Understanding the Influence of Target Acquisition on Survival, Integration, and Phenotypic Maturation of Dopamine Neurons within Stem Cell-Derived Neural Grafts in a Parkinson's Disease Model.

Midbrain dopaminergic (DA) neurons include many subtypes characterized by their location, connectivity and function. Surprisingly, mechanisms underpinning the specification of A9 neurons [responsible for motor function, including within ventral midbrain (VM) grafts for treating Parkinson's disease (PD)] over adjacent A10, remains largely speculated. We assessed the impact of synaptic targeting on survival, integration, and phenotype acquisition of dopaminergic neurons within VM grafts generated from fetal tissue or human pluripotent stem cells (PSCs). VM progenitors were grafted into female mice with 6OHDA-lesions of host midbrain dopamine neurons, with some animals also receiving intrastriatal quinolinic acid (QA) injections to ablate medium spiny neurons (MSN), the A9 neuron primary target. While loss of MSNs variably affected graft survival, it significantly reduced striatal yet increased cortical innervation. Consequently, grafts showed reduced A9 and increased A10 specification, with more DA neurons failing to mature into either subtype. These findings highlight the importance of target acquisition on DA subtype specification during development and repair.SIGNIFICANCE STATEMENT Parish and colleagues highlight, in a rodent model of Parkinson's disease (PD), the importance of synaptic target acquisition in the survival, integration and phenotypic specification of grafted dopamine neurons derived from fetal tissue and human stem cells. Ablation of host striatal neurons resulted in reduced dopamine neuron survival within grafts, re-routing of dopamine fibers from striatal to alternate cortical targets and a consequential reduced specification of A9 dopamine neurons (the subpopulation critical for restoration of motor function) and increase in A10 DA neurons.

Hemispheric cortical atrophy and chronic microglial activation following mild focal ischemic stroke in adult male rats.

Animal modeling has played an important role in our understanding of the pathobiology of stroke. The vast majority of this research has focused on the acute phase following severe forms of stroke that result in clear behavioral deficits. Human stroke, however, can vary widely in severity and clinical outcome. There is a rapidly building body of work suggesting that milder ischemic insults can precipitate functional impairment, including cognitive decline, that continues through the chronic phase after injury. Here we show that a small infarction localized to the frontal motor cortex of rats following injection of endothelin-1 results in an essentially asymptomatic state based on motor and cognitive testing, and yet produces significant histopathological change including remote atrophy and inflammation that persists up to 1 year. While there is understandably a major focus in stroke research on mitigating the acute consequences of primary infarction, these results point to progressive atrophy and chronic inflammation as additional targets for intervention in the chronic phase after injury. The present rodent model provides an important platform for further work in this area.

Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson's Disease.

Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for Parkinson's disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease.

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

Local Injection of Endothelin-1 in the Early Neonatal Rat Brain Models Ischemic Damage Associated with Motor Impairment and Diffuse Loss in Brain Volume.

Cerebral palsy is an irreversible movement disorder resulting from cerebral damage sustained during prenatal or neonatal brain development. As survival outcomes for preterm injury improve, there is increasing need to model ischemic injury at earlier neonatal time-points to better understand the subsequent pathological consequences. Here we demonstrate a novel neonatal ischemic model using focal administration of the potent vasoconstrictor peptide, endothelin-1 (ET-1), in newborn rats. The functional and histopathological outcomes compare favourably to those reported following the widely used hypoxic ischemia (HI) model. These include a robust motor deficit sustained into adulthood and recapitulation of hallmark features of preterm human brain injury, including atrophy of subcortical white matter and periventricular fiber bundles. Compared to procedures involving carotid artery manipulation and periods of hypoxia, the ET-1 ischemia model represents a rapid and technically simplified model more amenable to larger cohorts and with the potential to direct the locus of ischemic damage to specific brain areas.

Modelling the dopamine and noradrenergic cell loss that occurs in Parkinson's disease and the impact on hippocampal neurogenesis.

Key pathological features of Parkinson's Disease (PD) include the progressive degeneration of midbrain dopaminergic (DA) neurons and hindbrain noradrenergic (NA) neurons. The loss of DA neurons has been extensively studied and is the main cause of motor dysfunction. Importantly, however, there are a range of 'non-movement' related features of PD including cognitive dysfunction, sleep disturbances and mood disorders. The origins for these non-motor symptoms are less clear, but a possible substrate for cognitive decline may be reduced adult-hippocampal neurogenesis, which is reported to be impaired in PD. The mechanisms underlying reduced neurogenesis in PD are not well established. Here we tested the hypothesis that NA and DA depletion, as occurs in PD, impairs hippocampal neurogenesis. We used 6-hydroxydopamine or the immunotoxin dopamine-ß-hydroxylase-saporin to selectively lesion DA or NA neurons, respectively, in adult Sprague Dawley rats and assessed hippocampal neurogenesis through phenotyping of cells birth-dated using 5-bromo-2'-deoxyuridine. The results showed no difference in proliferation or differentiation of newborn cells in the subgranular zone of the dentate gyrus after NA or DA lesions. This suggests that impairment of hippocampal neurogenesis in PD likely results from mechanisms independent of, or in addition to degeneration of DA and NA neurons.

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

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.

Peptide-Based Scaffolds Support Human Cortical Progenitor Graft Integration to Reduce Atrophy and Promote Functional Repair in a Model of Stroke.

Stem cell transplants offer significant hope for brain repair following ischemic damage. Pre-clinical work suggests that therapeutic mechanisms may be multi-faceted, incorporating bone-fide circuit reconstruction by transplanted neurons, but also protection/regeneration of host circuitry. Here, we engineered hydrogel scaffolds to form "bio-bridges" within the necrotic lesion cavity, providing physical and trophic support to transplanted human embryonic stem cell-derived cortical progenitors, as well as residual host neurons. Scaffolds were fabricated by the self-assembly of peptides for a laminin-derived epitope (IKVAV), thereby mimicking the brain's major extracellular protein. Following focal ischemia in rats, scaffold-supported cell transplants induced progressive motor improvements over 9 months, compared to cell- or scaffold-only implants. These grafts were larger, exhibited greater neuronal differentiation, and showed enhanced electrophysiological properties reflective of mature, integrated neurons. Varying graft timing post-injury enabled us to attribute repair to both neuroprotection and circuit replacement. These findings highlight strategies to improve the efficiency of stem cell grafts for brain repair.

Long-Distance Axonal Growth and Protracted Functional Maturation of Neurons Derived from Human Induced Pluripotent Stem Cells After Intracerebral Transplantation.

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.

Functionalized composite scaffolds improve the engraftment of transplanted dopaminergic progenitors in a mouse model of Parkinson's disease.

With the brain's limited capacity for repair there is a need for new and innovative therapies to promote regeneration. Stem/progenitor cell transplantation has received increasing attention, and whilst clinical trials demonstrating functional integration exist, inherent variability between patients has hindered development of this therapy. Variable outcomes have largely been attributed to poor survival and insufficient reinnervation of target tissues due in part to the suboptimal host environment. Here we examined whether improving the physical properties of the host milieu, by way of bioengineered scaffolds, may enhance engraftment. We developed a composite scaffold, incorporating electrospun poly(l-lactic acid) short nanofibers embedded within a thermo-responsive xyloglucan hydrogel, which could be easily injected into the injured brain. Furthermore, to improve the trophic properties of the host brain, glial derived neurotrophic factor (GDNF), a protein known to promote cell survival and axonal growth, was blended into and/or covalently attached onto the composite scaffolds to provide controlled delivery. In vitro we confirmed the ability of the scaffolds to support ventral midbrain (VM) dopamine progenitors, and provide sustained delivery of GDNF - capable of eliciting effects on cell survival and dopaminergic axon growth. In Parkinsonian mice, we show that these composite scaffolds, whilst having no deleterious impact on the host immune response, enhanced the survival of VM grafts and reinnervation of the striatum, an effect that was augmented through the scaffold delivery of GDNF. Taken together, these functionalized composite scaffolds provide a means to significantly improve the milieu of the injured brain, enabling enhanced survival and integration of grafted neurons.

Chondroitinase improves midbrain pathway reconstruction by transplanted dopamine progenitors in Parkinsonian mice.

Within the adult central nervous system the lack of guidance cues together with the presence of inhibitory molecules produces an environment that is restrictive to axonal growth following injury. Consequently, while clinical trials in Parkinson's disease (PD) patients have demonstrated the capacity of fetal-derived dopamine neurons to survive, integrate and alleviate symptoms, the non-permissive host environment has contributed to the incomplete re-innervation of the target tissue by ectopic grafts, and even more noticeable, the poor reconstruction of the midbrain dopamine pathways following homotopic midbrain grafting. One such inhibitory molecule is the chondroitin sulfate proteoglycan (CSPG), a protein that has been shown to impede axonal growth during development and after injury. Digestion of CSPGs, by delivery of the bacterial enzyme chondroitinase ABC (ChABC), can improve axonal regrowth following a number of neural injuries. Here we examined whether ChABC could similarly improve axonal growth of transplanted dopamine neurons in an animal model of PD. Acute delivery of ChABC, into the medial forebrain bundle, degraded CSPGs along the nigrostriatal pathway. Simultaneous homotopic transplantation of dopaminergic progenitors, into the ventral midbrain of ChABC treated PD mice, had no effect on graft survival but resulted in enhanced axonal growth along the nigrostriatal pathway and reinnervation of the striatum, compared to control grafted mice. This study demonstrates that removal of axonal growth inhibitory molecules could significantly enhance dopaminergic graft integration, thereby holding implications for future approaches in the development of cell replacement therapies for Parkinsonian patients.