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Injectable biomimetic hydrogels have great potential for use in regenerative medicine as cellular delivery vectors. However, they can suffer from issues relating to hypoxia, including poor cell survival, differentiation, and functional integration owing to the lack of an established vascular network. Here we engineer a hybrid myoglobin:peptide hydrogel that can concomitantly deliver stem cells and oxygen to the brain to support engraftment until vascularisation can occur naturally. We show that this hybrid hydrogel can modulate cell fate specification within progenitor cell grafts, resulting in a significant increase in neuronal differentiation. We find that the addition of myoglobin to the hydrogel results in more extensive innervation within the host tissue from the grafted cells, which is essential for neuronal replacement strategies to ensure functional synaptic connectivity. This approach could result in greater functional integration of stem cell-derived grafts for the treatment of neural injuries and diseases affecting the central and peripheral nervous systems.

Long-read single-cell RNA sequencing (scRNA-seq) enables the quantification of RNA isoforms in individual cells. However, long-read scRNA-seq using the Oxford Nanopore platform has largely relied upon matched short-read data to identify cell barcodes. We introduce BLAZE, which accurately and efficiently identifies 10x cell barcodes using only nanopore long-read scRNA-seq data. BLAZE outperforms the existing tools and provides an accurate representation of the cells present in long-read scRNA-seq when compared to matched short reads. BLAZE simplifies long-read scRNA-seq while improving the results, is compatible with downstream tools accepting a cell barcode file, and is available at https://github.com/shimlab/BLAZE .

Bioinspired self-assembly is a bottom-up strategy enabling biologically sophisticated nanostructured biogels that can mimic natural tissue. Self-assembling peptides (SAPs), carefully designed, form signal-rich supramolecular nanostructures that intertwine to form a hydrogel material that can be used for a range of cell and tissue engineering scaffolds. Using the tools of nature, they are a versatile framework for the supply and presentation of important biological factors. Recent developments have shown promise for many applications such as therapeutic gene, drug and cell delivery and yet are stable enough for large-scale tissue engineering. This is due to their excellent programmability-features can be incorporated for innate biocompatibility, biodegradability, synthetic feasibility, biological functionality and responsiveness to external stimuli. SAPs can be used independently or combined with other (macro)molecules to recapitulate surprisingly complex biological functions in a simple framework. It is easy to accomplish localized delivery, since they can be injected and can deliver targeted and sustained effects. In this review, we discuss the categories of SAPs, applications for gene and drug delivery, and their inherent design challenges. We highlight selected applications from the literature and make suggestions to advance the field with SAPs as a simple, yet smart delivery platform for emerging BioMedTech applications.

The defined self-assembly of peptides (SAPs) into nanostructured bioactive hydrogels has great potential for repairing traumatic brain injuries, as they maintain a stable, homeostatic environment at an injury site, preventing further degeneration. They also present a bespoke platform to restore function via the naturalistic presentation of therapeutic proteins, such as stromal-cell-derived factor 1 (SDF-1), expressed by meningeal cells. A key challenge to the use of the SDF protein, however, is its rapid diffusion and degradation. Here, we engineered a homeostatic hydrogel produced by incorporating recombinant SDF-1 protein within a self-assembled peptide hydrogel to create a supportive milieu for transplanted cells. Our hydrogel can concomitantly deliver viable primary neural progenitor cells and sustained active SDF-1 to support the nascent graft, resulting in increased neuronal differentiation. Moreover, this homeostatic hydrogel can ensure a healthy and larger graft core without impeding neuronal fiber growth and innervation. These findings demonstrate the regenerative potential of these hydrogels to improve the integration of grafted cells to treat neural injuries and diseases.

Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson’s Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain’s main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin–integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.

Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.

Stem-cell-derived transplants may soon be a promising treatment option for Parkinson's disease. In preparation for clinical trial, Piao et al.1 report on generating a clinical-grade dopaminergic progenitor cell product and its rigorous testing to ensure safety and efficacy.

Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson's disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced-cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.

Neonatal arterial ischemic stroke is one of the more severe birth complications. The injury can result in extensive neurological damage and is robustly associated with later diagnoses of cerebral palsy (CP). An important part of efforts to develop new therapies include the on-going refinement and understanding of animal models that capture relevant clinical features of neonatal brain injury leading to CP. The potent vasoconstrictor peptide, Endothelin-1 (ET-1), has previously been utilised in animal models to reduce local blood flow to levels that mimic ischemic stroke. Our previous work in this area has shown that it is an effective and technically simple approach for modelling ischemic injury at very early neonatal ages, resulting in stable deficits in motor function. Here, we aimed to extend this model to also examine the impact on cognitive function. We show that focal delivery of ET-1 to the cortex of Sprague Dawley rats on postnatal day 0 (P0) resulted in impaired learning in a touchscreen-based test of visual discrimination and correlated with important clinical features of CP including damage to large white matter structures.

Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

Ischemic damage to the adult rodent forebrain has been widely used as a model system to study injury-induced neurogenesis, resulting in contradictory reports regarding the capacity of the postnatal brain to replace striatal projection neurons. Here we used a software-assisted, confocal approach to survey thousands of cells generated after striatal ischemic injury in rats and showed that injury fails not only to stimulate production of new striatal projection neurons in the adult brain but also to do so in the neonatal brain at early postnatal ages not previously explored. Conceptually this is significant, because it shows that even during periods of active striatal neurogenesis, injury is not a sufficient stimulus to promote replacement of these neurons. Understanding the intrinsic capacity of the postnatal brain to replace neurons in response to injury is fundamental to the development of "self-repair" therapies.

Here, we describe a xeno-free, feeder-free, and chemically defined protocol for the generation of ventral midbrain dopaminergic (vmDA) progenitors from human pluripotent stem cells (hPSCs). This simple-to-follow protocol results in high yields of cryopreservable dopamine neurons across multiple hPSC lines. Wnt signaling is the critical component of the differentiation and can be finely adjusted in a line-dependent manner to enhance production of dopamine neurons for the purposes of transplantation, studying development and homeostasis, disease modeling, drug discovery, and drug development. For complete details on the use and execution of this protocol, please refer to Gantner et al. (2020) and Niclis et al. (2017a).

The brain is a complex 3-dimensional structure, the organization of which provides a local environment that directly influences the survival, proliferation, differentiation, migration, and plasticity of neurons. To probe the effects of damage and disease on these cells, a synthetic environment is needed. Three-dimensional culturing of stem cells, neural progenitors, and neurons within fabricated biomaterials has demonstrated superior biomimetic properties over conventional 2-dimensional cultureware, offering direct recapitulation of both cell-cell and cell-extracellular matrix interactions. Within this review we address the benefits of deploying biomaterials as advanced cell culture tools capable of influencing neuronal fate and as in vitro models of the native in vivo microenvironment. We highlight recent and promising biomaterials approaches toward understanding neural network and their function relevant to neurodevelopment and provide our perspective on how these materials can be engineered and programmed to study both the healthy and diseased nervous system.

BACKGROUND: Gastrointestinal (GI) dysfunction, including constipation, is a common non-motor symptom of Parkinson's disease (PD). The toxin 6-hydroxydopamine (6OHDA) produces the symptoms of PD, surprisingly including constipation, after it is injected into the medial forebrain bundle (MFB). However, the mechanisms involved in PD-associated constipation caused by central application of 6OHDA remain unknown. We investigated effects of 6OHDA lesioning of the MFB on motor performance and GI function. METHODS: Male Sprague Dawley rats were unilaterally injected with 6OHDA in the MFB. Colorectal propulsion was assessed by bead expulsion after 4 weeks and by recording colorectal contractions and propulsion after 5 weeks. Enteric nervous system (ENS) neuropathy was examined by immunohistochemistry. KEY RESULTS: When compared to shams, 6OHDA-lesioned rats had significantly increased times of bead expulsion from the colorectum, indicative of colon dysmotility. Administration of the colokinetic, capromorelin, that stimulates defecation centers in the spinal cord, increased the number of contractions and colorectal propulsion in both groups compared to baseline; however, the effectiveness of capromorelin in 6OHDA-lesioned rats was significantly reduced in comparison with shams, indicating that 6OHDA animals have reduced responsiveness of the spinal defecation centers. Enteric neuropathy was observed in the distal colon, revealing that lesion of the MFB has downstream effects at the cellular level, remote from the site of 6OHDA administration. CONCLUSIONS & INFERENCES: We conclude that there are trans-synaptic effects of the proximal, forebrain, lesion of pathways from the brain that send signals down the spinal cord, at the levels of the defecation centers and the ENS.

Development of new stroke therapies requires animal models that recapitulate the pathophysiological and functional consequences of ischemic brain damage over time-frames relevant to the therapeutic intervention. This is particularly relevant for the rapidly developing area of stem cell therapies, where functional replacement of circuitry will require maturation of transplanted human cells over months. An additional challenge is the establishment of models of ischemia with stable behavioral phenotypes in chronically immune-suppressed animals to allow for long-term survival of human cell grafts. Here we report that microinjection of endothelin-1 into the sensorimotor cortex of athymic rats results in ischemic damage with a sustained deficit in function of the contralateral forepaw that persists for up to 9 months. The histological post-mortem analysis revealed chronic and diffuse atrophy of the ischemic cortical hemisphere that continued to progress over 9 months. Secondary atrophy remote to the primary site of injury and its relationship with long-term cognitive and functional decline is now recognized in human populations. Thus, focal cortical infarction in athymic rats mirrors important pathophysiological and functional features relevant to human stroke, and will be valuable for assessing efficacy of stem cell based therapies.

The dopamine precursor, levodopa, remains the "gold standard" treatment for Parkinson's disease, and, although it provides superlative efficacy in the early stages of the disease, its long-term use is limited by the development of severe motor side effects and a significant abating of therapeutic efficacy. Therefore, there remains a major unmet clinical need for the development of effective neuroprotective, neurorestorative or neuroreparatory therapies for this condition. The relatively selective loss of dopaminergic neurons from the nigrostriatal pathway makes Parkinson's disease an ideal candidate for reparative cell therapies, wherein the dopaminergic neurons that are lost in the condition are replaced through direct cell transplantation into the brain. To date, this approach has been developed, validated and clinically assessed using dopamine neuron-rich foetal ventral mesencephalon grafts which have been shown to survive and reinnervate the denervated brain after transplantation, and to restore motor function. However, despite long-term symptomatic relief in some patients, significant limitations, including poor graft survival and the impact this has on the number of foetal donors required, have prevented this therapy being more widely adopted as a restorative approach for Parkinson's disease. Injectable biomaterial scaffolds have the potential to improve the delivery, engraftment and survival of these grafts in the brain through provision of a supportive microenvironment for cell adhesion, growth and immune shielding. This article will briefly review the development of primary cell therapies for brain repair in Parkinson's disease and will consider the emerging literature which highlights the potential of using injectable biomaterial hydrogels in this context.

With the limited capacity for self-repair in the adult CNS, efforts to stimulate quiescent stem cell populations within discrete brain regions, as well as harness the potential of stem cell transplants, offer significant hope for neural repair. These new cells are capable of providing trophic cues to support residual host populations and/or replace those cells lost to the primary insult. However, issues with low-level adult neurogenesis, cell survival, directed differentiation and inadequate reinnervation of host tissue have impeded the full potential of these therapeutic approaches and their clinical advancement. Biomaterials offer novel approaches to stimulate endogenous neurogenesis, as well as for the delivery and support of neural progenitor transplants, providing a tissue-appropriate physical and trophic milieu for the newly integrating cells. In this review, we will discuss the various approaches by which bioengineered scaffolds may improve stem cell-based therapies for repair of the CNS.

Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation.

Neurotrophic growth factors are effective in slowing progressive degeneration and/or promoting neural repair through the support of residual host and/or transplanted neurons. However, limitations including short half-life and enzyme susceptibility of growth factors highlight the need for alternative strategies to prolong localised delivery at a site of injury. Here, we establish the utility of minimalist N-fluorenylmethyloxycarbonyl (Fmoc) self-assembling peptides (SAPs) as growth factor delivery vehicle, targeted at supporting neural transplants in an animal model of Parkinson's disease. The neural tissue-specific SAP, Fmoc-DIKVAV, demonstrated sustained release of glial cell line derived neurotrophic factor, up to 172 hr after gel loading. This represents a significant advance in drug delivery, because its lifetime in phosphate buffered saline was less than 1 hr. In vivo transplantation of neural progenitor cells, together with our growth factor-loaded material, into the injured brain improved graft survival compared with cell transplants alone. We show for the first time the use of minimalist Fmoc-SAP in an in vivo disease model for sustaining the delivery of neurotrophic growth factors, facilitating their spatial and temporal delivery in vivo, whilst also providing an enhanced niche environment for transplanted cells.

© 2017 The Authors Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re-construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547-1556.

Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. SIGNIFICANCE: The authors report the generation of highly pure midbrain dopamine cultures under feeder-free, fully defined, and xeno-free conditions from human pluripotent stem cells. Xeno-free differentiated cells display gene, protein, and electrophysiological properties of midbrain neurons, as well as improved grafting outcomes in Parkinsonian rodents, observations enhanced by the use of two novel reporter lines of interest t

Pluripotent stem cells (PSCs) are a valuable tool for interrogating development, disease modelling, drug discovery and transplantation. Despite the burgeoned capability to fate restrict human PSCs to specific neural lineages, comparative protocols for mouse PSCs have not similarly advanced. Mouse protocols fail to recapitulate neural development, consequently yielding highly heterogeneous populations, yet mouse PSCs remain a valuable scientific tool as differentiation is rapid, cost effective and an extensive repertoire of transgenic lines provides an invaluable resource for understanding biology. Here we developed protocols for neural fate restriction of mouse PSCs, using knowledge of embryonic development and recent progress with human equivalents. These methodologies rely upon naïve ground-state PSCs temporarily transitioning through LIF-responsive stage prior to neural induction and rapid exposure to regional morphogens. Neural subtypes generated included those of the dorsal forebrain, ventral forebrain, ventral midbrain and hindbrain. This rapid specification, without feeder layers or embryoid-body formation, resulted in high proportions of correctly specified progenitors and neurons with robust reproducibility. These generated neural progenitors/neurons will provide a valuable resource to further understand development, as well disorders affecting specific neuronal subpopulations.

Abnormal development of ventral midbrain (VM) dopaminergic (DA) pathways, essential for motor and cognitive function, may underpin a number of neurological disorders and thereby highlight the importance of understanding the birth and connectivity of the associated neurons. While a number of regulators of VM DA neurogenesis are known, processes involved in later developmental events, including terminal differentiation and axon morphogenesis, are less well understood. Recent transcriptional analysis studies of the developing VM identified genes expressed during these stages, including the cell adhesion molecule with homology to L1 (Chl1). Here, we map the temporal and spatial expression of CHL1 and assess functional roles of substrate-bound and soluble-forms of the protein during VM DA development. Results showed early CHL1 in the VM, corresponding with roles in DA progenitor migration and differentiation. Subsequently, we demonstrated roles for CHL1 in both axonal extension and repulsion, selectively of DA neurons, suggestive of a role in guidance towards forebrain targets and away from hindbrain nuclei. In part, CHL1 mediates these roles through homophilic CHL1-CHL1 interactions. Collectively, these findings enhance our knowledge of VM DA pathways development, and may provide new insights into understanding DA developmental conditions such as autism spectrum disorders.

Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.

Protein growth factors have demonstrated great potential for tissue repair, but their inherent instability and large size prevents meaningful presentation to biologically protected nervous tissue. Here, we create a nanofibrous network from a self-assembling peptide (SAP) hydrogel to carry and stabilize the growth factors. We significantly reduced growth factor degradation to increase their lifespan by over 40 times. To control the temporal release profile we covalently attached polysaccharide chitosan molecules to the growth factor to increase its interactions with the hydrogel nanofibers and achieved a 4 h delay, demonstrating the potential of this method to provide temporally controlled growth factor delivery. We also describe release rate based analysis to examine the growth factor delivery in more detail than standard cumulative release profiles allow and show that the chitosan attachment method provided a more consistent release profile with a 60% reduction in fluctuations. To prove the potential of this system as a complex growth factor delivery platform we demonstrate for the first time temporally distinct release of multiple growth factors from a single tissue specific SAP hydrogel: a significant goal in regenerative medicine.

A number of studies have shown that damage to brain structures adjacent to neurogenic regions can result in migration of new neurons from neurogenic zones into the damaged tissue. The number of differentiated neurons that survive is low, however, and this has led to the idea that the introduction of extrinsic signaling factors, particularly neurotrophic proteins, may augment the neurogenic response to a level that would be therapeutically relevant. Here we report on the impact of the relatively newly described neurotrophic factor, Meteorin, when over-expressed in the striatum following excitotoxic injury. Birth-dating studies using bromo-deoxy-uridine (BrdU) showed that Meteorin did not enhance injury-induced striatal neurogenesis but significantly increased the proportion of new cells with astroglial and oligodendroglial features. As a basis for comparison we found under the same conditions, glial derived neurotrophic factor significantly enhanced neurogenesis but did not effect gliogenesis. The results highlight the specificity of action of different neurotrophic factors in modulating the proliferative response to injury. Meteorin may be an interesting candidate in pathological settings involving damage to white matter, for example after stroke or neonatal brain injury.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.

Progressive cell loss is observed in the striatum, cerebral cortex, thalamus, hypothalamus, subthalamic nucleus and hippocampus in Huntington disease. In the striatum, dopamine-responsive medium spiny neurons are preferentially lost. Clinical features include involuntary movements, gait and orofacial impairments in addition to cognitive deficits and psychosis, anxiety and mood disorders. We utilized the Cre-LoxP system to generate mutant mice with selective postnatal ablation of D1 dopamine receptor-expressing striatal neurons to determine which elements of the complex Huntington disease phenotype relate to loss of this neuronal subpopulation. Mutant mice had reduced body weight, locomotor slowing, reduced rearing, ataxia, a short stride length wide-based erratic gait, impairment in orofacial movements and displayed haloperidol-suppressible tic-like movements. The mutation was associated with an anxiolytic profile. Mutant mice had significant striatal-specific atrophy and astrogliosis. D1-expressing cell number was reduced throughout the rostrocaudal extent of the dorsal striatum consistent with partial destruction of the striatonigral pathway. Additional striatal changes included up-regulated D2 and enkephalin mRNA, and an increased density of D2 and preproenkephalin-expressing projection neurons, and striatal neuropeptide Y and cholinergic interneurons. These data suggest that striatal D1-cell-ablation alone may account for the involuntary movements and locomotor, balance and orofacial deficits seen not only in HD but also in HD phenocopy syndromes with striatal atrophy. Therapeutic strategies would therefore need to target striatal D1 cells to ameliorate deficits especially when the clinical presentation is dominated by a bradykinetic/ataxic phenotype with involuntary movements.

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.

Cell replacement therapy for Parkinson's disease has predominantly focused on ectopic transplantation of fetal dopamine (DA) neurons into the striatum as a means to restore neurotransmission, rather than homotopic grafts into the site of cell loss, which would require extensive axonal growth. However, ectopic grafts fail to restore important aspects of DA circuitry necessary for controlled basal ganglia output, and this may underlie the suboptimal and variable functional outcomes in patients. We recently showed that DA neurons in homotopic allografts of embryonic ventral mesencephalon (VM) can send long axonal projections along the nigrostriatal pathway in order to innervate forebrain targets, although the extent of striatal reinnervation remains substantially less than can be achieved with ectopic placement directly into the striatal target. Here, we examined the possible benefits of using younger VM donor tissue and over-expression of glial cell-derived neurotrophic factor (GDNF) in the striatal target to improve the degree of striatal innervation from homotopic grafts. Younger donor tissue, collected on embryonic day (E)10, generated 4-fold larger grafts with greater striatal targeting, compared to grafts generated from more conventional E12 donor VM. Over-expression of GDNF in the host brain also significantly increased DA axonal growth and striatal innervation. Furthermore, a notable increase in the number and proportion of A9 DA neurons, essential for functional recovery, was observed in younger donor grafts treated with GDNF. Behavioural testing confirmed functional integration of younger donor tissue and demonstrated that improved motor function could be attributed to both local midbrain and striatal innervation. Together, these findings suggest there is significant scope for further development of intra-nigral grafting as a restorative approach for Parkinson's disease.

The male gender is determined by the sex-determining region on the Y chromosome (SRY) transcription factor. The unexpected action of SRY in the control of voluntary movement in male rodents suggests a role in the regulation of dopamine transmission and dopamine-related disorders with gender bias, such as Parkinson's disease. We investigated SRY expression in the human brain and function in vitro. SRY immunoreactivity was detected in the human male, but not female substantia nigra pars compacta, within a sub-population of tyrosine hydroxylase (TH) positive neurons. SRY protein also co-localized with TH positive neurons in the ventral tegmental area, and with GAD-positive neurons in the substantia nigra pars reticulata. Retinoic acid-induced differentiation of human precursor NT2 cells into dopaminergic cells increased expression of TH, NURR1, D2 R and SRY. In the human neuroblastoma cell line, M17, SRY knockdown resulted in a reduction in TH, DDC, DBH and MAO-A expression; enzymes which control dopamine synthesis and metabolism. Conversely, SRY over-expression increased TH, DDC, DBH, D2 R and MAO-A levels, accompanied by increased extracellular dopamine levels. A luciferase assay demonstrated that SRY activated a 4.6 kb 5' upstream regulatory region of the human TH promoter/nigral enhancer. Combined, these results suggest that SRY plays a role as a positive regulator of catecholamine synthesis and metabolism in the human male midbrain. This ancillary genetic mechanism might contribute to gender bias in fight-flight behaviours in men or their increased susceptibility to dopamine disorders, such as Parkinson's disease and schizophrenia.

Clinical trials have provided proof of principle that new dopamine neurons isolated from the developing ventral midbrain and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue is likely to be a contributing factor in the variable outcomes. Dissections of ventral midbrain used for transplantation will variously contain progenitors for different dopamine neuron subtypes as well as different neurotransmitter phenotypes. The overall impact of the resulting graft will be determined by the functional contribution from these different cell types. The A9 substantia nigra pars compacta dopamine neurons, for example, are known to be particularly important for motor recovery in animal models. Serotonergic neurons, on the other hand, have been implicated in unwanted dyskinesias. Currently little knowledge exists on how variables such as donor age, which have not been controlled for in clinical trials, will impact on the final neuronal composition of fetal grafts. Here we performed a birth dating study to identify the time-course of neurogenesis within the various ventral midbrain dopamine subpopulations in an effort to identify A9-enriched donor tissue for transplantation. The results show that A9 neurons precede the birth of A10 ventral tegmental area dopamine neurons. Subsequent grafting of younger ventral midbrain donor tissue revealed significantly larger grafts containing more mitotic dopamine neuroblasts compared to older donor grafts. These grafts were enriched with A9 neurons and showed significantly greater innervation of the target dorso-lateral striatum and DA release. Younger donor grafts also contained significantly less serotonergic neurons. These findings demonstrate the importance of

Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intra-cerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilized the human embryonic stem (ES) cell line Envy, which constitutively expresses green fluorescent protein (GFP), in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fiber patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.

During development, precise temporal and spatial gradients are responsible for guiding axons to their appropriate targets. Within the developing ventral midbrain (VM) the cues that guide dopaminergic (DA) axons to their forebrain targets remain to be fully elucidated. Wnts are morphogens that have been identified as axon guidance molecules. Several Wnts are expressed in the VM where they regulate the birth of DA neurons. Here, we describe that a precise temporo-spatial expression of Wnt5a accompanies the development of nigrostriatal projections by VM DA neurons. In mice at E11.5, Wnt5a is expressed in the VM where it was found to promote DA neurite and axonal growth in VM primary cultures. By E14.5, when DA axons are approaching their striatal target, Wnt5a causes DA neurite retraction in primary cultures. Co-culture of VM explants with Wnt5a-overexpressing cell aggregates revealed that Wnt5a is capable of repelling DA neurites. Antagonism experiments revealed that the effects of Wnt5a are mediated by the Frizzled receptors and by the small GTPase, Rac1 (a component of the non-canonical Wnt planar cell polarity pathway). Moreover, the effects were specific as they could be blocked by Wnt5a antibody, sFRPs and RYK-Fc. The importance of Wnt5a in DA axon morphogenesis was further verified in Wnt5a-/- mice, where fasciculation of the medial forebrain bundle (MFB) as well as the density of DA neurites in the MFB and striatal terminals were disrupted. Thus, our results identify a novel role of Wnt5a in DA axon growth and guidance.

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.