Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. METHODS: We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. RESULTS: Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. CONCLUSIONS: These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.

Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Invasive spread of glioblastoma (GBM) is linked to changes in chondroitin sulfate (CS) proteoglycan (CSPG)-associated sulfated glycosaminoglycans (GAGs) that are selectively up-regulated in the tumor microenvironment (TME). We hypothesized that inhibiting CS-GAG signaling in the TME would stem GBM invasion. Rat F98 GBM cells demonstrated enhanced preferential cell invasion into oversulfated 3-dimensional composite of CS-A and CS-E [4- and 4,6-sulfated CS-GAG (COMP)] matrices compared with monosulfated (4-sulfated) and unsulfated hyaluronic acid matrices in microfluidics-based choice assays, which is likely influenced by differential GAG receptor binding specificities. Both F98 and human patient-derived glioma stem cells (GSCs) demonstrated a high degree of colocalization of the GSC marker CD133 and CSPGs. The small molecule sulfated GAG antagonist bis-2-methyl-4-amino-quinolyl-6-carbamide (surfen) reduced invasion and focal adhesions in F98 cells encapsulated in COMP matrices and blocked CD133 and antichondroitin sulfate antibody (CS-56) detection of respective antigens in F98 cells and human GSCs. Surfen-treated F98 cells down-regulated CSPG-binding receptor transcripts and protein, as well as total and activated ERK and protein kinase B. Lastly, rats induced with frontal lobe tumors and treated with a single intratumoral dose of surfen demonstrated reduced tumor burden and spread compared with untreated controls. These results present a first demonstration of surfen as an inhibitor of sulfated GAG signaling to stem GBM invasion.-Logun, M. T., Wynens, K. E., Simchick, G., Zhao, W., Mao, L., Zhao, Q., Mukherjee, S., Brat, D. J., Karumbaiah, L. Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Expanding Hydrophobically Modified Chitosan Foam for Internal Surgical Hemostasis: Safety Evaluation in a Murine Model.

BACKGROUND: A novel injectable expanding foam based on hydrophobically modified chitosan (HM-CS) was developed to improve hemostasis during surgeries. HM-CS is an amphiphilic derivative of the natural biopolymer chitosan (CS); HM-CS has been shown to improve the natural hemostatic characteristics of CS, but its internal safety has not been systematically evaluated. The goal of this study was to compare the long-term in vivo safety of HM-CS relative to a commonly used fibrin sealant (FS), TISSEEL (Baxter). METHODS: Sixty-four Sprague-Dawley rats (275-325 g obtained from Charles River Laboratories) were randomly assigned to control (n = 16) or experimental (n = 48) groups. Samples of the test materials (HM-CS [n = 16], CS [n = 16], and FS [n = 16]) applied to a nonlethal liver excision (0.4 ± 0.3 g of the medial lobe) in rats were left inside the abdomen to degrade. Animals were observed daily for signs of morbidity and mortality. Surviving animals were sacrificed at 1 and 6 wk; the explanted injury sites were microscopically assessed. RESULTS: All animals (64/64) survived both the 1- and 6-wk time points without signs of morbidity. Histological examination showed a comparable pattern of degradation for the various test materials. FS remnants and significant adhesions to neighboring tissues were observed at 6 wk. Residual CS and HM-CS were observed at the 6 wk with fatty deposits at the site of injury. Minimal adhesions were observed for CS and HM-CS. CONCLUSIONS: The internal safety observed in the HM-CS test group after abdominal implantation indicates that injectable HM-CS expanding foam may be an appropriate internal use hemostatic candidate.

Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells.

Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.

Neuritogenic glycosaminoglycan hydrogels promote functional recovery after severe traumatic brain injury.

Objective.Severe traumatic brain injury (sTBI) induced neuronal loss and brain atrophy contribute significantly to long-term disabilities. Brain extracellular matrix (ECM) associated chondroitin sulfate (CS) glycosaminoglycans promote neural stem cell (NSC) maintenance, and CS hydrogel implants have demonstrated the ability to enhance neuroprotection, in preclinical sTBI studies. However, the ability of neuritogenic chimeric peptide (CP) functionalized CS hydrogels in promoting functional recovery, after controlled cortical impact (CCI) and suction ablation (SA) induced sTBI, has not been previously demonstrated. We hypothesized that neuritogenic (CS)CP hydrogels will promote neuritogenesis of human NSCs, and accelerate brain tissue repair and functional recovery in sTBI rats.Approach.We synthesized chondroitin 4-Osulfate (CS-A)CP, and 4,6-O-sulfate (CS-E)CP hydrogels, using strain promoted azide-alkyne cycloaddition (SPAAC), to promote cell adhesion and neuritogenesis of human NSCs,in vitro; and assessed the ability of (CS-A)CP hydrogels in promoting tissue and functional repair, in a novel CCI-SA sTBI model,in vivo. Main results.Results indicated that (CS-E)CP hydrogels significantly enhanced human NSC aggregation and migration via focal adhesion kinase complexes, when compared to NSCs in (CS-A)CP hydrogels,in vitro. In contrast, NSCs encapsulated in (CS-A)CP hydrogels differentiated into neurons bearing longer neurites and showed greater spontaneous activity, when compared to those in (CS-E)CP hydrogels. The intracavitary implantation of (CS-A)CP hydrogels, acutely after CCI-SA-sTBI, prevented neuronal and axonal loss, as determined by immunohistochemical analyses. (CS-A)CP hydrogel implanted animals also demonstrated the significantly accelerated recovery of 'reach-to-grasp' function when compared to sTBI controls, over a period of 5-weeks.Significance.These findings demonstrate the neuritogenic and neuroprotective attributes of (CS)CP 'click' hydrogels, and open new avenues for the development of multifunctional glycomaterials that are functionalized with biorthogonal handles for sTBI repair.

AutoRG: An automatized reach-to-grasp platform technology for assessing forelimb motor function, neural circuit activation, and cognition in rodents.

BACKGROUND: Rodent reach-to-grasp function assessment is a translationally powerful model for evaluating neurological function impairments and recovery responses. Existing assessment platforms are experimenter-dependent, costly, or low-throughput with limited output measures. Further, a direct histologic comparison of neural activation has never been conducted between any novel, automated platform and the well-established single pellet skilled reach task (SRT). NEW METHOD: To address these technological and knowledge gaps, we designed an open-source, low-cost Automatized Reach-to-Grasp (AutoRG) pull platform that reduces experimenter interventions and variability. We assessed reach-to-grasp function in rats across seven progressively difficult stages using AutoRG. We mapped AutoRG and SRT-activated motor circuitries in the rat brain using volumetric imaging of the immediate early gene-encoded Arc (activity-regulated cytoskeleton-associated) protein. RESULTS: Rats demonstrated robust forelimb reaching and pulling behavior after training in AutoRG. Reliable force versus time responses were recorded for individual reach events in real time, which were used to derive several secondary functional measures of performance. Moreover, we provide the first demonstration that for a training period of 30 min, AutoRG and SRT both engage similar neural responses in the caudal forelimb area (CFA), rostral forelimb area (RFA), and sensorimotor area (S1). CONCLUSION: AutoRG is the first low-cost, open-source pull system designed for the scale-up of volitional forelimb motor function testing and characterization of rodent reaching behavior. The similarities in neuronal activation patterns observed in the rat motor cortex after SRT and AutoRG assessments validate the AutoRG as a rigorously characterized, scalable alternative to the conventional SRT and expensive commercial systems.

Photocrosslinkable chitosan based hydrogels for neural tissue engineering.

Hydrogel based scaffolds for neural tissue engineering can provide appropriate physico-chemical and mechanical properties to support neurite extension and facilitate transplantation of cells by acting as 'cell delivery vehicles'. Specifically, in situ gelling systems such as photocrosslinkable hydrogels can potentially conformally fill irregular neural tissue defects and serve as stem cell delivery systems. Here, we report the development of a novel chitosan based photocrosslinkable hydrogel system with tunable mechanical properties and degradation rates. A two-step synthesis of amino-ethyl methacrylate derivitized, degradable, photocrosslinkable chitosan hydrogels is described. When human mesenchymal stem cells were cultured in photocrosslinkable chitosan hydrogels, negligible cytotoxicity was observed. Photocrosslinkable chitosan hydrogels facilitated enhanced neurite differentiation from primary cortical neurons and enhanced neurite extension from dorsal root ganglia (DRG) as compared to agarose based hydrogels with similar storage moduli. Neural stem cells (NSCs) cultured within photocrosslinkable chitosan hydrogels facilitated differentiation into tubulin positive neurons and astrocytes. These data demonstrate the potential of photocrosslinked chitosan hydrogels, and contribute to an increasing repertoire of hydrogels designed for neural tissue engineering.

Glycomaterials to Investigate the Functional Role of Aberrant Glycosylation in Glioblastoma.

Glioblastoma (GBM) is a stage IV astrocytoma that carries a dismal survival rate of ≈10 months postdiagnosis and treatment. The highly invasive capacity of GBM and its ability to escape therapeutic challenges are key factors contributing to the poor overall survival rate. While current treatments aim to target the cancer cell itself, they fail to consider the significant role that the GBM tumor microenvironment (TME) plays in promoting tumor progression and therapeutic resistance. The GBM tumor glycocalyx and glycan-rich extracellular matrix (ECM), which are important constituents of the TME have received little attention as therapeutic targets. A wide array of aberrantly modified glycans in the GBM TME mediate tumor growth, invasion, therapeutic resistance, and immunosuppression. Here, an overview of the landscape of aberrant glycan modifications in GBM is provided, and the design and utility of 3D glycomaterials are discussed as a tool to evaluate glycan-mediated GBM progression and therapeutic efficacy. The development of alternative strategies to target glycans in the TME can potentially unveil broader mechanisms of restricting tumor growth and enhancing the efficacy of tumor-targeting therapeutics.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Glycosaminoglycan scaffolding and neural progenitor cell transplantation promotes regenerative immunomodulation in the mouse ischemic brain.

Ischemic stroke is a leading cause of morbidity and mortality, with limited treatments that can facilitate brain regeneration. Neural progenitor cells (NPCs) hold promise for replacing tissue lost to stroke, and biomaterial approaches may improve their efficacy to overcome hurdles in clinical translation. The immune response and its role in stroke pathogenesis and regeneration may interplay with critical mechanisms of stem cell and biomaterial therapies. Cellular therapy can modulate the immune response to reduce toxic neuroinflammation early after ischemia. However, few studies have attempted to harness the regenerative effects of neuroinflammation to augment recovery. Our previous studies demonstrated that intracerebrally transplanted NPCs encapsulated in a chondroitin sulfate-A hydrogel (CS-A + NPCs) can improve vascular regeneration after stroke. In this paper, we found that CS-A + NPCs affect the microglia/macrophage response to promote a regenerative phenotype following stroke in mice. Following transplantation, PPARγ-expressing microglia/macrophages, and MCP-1 and IL-10 protein levels are enhanced. Secreted immunomodulatory factor expression of other factors was altered compared to NPC transplantation alone. Post-stroke depression-like behavior was reduced following cellular and material transplantation. Furthermore, we showed in cultures that microglia/macrophages encapsulated in CS-A had increased expression of angiogenic and arteriogenic mediators. Neutralization with anti-IL-10 antibody negated these effects in vitro. Cumulatively, this work provides a framework for understanding the mechanisms by which immunomodulatory biomaterials can enhance the regenerative effects of cellular therapy for ischemic stroke and other brain injuries.

Targeted downregulation of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase significantly mitigates chondroitin sulfate proteoglycan-mediated inhibition.

Chondroitin sulfate-4,6 (CS-E) glycosaminoglycan (GAG) upregulation in astroglial scars is a major contributor to chondroitin sulfate proteoglycan (CSPG)-mediated inhibition [Gilbert et al. (2005) Mol Cell Neurosci 29:545­558]. However, the role of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S6ST) catalyzed sulfation of CS-E, and its contribution to CSPG-mediated inhibition of CNS regeneration remains to be fully elucidated. Here, we used in situ hybridization to show localized upregulation of GalNAc4S6ST mRNA after CNS injury. Using in vitro spot assays with immobilized CS-E, we demonstrate dose-dependent inhibition of rat embryonic day 18 (E18) cortical neurons. To determine whether selective downregulation of CS-E affected the overall inhibitory character of extracellular matrix produced by reactive astrocytes, single [against (chondroitin 4) sulfotransferase 11 (C4ST1) or GalNAc4S6ST mRNA] or double [against C4ST1 and GalNAc4S6ST mRNA] siRNA treatments were conducted and assayed using quantitative real-time polymerase chain reaction and high-performance liquid chromatography to confirm the specific downregulation of CS-4S GAG (CS-A) and CS-E. Spot and Bonhoeffer stripe assays using astrocyte-conditioned media from siRNA-treated rat astrocytes showed a significant decrease in inhibition of neuronal attachment and neurite extensions when compared with untreated and TGF-treated astrocytes. These findings reveal that selective attenuation of CS-E via siRNA targeting of GalNAc4S6ST significantly mitigates CSPG-mediated inhibition of neurons, potentially offering a novel intervention strategy for CNS injury.

Cortical Laminar Recording of Multi-unit Response to Distal Forelimb Electrical Stimulation in Rats.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. In this protocol, we describe how to perform recordings to quantify the neuroprotective and regenerative effect of implanted engineered CS-GAG hydrogel (eCS) on brain tissue. This experiment was performed in rats under three conditions: healthy without injury (Sham), controlled cortical impact (CCI) injury on the rostral forelimb area (RFA), and CCI-RFA with eCS implants. This protocol describes the procedure used to perform the craniotomy, the positioning of the cortical recording electrode, the positioning of the stimulation electrode (contralateral paw), and the recording procedure. In addition, a description of the exact electrical setup is provided. This protocol details the recordings in the brain of injured animals while preserving most of the uninjured tissue intact, with additional considerations for intralesional and laminar recordings of multi-unit response. Graphic abstract: Sensorimotor response to paw stimulation using cortical laminar recordings.

Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of "reach-to-grasp" function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI.

Neurostimulation and Reach-to-Grasp Function Recovery Following Acquired Brain Injury: Insight From Pre-clinical Rodent Models and Human Applications.

Reach-to-grasp is an evolutionarily conserved motor function that is adversely impacted following stroke and traumatic brain injury (TBI). Non-invasive brain stimulation (NIBS) methods, such as transcranial magnetic stimulation and transcranial direct current stimulation, are promising tools that could enhance functional recovery of reach-to-grasp post-brain injury. Though the rodent literature provides a causal understanding of post-injury recovery mechanisms, it has had a limited impact on NIBS protocols in human research. The high degree of homology in reach-to-grasp circuitry between humans and rodents further implies that the application of NIBS to brain injury could be better informed by findings from pre-clinical rodent models and neurorehabilitation research. Here, we provide an overview of the advantages and limitations of using rodent models to advance our current understanding of human reach-to-grasp function, cortical circuitry, and reorganization. We propose that a cross-species comparison of reach-to-grasp recovery could provide a mechanistic framework for clinically efficacious NIBS treatments that could elicit better functional outcomes for patients.

Label-free ferrohydrodynamic separation of exosome-like nanoparticles.

Isolation of exosomes from biological samples provides a minimally-invasive alternative for basic understanding, diagnosis, and prognosis of metastatic cancers. The biology and clinical values of exosomes are under intensive investigation, yet most studies are limited by technical challenges in recovering these exosomes with heterogeneous sizes and cargos from biological samples. We report a novel method based on "particle ferrohydrodynamics" and its associated microfluidic device, termed as the FerroChip, which can separate exosome-like nanoparticles from microliters of cell culture media and human serum in a label-free, continuous-flow and size-dependent manner, and achieves a high recovery rate (94.3%) and a high purity (87.9%). Separated exosome-like nanoparticles had diameters, morphology, and protein expressions that were consistent with other reports. This method, upon further molecular characterization, could potentially facilitate basic understanding of exosomes and its clinical application in blood liquid biopsy.

Cortical Transplantation of Brain-Mimetic Glycosaminoglycan Scaffolds and Neural Progenitor Cells Promotes Vascular Regeneration and Functional Recovery after Ischemic Stroke in Mice.

Stroke causes significant mortality and morbidity. Currently, there are no treatments which can regenerate brain tissue lost to infarction. Neural progenitor cells (NPCs) are at the forefront of preclinical studies for regenerative stroke therapies. NPCs can differentiate into and replace neurons and promote endogenous recovery mechanisms such as angiogenesis via trophic factor production and release. The stroke core is hypothetically the ideal location for replacement of neural tissue since it is in situ and develops into a potential space where injections may be targeted with minimal compression of healthy peri-infarct tissue. However, the compromised perfusion and tissue degradation following ischemia create an inhospitable environment resistant to cellular therapy. Overcoming these limitations is critical to advancing cellular therapy. In this work, the therapeutic potential of mouse-induced pluripotent stem cell derived NPCs is tested encapsulated in a basic fibroblast growth factor (bFGF) binding chondroitin sulfate-A (CS-A) hydrogel transplanted into the infarct core in a mouse sensorimotor cortex mini-stroke model. It is shown that CS-A encapsulation significantly improves vascular remodeling, cortical blood flow, and sensorimotor behavioral outcomes after stroke. It is found these improvements are negated by blocking bFGF, suggesting that the sustained trophic signaling endowed by the CS-A hydrogel combined with NPC transplantation can promote tissue repair.

Extracellular Vesicles Mediate Neuroprotection and Functional Recovery after Traumatic Brain Injury.

The lack of effective therapies for moderate-to-severe traumatic brain injuries (TBIs) leaves patients with lifelong disabilities. Neural stem cells (NSCs) have demonstrated great promise for neural repair and regeneration. However, direct evidence to support their use as a cell replacement therapy for neural injuries is currently lacking. We hypothesized that NSC-derived extracellular vesicles (NSC EVs) mediate repair indirectly after TBI by enhancing neuroprotection and therapeutic efficacy of endogenous NSCs. We evaluated the short-term effects of acute intravenous injections of NSC EVs immediately following a rat TBI. Male NSC EV-treated rats demonstrated significantly reduced lesion sizes, enhanced presence of endogenous NSCs, and attenuated motor function impairments 4 weeks post-TBI, when compared with vehicle- and TBI-only male controls. Although statistically not significant, we observed a therapeutic effect of NSC EVs on brain lesion volume, nestin expression, and behavioral recovery in female subjects. Our study demonstrates the neuroprotective and functional benefits of NSC EVs for treating TBI and points to gender-dependent effects on treatment outcomes, which requires further investigation.

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells.

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate.

Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing.

Multi-photon scanning microscopy provides a robust tool for optical sectioning, which can be used to capture fast biological events such as blood flow, mitochondrial activity, and neuronal action potentials. For many studies, it is important to visualize several different focal planes at a rate akin to the biological event frequency. Typically, a microscope is equipped with mechanical elements to move either the sample or the objective lens to capture volumetric information, but these strategies are limited due to their slow speeds or inertial artifacts. To overcome this problem, remote focusing methods have been developed to shift the focal plane axially without physical movement of the sample or the microscope. Among these methods is liquid lens technology, which adjusts the focus of the lens by changing the wettability of the liquid and hence its curvature. Liquid lenses are inexpensive active optical elements that have the potential for fast multi-photon volumetric imaging, hence a promising and accessible approach for the study of biological systems with complex dynamics. Although remote focusing using liquid lens technology can be used for volumetric point scanning multi-photon microscopy, optical aberrations and the effects of high energy laser pulses have been concerns in its implementation. In this paper, we characterize a liquid lens and validate its use in relevant biological applications. We measured optical aberrations that are caused by the liquid lens, and calculated its response time, defocus hysteresis, and thermal response to a pulsed laser. We applied this method of remote focusing for imaging and measurement of multiple in-vivo specimens, including mesenchymal stem cell dynamics, mouse tibialis anterior muscle mitochondrial electrical potential fluctuations, and mouse brain neural activity. Our system produces 5 dimensional (x,y,z,λ,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 µm3.

Chondroitin Sulfate Glycosaminoglycan Scaffolds for Cell and Recombinant Protein-Based Bone Regeneration.

Bone morphogenetic protein 2 (BMP-2)-loaded collagen sponges remain the clinical standard for treatment of large bone defects when there is insufficient autograft, despite associated complications. Recent efforts to negate comorbidities have included biomaterials and gene therapy approaches to extend the duration of BMP-2 release and activity. In this study, we compared the collagen sponge clinical standard to chondroitin sulfate glycosaminoglycan (CS-GAG) scaffolds as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) and rhBMP-2 expression via human BMP-2 gene inserted into mesenchymal stem cells (BMP-2 MSC). We demonstrated extended release of rhBMP-2 from CS-GAG scaffolds compared to their collagen sponge counterparts, and further extended release from CS-GAG gels seeded with BMP-2 MSC. When used to treat a challenging critically sized femoral defect model in rats, both rhBMP-2 and BMP-2 MSC in CS-GAG induced comparable bone formation to the rhBMP-2 in collagen sponge, as measured by bone volume, strength, and stiffness. We conclude that CS-GAG scaffolds are a promising delivery vehicle for controlling the release of rhBMP-2 and to mediate the repair of critically sized segmental bone defects. Stem Cells Translational Medicine 2019;8:575-585.