Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI)

Spinal cord injury, a traumatic disease characterised by a massive degeneration of neural tissue, was recently targeted for neuroregenerative interventions. Our approach is the development of pharmacologically pulsed neuroimplants using 3D collagen scaffolds hosting NSCs. We aim to monitor the properties of NSCs ex vivo and in vivo, using synthetic small molecules with neuroprotective and neurogenic properties. Synthetic, highly lipophilic CNS bioavailable small molecules, synthesized by our group (microneurotrophins), bind to neurotrophins receptors (Gravanis et al, Science Signaling, 2012, Calogeropoulou et al., J Med Chem., 2009). BNN27 can specifically interact with TrkA and p75NTR receptors activating specific signalling pathways controlling neuronal cell survival and neurogenesis (Charalampopoulos et al, PNAS, 2004, Lazaridis et al., PLoS Biol., 2011). We are seeding embryonic and adult mouse NSC on collagen 3D scaffolds of different composition (collagen, chondroitin-6-sulphate and gelatin) and construction (size of pores and stiffness), testing cell behaviour (survival, proliferation or differentiation) in basal conditions or pulsed with neurotrophins and/or microneurotrophins. Using the knock in sox2-egfp mice strain and fluorescence activated cell sorting (FACS) analysis, we obtain NSCs cultures with a sox2-positive population more than 90% pure. We evaluate specific markers of proliferation (ki67) and/or differentiation (GFAP for glial cells, Tuj1 for mature neurons and O4 for oligodendrocytes): we are currently testing the possible effect of BNN27 on proliferation of cortical NSCs in 2D cultures (increased numbers of ki67 positive cells up to 12%). The composition and the structure of 3D scaffolds seem to play a significant functional role: scaffolds with a combined composition such as 50% collagen/50% gelatin and 92% collagen/8% chondroitin-6-sulphate support NSC survival since they sustain sox2 expression and propagate neurosphere formation. On the other hand, sensory neurons (Dorsal Root Ganglia, DRGs) grew better in pure collagen scaffolds with less rigid structure. Our preliminary findings suggest that microneurotrophin BNN27 enhances the proliferation of NSCs in culture. Furthermore, the structure of collagen scaffolds heavily affects cellular properties, like stemness or differentiation

Μελέτη των βιολογικών ιδιοτήτων και των θεραπευτικών χρήσεων εμβρυονικών και ενήλικων νευρικών βλαστικών κυττάρων, καλλιεργούμενα σε τρισδιάστατα ικριώματα κολλαγόνου

Our work combined technologies of biology of Central Nervous System (CNS), material science and pharmacology for the development of novel approaches to develop new treatments of neurodegenerative diseases and CNS injury, based on neural tissue regeneration and neural stem cells. We aimed to combine matrices (3D collagen scaffolds), neural stem cells (NSCs) and neurotrophic compounds for spinal cord regeneration. We thus cultured NSCs in 3D collagen scaffolds for the development of an ex vivo niche which could be transplanted in a spinal cord injury model to induce regeneration. Secondly, we assessed the effects of synthetic microneurotrophins on neurogenesis and neuroprotection aiming to their exogenous administration for the pharmacological control of the implanted biological device.Η διατριβή είχε ως στόχο την αλληλεπίδραση μεταξύ της βασικής βιολογίας του κεντρικού νευρικού συστήματος, της τεχνολογίας υλικών και της φαρμακολογίας με απώτερο σκοπό την ανάπτυξη νέων θεραπευτικών προσεγγίσεων νευροεκφυλιστικών νόσων και τραυματισμών. Καλλιεργούμε νευρικά βλαστικά κύτταρα σε ικρώματα κολλαγόνου τριών διαστάσεων στοχεύοντας στην δημιουργία ex vivo μικροπεριβάλλοντος καλλιέργειας βλαστοκυττάρων ικανό για την μεταμόσχευση του σε μοντέλο τραυματισμού της σπονδυλικής στήλης επίμυων. Επιπλέον, εξετάζουμε τις νευροαναγγενητικές και νευροπροστατευτικές δράσεις συνθετικών αναλόγων νευροτροφινών σε ενήλικα νευρικά βλαστικά κύτταρα με απώτερο σκοπό την φαρμακολογική υποστήριξη του μοσχεύματος

An optogenetic cell therapy to restore control of target muscles in an aggressive mouse model of amyotrophic lateral sclerosis

Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, no therapies exist that can prevent progressive motor neuron degeneration, muscle denervation, or paralysis in ALS. Here, we report important advances in the development of an optogenetic, neural replacement strategy that can effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1G93A mouse model of ALS, thus providing an interface to selectively control the function of targeted muscles using optical stimulation. We also identify a specific approach to confer complete survival of allogeneic replacement motor neurons. Furthermore, we demonstrate that an optical stimulation training paradigm can prevent atrophy of reinnervated muscle fibers and results in a tenfold increase in optically evoked contractile force. Together, these advances pave the way for an assistive therapy that could benefit all ALS patients

Neural stem cell delivery via porous collagen scaffolds promotes neuronal differentiation and locomotion recovery in spinal cord injury

Neural stem cell (NSC) grafts have demonstrated significant effects in animal models of spinal cord injury (SCI), yet their clinical translation remains challenging. Significant evidence suggests that the supporting matrix of NSC grafts has a crucial role in regulating NSC effects. Here we demonstrate that grafts based on porous collagen-based scaffolds (PCSs), similar to biomaterials utilized clinically in induced regeneration, can deliver and protect embryonic NSCs at SCI sites, leading to significant improvement in locomotion recovery in an experimental mouse SCI model, so that 12 weeks post-injury locomotion performance of implanted animals does not statistically differ from that of uninjured control animals. NSC-seeded PCS grafts can modulate key processes required to induce regeneration in SCI lesions including enhancing NSC neuronal differentiation and functional integration in vivo, enabling robust axonal elongation, and reducing astrogliosis. Our findings suggest that the efficacy and translational potential of emerging NSC-based SCI therapies could be enhanced by delivering NSC via scaffolds derived from well-characterized clinically proven PCS