A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Induced pluripotent stem cells derived from the developing striatum as a potential donor source for cell replacement therapy for Huntington disease.

BACKGROUND: Cell replacement therapy (CRT) for Huntington disease (HD) requires a source of striatal (STR) progenitors capable of restoring the function lost due to STR degeneration. Authentic STR progenitors can be collected from the fetal putative striatum, or whole ganglionic eminence (WGE), but these tissues remain impractical for widespread clinical application, and alternative donor sources are required. Here we begin exploring the possibility that induced pluripotent stem cells (iPSC) derived from WGE may retain an epigenetic memory of their tissue of origin, which could enhance their ability to differentiate into STR cells. RESULTS: We generate four iPSC lines from human WGE (hWGE) and establish that they have a capacity similar to human embryonic stem cells with regard to their ability to differentiate toward an STR phenotype, as measured by expression and demethylation of key STR genes, while maintaining an overall different methylome. Finally, we demonstrate that these STR-differentiated hWGE iPSCs share characteristics with hWGE (i.e., authentic STR tissues) both in vitro and following transplantation into an HD model. Overall, iPSCs derived from human WGE show promise as a donor source for CRT for HD.

Temporal dynamics of lactate concentration in the human brain during acute inspiratory hypoxia.

PURPOSE: To demonstrate the feasibility of measuring the temporal dynamics of cerebral lactate concentration and examine these dynamics in human subjects using magnetic resonance spectroscopy (MRS) during hypoxia. MATERIALS AND METHODS: A respiratory protocol consisting of 10-minute baseline normoxia, 20-minute inspiratory hypoxia, and ending with 10-minute normoxic recovery was used, throughout which lactate-edited MRS was performed. This was repeated four times in three subjects. A separate session was performed to measure blood lactate. Impulse response functions using end-tidal oxygen and blood lactate as system inputs and cerebral lactate as the system output were examined to describe the dynamics of the cerebral lactate response to a hypoxic challenge. RESULTS: The average lactate increase was 20% ± 15% during the last half of the hypoxic challenge. Significant changes in cerebral lactate concentration were observed after 400 seconds. The average relative increase in blood lactate was 188% ± 95%. The temporal dynamics of cerebral lactate concentration was reproducibly demonstrated with 200-second time bins of MRS data (coefficient of variation 0.063 ± 0.035 between time bins in normoxia). The across-subject coefficient of variation was 0.333. CONCLUSION: The methods for measuring the dynamics of the cerebral lactate response developed here would be useful to further investigate the brain's response to hypoxia.

Perspectives on optimizing local delivery of drugs to peripheral nerves using mathematical models.

Drug therapies for treating peripheral nerve injury repair have shown significant promise in preclinical studies. Despite this, drug treatments are not used routinely clinically to treat patients with peripheral nerve injuries. Drugs delivered systemically are often associated with adverse effects to other tissues and organs; it remains challenging to predict the effective concentration needed at an injured nerve and the appropriate delivery strategy. Local drug delivery approaches are being developed to mitigate this, for example via injections or biomaterial-mediated release. We propose the integration of mathematical modeling into the development of local drug delivery protocols for peripheral nerve injury repair. Mathematical models have the potential to inform understanding of the different transport mechanisms at play, as well as quantitative predictions around the efficacy of individual local delivery protocols. We discuss existing approaches in the literature, including drawing from other research fields, and present a process for taking forward an integrated mathematical-experimental approach to accelerate local drug delivery approaches for peripheral nerve injury repair. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Computational Models Neurological Diseases > Biomedical Engineering.

Engineered neural tissue made using hydrogels derived from decellularised tissues for the regeneration of peripheral nerves.

Engineered neural tissue (EngNT) promotes in vivo axonal regeneration. Decellularised materials (dECM) are complex biologic scaffolds that can improve the cellular environment and also encourage positive tissue remodelling in vivo. We hypothesised that we could incorporate a hydrogel derived from a decellularised tissue (dECMh) into EngNT, thereby providing an alternative to the currently used purified collagen I hydrogel for the first time. Decellularisation was carried out on bone (B-ECM), liver (LIV-ECM), and small intestinal (SIS-ECM) tissues and the resultant dECM was biochemically and mechanically characterised. dECMh differed in mechanical and biochemical properties that likely had an effect on Schwann cell behaviour observed in metabolic activity and contraction profiles. Cellular alignment was observed in tethered moulds within the B-ECM and SIS-ECM derived hydrogels only. No difference was observed in dorsal root ganglia (DRG) neurite extension between the dECMh groups and collagen I groups when applied as a coverslip coating, however, when DRG were seeded atop EngNT constructs, only the B-ECM derived EngNT performed similarly to collagen I derived EngNT. B-ECM EngNT further exhibited similar axonal regeneration to collagen I EngNT in a 10 mm gap rat sciatic nerve injury model after 4 weeks. Our results have shown that various dECMh can be utilised to produce EngNT that can promote neurite extension in vitro and axonal regeneration in vivo. STATEMENT OF SIGNIFICANCE: Nerve autografts are undesirable due to the sacrifice of a patient's own nerve tissue to repair injuries. Engineered neural tissue (EngNT) is a type of living artificial tissue that has been developed to overcome this. To date, only a collagen hydrogel has been shown to be effective in the production and utilisation of EngNT in animal models. Hydrogels may be made from decellularised extracellular matrix derived from many tissues. In this study we showed that hydrogels from various tissues may be used to create EngNT and one was shown to comparable to the currently used collagen based EngNT in a rat sciatic nerve injry model.

Development of ibuprofen-loaded electrospun materials suitable for surgical implantation in peripheral nerve injury.

The development of nerve wraps for use in the repair of peripheral nerves has shown promise over recent years. A pharmacological effect to improve regeneration may be achieved by loading such materials with therapeutic agents, for example ibuprofen, a non-steroidal anti-inflammatory drug with neuroregenerative properties. In this study, four commercially available polymers (polylactic acid (PLA), polycaprolactone (PCL) and two co-polymers containing different ratios of PLA to PCL) were used to fabricate ibuprofen-loaded nerve wraps using blend electrospinning. In vitro surgical handling experiments identified a formulation containing a PLA/PCL 70/30 molar ratio co-polymer as the most suitable for in vivo implantation. In a rat model, ibuprofen released from electrospun materials significantly improved the rate of axonal growth and sensory recovery over a 21-day recovery period following a sciatic nerve crush. Furthermore, RT-qPCR analysis of nerve segments revealed that the anti-inflammatory and neurotrophic effects of ibuprofen may still be observed 21 days after implantation. This suggests that the formulation developed in this work could have potential to improve nerve regeneration in vivo.

Considerations for the use of biomaterials to support cell therapy in neurodegenerative disease.

Using biomaterials to complement cell therapies for a range of neurodegenerative disorders provides a potential opportunity to improve cell survival, integration and regeneration. Materials can be developed to serve as cell or drug delivery systems, temporary scaffolds or longer-term encapsulation structures. However, as yet clinical translation has been limited, with much work still required to achieve this. This chapter discusses a number of considerations for using biomaterials to support cell therapies, which are likely to be essential to achieve successful translation.

An alginate-based encapsulation system for delivery of therapeutic cells to the CNS.

Treatment options for neurodegenerative conditions such as Parkinson's disease have included the delivery of cells which release dopamine or neurotrophic factors to the brain. Here, we report the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner. An optimal alginate formulation was selected with a view to providing a sustained physical barrier between engrafted cells and host tissue, enabling exchange of small molecules while blocking components of the host immune response. In addition, a potent immunosuppressant, FK506, was incorporated into the outer layer of alginate beads using electrosprayed poly-ε-caprolactone core-shell nanoparticles with prolonged release profiles. The stiffness, porosity, stability and ability of the alginate beads to support and protect encapsulated SH-SY5Y cells was demonstrated, and the release profile of FK506 and its effect on T-cell proliferation in vitro was characterized. Collectively, our results indicate this multi-layer encapsulation technology has the potential to be suitable for use in CNS cell delivery, to protect implanted cells from host immune responses whilst providing permeability to nutrients and released therapeutic molecules.

Engineered aligned endothelial cell structures in tethered collagen hydrogels promote peripheral nerve regeneration.

Vascularisation is important in nerve tissue engineering to provide blood supply and nutrients for long-term survival of implanted cells. Furthermore, blood vessels in regenerating nerves have been shown to serve as tracks for Schwann cells to migrate along and thus form Bands of Büngner which promote axonal regeneration. In this study, we have developed tissue-engineered constructs containing aligned endothelial cells, or co-cultures of both endothelial cells and Schwann cells to test whether these structures could promote regeneration across peripheral nerve gaps. Type I rat tail collagen gels containing HUVECs (Human Umbilical Vein Endothelial Cells, 4 × 106 cells/ml) were cast in perforated tethering silicone conduits to facilitate cellular self-alignment and tube formation for 4 days of culture. For co-culture constructs, optimal tube formation and cellular alignment was achieved with a ratio of 4:0.5 × 106 cells/ml (HUVECs:Schwann cells). An in vivo test of the engineered constructs to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that constructs containing only HUVECs significantly promoted axonal regeneration and vascularisation across the gap, as compared to conventional aligned Schwann cell constructs and those containing co-cultured HUVECs and Schwann cells. Our results suggest that tissue-engineered constructs containing aligned endothelial cells within collagen matrix could be good candidates to treat peripheral nerve injury. STATEMENT OF SIGNIFICANCE: Nerve tissue engineering provides a potential way to overcome the limitations associated with current clinical grafting techniques for the repair of severe peripheral nerve injuries. However, the therapeutic cells within engineered nerve tissue require effective vascularisation in order to survive. This work therefore aimed to develop engineered nerve constructs containing aligned tube-like structures made from endothelial cells. Not only did this provide a method to improve vascularisation, it demonstrated for the first time that aligned endothelial cells can outperform Schwann cells in promoting nerve regeneration in the rat sciatic nerve model. This has introduced the concept of developing pre-vascularised engineered nerve tissues, and indicated the potential usefulness of endothelial cell structures in tissue engineering for peripheral nerve repair.

Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications.

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive "off-the-shelf" alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.

Dissection and Preparation of Human Primary Fetal Ganglionic Eminence Tissue for Research and Clinical Applications.

Here, we describe detailed dissection and enzymatic dissociation protocols for the ganglionic eminences from the developing human brain to generate viable quasi-single cell suspensions for subsequent use in transplantation or cell culture. These reliable and reproducible protocols can provide tissue for use in the study of the developing human brain, as well as for the preparation of donor cells for transplantation in Huntington's disease (HD). For use in the clinic as a therapy for HD, the translation of these protocols from the research laboratory to the GMP suite is described, including modification to reagents used and appropriate monitoring and tissue release criteria.

The Effect of Tissue Preparation and Donor Age on Striatal Graft Morphology in the Mouse.

Huntington's disease (HD) is a progressive neurodegenerative disease in which striatal medium spiny neurons (MSNs) are lost. Neuronal replacement therapies aim to replace MSNs through striatal transplantation of donor MSN progenitors, which successfully improve HD-like deficits in rat HD models and have provided functional improvement in patients. Transplants in mouse models of HD are more variable and have lower cell survival than equivalent rat grafts, yet mice constitute the majority of transgenic HD models. Improving the quality and consistency of mouse transplants would open up access to this wider range of rodent models and facilitate research to increase understanding of graft mechanisms, which is essential to progress transplantation as a therapy for HD. Here we determined how donor age, cell preparation, and donor/host strain choice influenced the quality of primary embryonic grafts in quinolinic acid lesion mouse models of HD. Both a within-strain (W-S) and a between-strain (B-S) donor/host paradigm were used to compare transplants of donor tissues derived from mice at embryonic day E12 and E14 prepared either as dissociated suspensions or as minimally manipulated tissue pieces (TP). Good graft survival was observed, although graft volume and cellular composition were highly variable. The effect of cell preparation on grafts differed significantly depending on donor age, with E14 cell suspensions yielding larger grafts compared to TP. Conversely, TP were more effective when derived from E12 donor tissue. A W-S model produced larger grafts with greater MSN content, and while high levels of activated microglia were observed across all groups, a greater number was found in B-S transplants. In summary, we show that the effect of tissue preparation on graft morphology is contingent on the age of donor tissue used. The presence of microglial activation in all groups highlights the host immune response as an important consideration in mouse transplantation.

Bioprocessing strategies to enhance the challenging isolation of neuro-regenerative cells from olfactory mucosa.

Olfactory ensheathing cells (OECs) are a promising potential cell therapy to aid regeneration. However, there are significant challenges in isolating and characterizing them. In the current study, we have explored methods to enhance the recovery of cells expressing OEC marker p75NTR from rat mucosa. With the addition of a 24-hour differential adhesion step, the expression of p75NTR was significantly increased to 73 ± 5% and 46 ± 18% on PDL and laminin matrices respectively. Additionally, the introduction of neurotrophic factor NT-3 and the decrease in serum concentration to 2% FBS resulted in enrichment of OECs, with p75NTR at nearly 100% (100 ± 0% and 98 ± 2% on PDL and laminin respectively), and candidate fibroblast marker Thy1.1 decreased to zero. Culturing OECs at physiologically relevant oxygen tension (2-8%) had a negative impact on p75NTR expression and overall cell survival. Regarding cell potency, co-culture of OECs with NG108-15 neurons resulted in more neuronal growth and potential migration at atmospheric oxygen. Moreover, OECs behaved similarly to a Schwann cell line positive control. In conclusion, this work identified key bioprocessing fundamentals that will underpin future development of OEC-based cell therapies for potential use in spinal cord injury repair. However, there is still much work to do to create optimized isolation methods.

Male and Female Mice Lacking Neuroligin-3 Modify the Behavior of Their Wild-Type Littermates.

In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of Nlgn3, a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other's behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals' behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers' behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3, being more influential in males than females.

Direct Comparison of Rat- and Human-Derived Ganglionic Eminence Tissue Grafts on Motor Function.

Huntington's disease (HD) is a debilitating, genetically inherited neurodegenerative disorder that results in early loss of medium spiny neurons from the striatum and subsequent degeneration of cortical and other subcortical brain regions. Behavioral changes manifest as a range of motor, cognitive, and neuropsychiatric impairments. It has been established that replacement of the degenerated medium spiny neurons with rat-derived fetal whole ganglionic eminence (rWGE) tissue can alleviate motor and cognitive deficits in preclinical rodent models of HD. However, clinical application of this cell replacement therapy requires the use of human-derived (hWGE), not rWGE, tissue. Despite this, little is currently known about the functional efficacy of hWGE. The aim of this study was to directly compare the ability of the gold standard rWGE grafts, against the clinically relevant hWGE grafts, on a range of behavioral tests of motor function. Lister hooded rats either remained as unoperated controls or received unilateral excitotoxic lesions of the lateral neostriatum. Subsets of lesioned rats then received transplants of either rWGE or hWGE primary fetal tissue into the lateral striatum. All rats were tested postlesion and postgraft on the following tests of motor function: staircase test, apomorphine-induced rotation, cylinder test, adjusting steps test, and vibrissae-evoked touch test. At 21 weeks postgraft, brain tissue was taken for histological analysis. The results revealed comparable improvements in apomorphine-induced rotational bias and the vibrissae test, despite larger graft volumes in the hWGE cohort. hWGE grafts, but not rWGE grafts, stabilized behavioral performance on the adjusting steps test. These results have implications for clinical application of cell replacement therapies, as well as providing a foundation for the development of stem cell-derived cell therapy products.

Neonatal desensitization for the study of regenerative medicine.

Cell replacement is a therapeutic option for numerous diseases of the CNS. Current research has identified a number of potential human donor cell types, for which preclinical testing through xenotransplantation in animal models is imperative. Immune modulation is necessary to promote donor cell survival for sufficient time to assess safety and efficacy. Neonatal desensitization can promote survival of human donor cells in adult rat hosts with little impact on the health of the host and for substantially longer than conventional methods, and has subsequently been applied in a range of studies with variable outcomes. Reviewing these findings may provide insight into the method and its potential for use in preclinical studies in regenerative medicine.

Is the adult mouse striatum a hostile host for neural transplant survival?

Human donor cells, including neurally directed embryonic stem cells and induced pluripotent stem cells with the potential to be used for neural transplantation in a range of neurodegenerative disorders, must first be tested preclinically in rodent models of disease to demonstrate safety and efficacy. One strategy for circumventing the rejection of xenotransplanted human cells is to desensitize the host animal to human cells in the early neonatal period so that a subsequent transplant in adulthood is not immunorejected. This method has been robustly validated in the rat, but currently not in the mouse in which most transgenic models of neurodegeneration have been generated. Thus, we set out to determine whether this could be achieved through modification of the existing rat protocol. Mice were inoculated in the neonatal period with a suspension of human embryonic cortical tissue of varying cell numbers, and received a subsequent human embryonic cortical tissue cell transplant in adulthood. Graft survival was compared with those in mice immunosuppressed with cyclosporine A and those receiving allografts of mouse whole ganglionic eminence tissue. Poor survival was found across all groups, suggesting a general problem with the use of mouse hosts for testing human donor cells.