the capsules. One week after intrathecal lumbar injection of human bone marrow MSC encapsulated in alginate (eMSC), rat IL-10 expression was upregulated in distant rat spinal cord injury sites. Detection of human IL-10 protein in rostrally derived cerebrospinal fluid (CSF) indicated distribution of this human MSC-secreted cytokine throughout rat spinal cord CSF. Intraperitoneal (IP) injection of eMSC in a rat model for endotoxemia reduced serum levels of inflammatory cytokines within 5 h. Detection of human IL-6 in sera after injection of human eMSC indicates rapid systemic distribution of this human MSC-secreted cytokine. Despite proof of concept for eMSC in various disorders using animal models, translation of encapsulation technology has not been feasible primarily because methods for scale-up are not available. To scale-up production of eMSC, we developed a rapid, semi-continuous, capsule collection system coupled to an electrosprayer. This system can produce doses of encapsulated cells sufficient for use in clinical translation.

Efficacy of MSC in Patients with Severe COVID-19: Analysis of the Literature and a Case Study.

Patients with severe COVID-19 experience cytokine storm, an uncontrolled upregulation of pro-inflammatory cytokines, which if unresolved leads to acute respiratory distress syndrome (ARDS), organ damage, and death. Treatments with mesenchymal stromal cells (MSC) [Viswanathan S, Shi Y, Galipeau J, et al. Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy. 2019;21:1019-1024] appear to be effective in reducing morbidity and mortality. MSC respond to pro-inflammatory cytokines by releasing anti-inflammatory factors and mobilizing immune cells. We analyzed 82 COVID-19 clinical trials registered at ClinicalTrials.gov to determine MSC dosing, routes of administration, and outcome measures. Nearly all trials described the use of intravenous delivery with most doses ranging between 50 and 125 million MSC/treatment, which overlaps with a minimal effective dose range that we described previously. We also searched the literature to analyze clinical trial reports that used MSC to treat COVID-19. MSC were found to improve survival and oxygenation, increase discharge from intensive care units and hospitals, and reduce levels of pro-inflammatory markers. We report on a 91-year-old man with severe COVID-19 who responded rapidly to MSC treatment with transient reductions in several pro-inflammatory markers and delayed improvement in oxygenation. The results suggest that frequent monitoring of pro-inflammatory markers for severe COVID-19 will provide improved treatment guidelines by determining relationships between cytokine storms and ARDS. We propose that markers for cytokine storm are leading indicators for ARDS and that measurement of cytokines will indicate earlier treatment with MSC than is performed now for ARDS in severe COVID-19.

Trends in mesenchymal stem cell clinical trials 2004-2018: Is efficacy optimal in a narrow dose range?

The number of clinical trials using mesenchymal stem cells (MSCs) has increased since 2008, but this trend slowed in the past several years and dropped precipitously in 2018. Previous reports have analyzed MSC clinical trials by disease, phase, cell source, country of origin, and trial initiation date, all of which can be downloaded directly from ClinicalTrials.gov. We have extended analyses to a larger group of 914 MSC trials reported through 2018. To search for potential factors that may influence the design of new trials, we extracted data on routes of administration and dosing from individual ClinicalTrials.gov records as this information cannot be downloaded directly from the database. Intravenous (IV) injection is the most common, least invasive and most reproducible method, accounting for 43% of all trials. The median dose for IV delivery is 100 million MSCs/patient/dose. Analysis of all trials using IV injection that reported positive outcomes indicated minimal effective doses (MEDs) ranging from 70 to 190 million MSCs/patient/dose in 14/16 trials with the other two trials administering much higher doses of at least 900 million cells. Dose-response data showing differential efficacy for improved outcomes were reported in only four trials, which indicated a narrower MED range of 100-150 million MSCs/patient with lower and higher IV doses being less effective. The results suggest that it may be critical to determine MEDs in early trials before proceeding with large clinical trials.

Potential risk of clonally expanded amnion mesenchymal stem cell transplants in contused spinal cords.

OBJECTIVE: Mesenchymal stem/stromal cells (MSC) promote recovery after spinal cord injury (SCI) using adult bone marrow MSC (BM-MSC). Newborn tissues are a convenient source of MSC that does not involve an invasive procedure for cell collection. In this study the authors tested the effects of rat amnion MSC clone (rAM-MSC) in SCI. METHODS: We tested intra-parenchymal injection of a GFP+ rat rAM-MSC clone derived from E18.5 rats in rat SCI and measured behavioral recovery (BBB scores), histology and X-ray opacity. Expression of aggrecan was measured in culture after treatment with TGFß. RESULTS: Injection of rAM-MSC after SCI did not improve BBB scores compared to control vehicle injections; rather they reduced scores significantly over 6 weeks. Spinal cords injected with rAM-MSC were hard in regions surrounding the SCI site, which was confirmed by X-ray opacity. Whole mount imaging of these cords showed minimal tissue loss in the SCI site that occurred in SCI controls, and persistence of GFP+ rAM-MSC. Mason's Trichrome staining of tissue sections showed more intense staining for extracellular matrix (ECM) surrounding and extending beyond the SCI site with injections of rAM-MSC but not in controls. In response to TGF-ß treatment in culture, chondrogenic aggrecan was expressed at higher levels in rAM-MSC than in rBM-MSC, suggesting that the upregulation of TGF-ß in SCI sites may promote chondrogenic differentiation. CONCLUSION: Acute injection after SCI of a clonally expanded rAM-MSC resulted in aberrant differentiation towards a chondrocytic phenotype that disrupts the spinal cord and inhibits behavioral recovery after SCI. It will be critical to ensure that injection of extensively expanded neonatal cells do not differentiate aberrantly in traumatic CNS tissue and disrupt recovery.

Nitration of Hsp90 induces cell death.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

E-cadherin-expressing feeder cells promote neural lineage restriction of human embryonic stem cells.

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.

Encapsulated mesenchymal stromal cells for in vivo transplantation.

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.

Single, high-dose intraspinal injection of chondroitinase reduces glycosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after hemisection but not contusion.

Chondroitin sulfate proteoglycans (CSPGs) inhibit axonal growth, and treatment with chondroitinase ABC promotes axonal regeneration in some models of central nervous system (CNS) injury. The aims of this study were (1) to compare the spatiotemporal appearance of CSPG expression between spinal cord contusion and hemisection models, and (2) to evaluate chondroitinase treatment effects on axonal regrowth in the two injury models. After hemisection, CSPG-immunoreactivity (IR) in the injury site rose to peak levels at 18 days but then decreased dramatically by 49 days; in contrast, CSPG-IR remained high for at least 49 days after contusion. After hemisection, many anterogradely labeled corticospinal tract (CST) axons remained close to CSPG-rich lesion sites, but after contusion, most CST axons retracted by approximately 1 mm rostral from the rostral-most CSPG-rich cyst. Intraspinal injection of chondroitinase at 0, 1, 2, and 4 weeks following injury dramatically reduced CSPG-IR in both injury models within 4 days, and CSPG-IR remained low for at least 3 weeks. After the chondroitinase treatment, many axons grew around the lesion site in hemisected spinal cords but not in contused spinal cords. We propose that improved axonal growth in hemisected spinal cords is due to decreased inhibition resulting from degradation of CSPGs located adjacent to severed CST axons. However, in spinal cord contusions, retracted CST axons fail to grow across gliotic regions that surround CSPG-rich injury sites despite efficient degradation with chondroitinase, suggesting that other inhibitors of axonal growth persist in the gliotic regions.

ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension.

Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P(2)-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.

Activated Notch1 maintains the phenotype of radial glial cells and promotes their adhesion to laminin by upregulating nidogen.

Radial glia are neural stem cells that exist only transiently during central nervous system (CNS) development, where they serve as scaffolds for neuronal migration. Their instability makes them difficult to study, and therefore we have isolated stabilized radial glial clones from E14.5 cortical progenitors (e.g., L2.3) after expression of v-myc. Activated Notch1 intracellular region (actNotch1) promotes radial glia in the embryonic mouse forebrain (Gaiano et al., (2000), and when it was introduced into E14.5 cortical progenitors or radial glial clone L2.3, the cells exhibited enhanced radial morphology and increased expression of the radial glial marker BLBP. A representative clone of L2.3 cells expressing actNotch1 called NL2.3-4 migrated more extensively than L2.3 cells in culture and in white matter of the adult rat spinal cord. Microarray and RT-PCR comparisons of mRNAs expressed in these closely related clones showed extensive similarities, but differed significantly for certain mRNAs including several cell adhesion molecules. Cell adhesion assays demonstrated significantly enhanced adhesion to laminin of NL2.3-4 by comparison to L2.3 cells. The laminin binding protein nidogen was the most highly induced adhesion molecule in NL2.3-4, and immunological analyses indicated that radial glia synthesize and secrete nidogen. Adhesion of NL2.3-4 cells to laminin was inhibited by anti-nidogen antibodies and required the nidogen binding region in laminin, indicating that nidogen promotes cell adhesion to laminin. The combined results indicate that persistent expression of activated Notch1 maintains the phenotype of radial glial cells, inhibits their differentiation, and promotes their adhesion and migration on a laminin/nidogen complex.

BMP and LIF signaling coordinately regulate lineage restriction of radial glia in the developing forebrain.

The earliest radial glia are neural stem cells that guide neural cell migration away from ventricular zones. Subsequently, radial glia become lineage restricted during development before they differentiate into more mature cell types in the CNS. We have previously shown that subpopulations of radial glial cells express markers for glial and neuronal restricted precursors (GRPs and NRPs) in expression patterns that are temporally and spatially regulated during CNS development. To characterize further the mechanism of this regulation in rat forebrain, we tested whether secreted factors that are present during development effect lineage restriction of radial glia. We show here that in radial glial cultures LIF/CNTF up-regulates, whereas BMP2 down-regulates GRP antigens recognized by monoclonal antibodies A2B5/4D4. These activities combined with secretion of BMPs dorsally and LIF/CNTF from the choroid plexus provide an explanation for the graded distribution pattern of A2B5/4D4 in dorso-lateral ventricular regions in vivo. The regulation by LIF/CNTF of A2B5/4D4 is mediated through the JAK-STAT pathway. BMP2 promotes expression on radial glial cells of the NRP marker polysialic acid most likely by regulating N-CAM expression itself, as well as at least one polysialyl transferase responsible for synthesis of polysialic acid on N-CAM. Taken together, these results suggest that generation of lineage-restricted precursors is coordinately regulated by gradients of the secreted factors BMPs and LIF/CNTF during development of dorsal forebrain.

Embryonic radial glia bridge spinal cord lesions and promote functional recovery following spinal cord injury.

Radial glial cells are neural stem cells (NSC) that are transiently found in the developing CNS. To study radial glia, we isolated clones following immortalization of E13.5 GFP rat neurospheres with v-myc. Clone RG3.6 exhibits polarized morphology and expresses the radial glial markers nestin and brain lipid binding protein. Both NSC and RG3.6 cells migrated extensively in the adult spinal cord. However, RG3.6 cells differentiated into astroglia slower than NSC, suggesting that immortalization can delay differentiation of radial glia. Following spinal cord contusion, implanted RG3.6 cells migrated widely in the contusion site and into spared white matter where they exhibited a highly polarized morphology. When injected immediately after injury, RG3.6 cells formed cellular bridges surrounding spinal cord lesion sites and extending into spared white matter regions in contrast to GFP fibroblasts that remained in the lesion site. Behavioral analysis indicated higher BBB scores in rats injected with RG3.6 cells than rats injected with fibroblasts or medium as early as 1 week after injury. Spinal cords transplanted with RG3.6 cells or dermal fibroblasts exhibited little accumulation of chondroitin sulfate proteoglycans (CSPG) including NG2 proteoglycans that are known to inhibit axonal growth. Reduced levels of CSPG were accompanied by little accumulation in the injury site of activated macrophages, which are a major source of CSPG. However, increased staining and organization of neurofilaments were found in injured rats transplanted with RG3.6 cells suggesting neuroprotection or regrowth. The combined results indicate that acutely transplanted radial glia can migrate to form bridges across spinal cord lesions in vivo and promote functional recovery following spinal cord injury by protecting against macrophages and secondary damage.

Signal transduction pathways implicated in neural recognition molecule L1 triggered neuroprotection and neuritogenesis.

The signal transduction pathways involved in adhesion molecule L1-triggered neuritogenesis and neuroprotection were investigated using the extracellular domain of mouse or human L1 in fusion with the Fc portion of human immunoglobulin G or L1 purified from mouse brain by affinity chromatography. Substrate L1-triggered neuritogenesis and neuroprotection depended on distinct but also overlapping signal transduction pathways and on the expression of L1 at the neuronal cell surface. PI3 kinase inhibitors, Src family kinase inhibitors as well as mitogen-activated protein kinase kinase inhibitors reduced both L1-triggered neuritogenesis and neuroprotection. In contrast, fibroblast growth factor receptor inhibitors, a protein kinase A inhibitor, and an inhibitor of cAMP-mediated signal transduction pathways, blocked neuritogenesis, but did not affect L1-triggered neuroprotection. Proteolytic cleavage of L1 or its interaction partners is necessary for both L1-mediated neuritogensis and neuroprotection. Furthermore, L1-triggered neuroprotection was found to be associated with increased phosphorylation of extracellular signal-regulated kinases 1/2, Akt and Bad, and inhibition of caspases. These observations suggest possibilities of differentially targeting signal transduction pathways for L1-dependent neuritogenesis and neuroprotection.

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors.

Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development.

In vitro analysis of mechanisms underlying age-dependent failure of axon regeneration.

Severed axons of the inferior colliculus (IC) commissure can regenerate across a lesion in organotypic cultures from postnatal day (P) 6 gerbils, but this regenerative capacity is lost by P12 (Hafidi et al. [ 1995] J Neurosci 15:1298-1307, [1999] J Neurobiol 41:267-280). In the present study, we examined the mechanisms underlying this age-dependent failure of axons to regenerate. In P6-P12 heterochronic cultures, the P12 axons failed to cross the lesion site and project to the contralateral P6 IC lobe. In contrast, axons originating from the P6 lobe could regenerate through the lesion and invade the contralateral P12 IC lobe. To determine whether this age-dependent change in regenerative capacity can develop in organotypic cultures, IC slices with an intact commissure were obtained from P6 animals, grown in vitro for 6 days, and then lesioned at the commissure. In these slices, axon regeneration failure was similar to that observed in normal P12 tissue. Several in vitro treatments enhanced axon regeneration: removal of the entire midline region, inhibition of protein synthesis at the lesion site, and exposure to ABC chondroitinase. Furthermore, when the injured commissural axons were provided with a carpet of C6-R cells (a radial glia-like cell line), significantly more axons projected to the contralateral lobe of the IC. Taken together, these results suggest that the maturation of nonneuronal cells within the lesion site lead to failed axon regeneration in mature animals, and show that ameliorative strategies can be evaluated in vitro.

Trauma-induced tumorigenesis of cells implanted into the rat spinal cord.

OBJECT: Findings in several clinical cases have suggested a correlation between tumor formation and previous injury to the central nervous system (CNS); however, the relationship between trauma and tumorigenesis has not been investigated well experimentally. In this study the authors provide evidence correlating tumorigenesis with trauma in the rat spinal cord. METHODS: A glial cell line, C6R-G/H, which expresses green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT), was implanted into normal and injured rat spinal cords. In all rats in which the cells were implanted into an injured site, locomotor function deteriorated and histological analysis demonstrated glioblastoma multiforme by 6 weeks; tumorigenesis was correlated with a loss of both GFP expression and resistance to hygromycin treatment. In contrast, no evidence of tumor formation was found at 6 weeks in rats in which the cells were implanted into healthy tissue. When C6R-G/H cells were treated with contused spinal cord extract in culture before implantation, they lost GFP expression and hygromycin resistance, and later formed tumors after implantation into normal spinal cord. CONCLUSIONS: The findings of this study indicate that trauma can induce tumorigenesis. Implantation of C6R-G/H cells into traumatized spinal cords resulted in their transformation, which was signaled by loss of GFP expression and hygromycin resistance accompanied by tumor formation. Exposure to extracts derived from injured spinal cord produced similar transformation and gene expression changes, as well as tumor formation after such cells were implanted into normal cords. Care, therefore, should be taken when cells are implanted into an injured CNS because of potential mutagenesis due to trauma-induced factors.