In vivo study to assess dosage of allogeneic pig retinal progenitor cells: Long-term survival, engraftment, differentiation and safety.

Despite notable efforts and significant therapeutical advances, age-related macular degeneration remains the single most common reason for vision loss. Retinal progenitor cells (RPCs) are considered promising candidates for cellular treatments that repair and restore vision. In this allogenic study, the phenotypic profile of pig and human RPCs derived using similar manufacturing processes is compared. The long-term (12-week) survival of green fluorescent protein-pig retinal progenitor cells GFP-pRPC after subretinal transplantation into normal miniature pig (mini-pig) retina is investigated. Human eyes are both anatomically and physiologically mimicked by pig eyes, so the pig is an ideal model to show an equivalent way of delivering cells, immunological response and dosage. The phenotypic equivalency of porcine and clinically intended human RPCs was established. Thirty-nine mini-pigs are used in this study, and vehicle-injected eyes and non-injected eyes serve as controls. Six groups are given different dosages of pRPCs, and the cells are found to survive well in all groups. At 12 weeks, strong evidence of integration is indicated by the location of the grafted cells within the neuro-retina, extension of processes to the plexiform layers and expression of key retinal markers such as recoverin, rhodopsin and synaptophysin. No immunosuppression is used, and no immune response is found in any of the groups. No pRPC-related histopathology findings are reported in the major organs investigated. An initial dose of 250 k cells in 100 µl of buffer is established as an appropriate initial dose for future human clinical trials.

Implantation of the clinical-grade human neural stem cell line, CTX0E03, rescues the behavioral and pathological deficits in the quinolinic acid-lesioned rodent model of Huntington's disease.

Huntington's disease (HD) is a devastating, autosomal-dominant neurodegenerative disease, for which there are currently no disease-modifying therapies. Clinical trials to replace the damaged striatal medium spiny neurons (MSNs) have been attempted in the past two decades but have met with only limited success. In this study, we investigated whether a clonal, conditionally immortalized neural stem cell line (CTX0E03), which has already shown safety and signals of efficacy in chronic ischemic stroke patients, could rescue deficits seen in an animal model of HD. After CTX0E03 transplantation into the quinolinic acid-lesioned rat model of HD, behavioral changes were measured using the rotarod, stepping, and staircase tests. In vivo differentiation and neuronal connections of the transplanted CTX0E03 cells were evaluated with immunohistochemical staining and retrograde tracing with Fluoro-Gold. We found that transplantation of CTX0E03 gave rise to a significant behavioral improvement compared with the sham- or fibroblast-transplanted group. Transplanted CTX0E03 formed MSNs (DARPP-32) and GABAergic neurons (GABA, GAD65/67) with BDNF expression in the striatum, while cortically transplanted cells formed Tbr1-positive neurons. Using a retrograde label, we also found stable engraftment and connection of the transplanted cells with host brain tissues. CTX0E03 transplantation also reduced glial scar formation and inflammation, as well as increasing endogenous neurogenesis and angiogenesis. Overall, our results demonstrate that CTX0E03, a clinical-grade neural stem cell line, is effective for preclinical test in HD, and, therefore, will be useful for clinical development in the treatment of HD patients.

Human Neural Stem Cell Therapy for Chronic Ischemic Stroke: Charting Progress from Laboratory to Patients.

Chronic disability after stroke represents a major unmet neurologic need. ReNeuron's development of a human neural stem cell (hNSC) therapy for chronic disability after stroke is progressing through early clinical studies. A Phase I trial has recently been published, showing no safety concerns and some promising signs of efficacy. A single-arm Phase II multicenter trial in patients with stable upper-limb paresis has recently completed recruitment. The hNSCs administrated are from a manufactured, conditionally immortalized hNSC line (ReNeuron's CTX0E03 or CTX), generated with c-mycERTAM technology. This technology has enabled CTX to be manufactured at large scale under cGMP conditions, ensuring sufficient supply to meets the demands of research, clinical development, and, eventually, the market. CTX has key pro-angiogenic, pro-neurogenic, and immunomodulatory characteristics that are mechanistically important in functional recovery poststroke. This review covers the progress of CTX cell therapy from its laboratory origins to the clinic, concluding with a look into the late stage clinical future.

The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants.

PURPOSE: The development of photoreceptor replacement therapy for retinal degenerative disorders requires the identification of the optimal cell source and immunosuppressive regimen in a large animal model. Allotransplants are not acutely rejected in swine subretinal space, although it is not known if survival can be improved with immunosuppression. Here we investigated the survival and integration of expanded pig retinal progenitor cells (pRPCs) in normal recipients with and without transient anti-inflammatory suppression. METHODS: pRPCs were derived from the neural retina of E60 GFP transgenic pigs, expanded for six passages, characterized, and transplanted into the subretinal space of 12 pigs. Six recipients received a single intravitreal injection of rapamycin and dexamethasone. RESULTS: pRPCs expressed the photoreceptor development genes Sox2, Pax6, Lhx2, Crx, Nrl, and Recoverin in vitro. Transplanted cells were identified in 9 out of 12 recipients 4 weeks after the injection. pRPCs integrated primarily into the photoreceptor inner segment layer and outer nuclear layer with single cells present in the inner nuclear layer. Donor cells remained recoverin-positive and acquired rhodopsin. We did not observe any signs of graft proliferation. The immunosuppression did not affect the survival or distribution of grafts. No macrophage infiltration or loss of retinal structure was observed in either group. CONCLUSIONS: Local immunosuppression with rapamycin and dexamethasone does not improve the outcome of pRPC allotransplantation into the subretinal space. TRANSLATIONAL RELEVANCE: Survival and integration of pRPC together with the lack of graft proliferation suggests that allogeneic RPC transplantation without transient immunosuppression is a favorable approach for photoreceptor cell replacement.

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER(TAM)construct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3'UTRs by dual reporter plasmid transfection and dual luciferase assay.

Human retinal progenitor cell transplantation preserves vision.

Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.

Clinical-grade human neural stem cells promote reparative neovascularization in mouse models of hindlimb ischemia.

OBJECTIVE: CTX0E03 (CTX) is a clinical-grade human neural stem cell (hNSC) line that promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now under clinical development for stroke disability. We evaluated the therapeutic activity of intramuscular CTX hNSC implantation in murine models of hindlimb ischemia for potential translation to clinical studies in critical limb ischemia. APPROACH AND RESULTS: Immunodeficient (CD-1 Fox(nu/nu)) mice acutely treated with hNSCs had overall significantly increased rates and magnitude of recovery of surface blood flow (laser Doppler), limb muscle perfusion (fluorescent microspheres, P<0.001), and capillary and small arteriole densities in the ischemic limb (fluorescence immunohistochemistry, both P<0.001) when compared with the vehicle-treated group. Hemodynamic and anatomic improvements were dose related and optimal at a minimum dose of 3×10(5) cells. Dose-dependent improvements in blood flow and increased vessel densities by hNSC administration early after ischemia were confirmed in immunocompetent CD-1 and streptozotocin-induced diabetic mice, together with marked reductions in the incidence of necrotic toes (P<0.05). Delayed administration of hNSCs, 7 days after occlusion, produced restorative effects when comparable with acute treatment of 35 days after hindlimb ischemia. Histological studies in hindlimb ischemia immunocompetent mice for the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory, and angiogenic responses and acceleration of myogenesis. CONCLUSIONS: hNSC therapy represents a promising treatment option for critical limb ischemia.

In vivo and in vitro characterization of the angiogenic effect of CTX0E03 human neural stem cells.

CTX0E03 is a human neural stem cell line previously reported to reduce sensory motor deficits in a middle cerebral artery occlusion (MCAo) model of stroke. The objective of this study was to investigate if CTX0E03 treatment promotes angiogenesis. As stroke leads to damage of the vasculature in the brain, angiogenesis may contribute to the functional recovery. To test this hypothesis, the angiogenic activity of CTX0E03 was assessed both in vitro and in vivo. In vitro, CTX0E03 expression of trophic and proangiogenic factors was determined by real-time RT-PCR, Western blot, and ELISA, and its angiogenic activity was investigated in well-established angiogenesis assays. In vivo, angiogenesis was investigated in naive mice and MCAo rat brain and was evaluated by immunohistochemistry (IHC) using Von Willebrand factor (VWF), a marker of blood vessel formation, and BrdU/CD31 double labeling in naive mice only. In vitro results showed that CTX0E03-conditioned medium and coculture significantly increased total tubule formation compared with controls (p=0.002 and p=0.0008, respectively). Furthermore, CTX0E03 cells were found to be in direct association with the tubules by ICC. In vivo CTX0E03-treated brains demonstrated a significant increase in areas occupied by VWF-positive microvessels compared with vehicle-treated naive mice (two-way ANOVA, Interaction p<0.05, Treatment p<0.0001, Time p<0.0) and MCAo rat (p=0.001 unpaired t test, Welch's correction). CTX0E03-treated naive mouse brains showed an increase in BrdU/CD31 colabeling. In conclusion, in vitro CTX0E03 cells express proangiogenic factors and may promote angiogenesis by both release of paracrine factors and direct physical interaction. Furthermore, in vivo CTX0E03-treated rodent brains exhibited a significant increase in microvessels at the site of implantation compared with vehicle-injected groups. Taken together these data suggest that CTX0E03 cell therapy may provide significant benefit to stroke patients through upregulation of angiogenesis in the ischemic brain.

Implantation of c-mycER TAM immortalized human mesencephalic-derived clonal cell lines ameliorates behavior dysfunction in a rat model of Parkinson's disease.

Human neural stem cells offer the hope that a cell therapy treatment for Parkinson's disease (PD) could be made widely available. In this study, we describe two clonal human neural cell lines, derived from two different 10-week-old fetal mesencephalic tissues and immortalized with the c-mycER(TAM) transgene. Under the growth control of 4-hydroxytamoxifen, both cell lines display stable long-term growth in culture with a normal karyotype. In vitro, these nestin-positive cells are able to differentiate into tyrosine hydroxylase (TH)-positive neurons and are multipotential. Implantation of the undifferentiated cells into the 6-OHDA substantia nigral lesioned rat model displayed sustained improvements in a number of behavioral tests compared with noncell-implanted, vehicle-injected controls over the course of 6 months. Histological analysis of the brains showed survival of the implanted cells but no evidence of differentiation into TH-positive neurons. An average increase of approximately 26% in host TH immunoreactivity in the lesioned dorsal striatum was observed in the cell-treated groups compared to controls, with no difference in loss of TH cell bodies in the lesioned substantia nigra. Further analysis of the cell lines identified a number of expressed trophic factors, providing a plausible explanation for the effects observed in vivo. The exact mechanisms by which the implanted human neural cell lines provide behavioral improvements in the PD model are not completely understood; however, these findings provide evidence that cell therapy can be a potent treatment for PD acting through a mechanism independent of dopaminergic neuronal cell replacement.

Neurotrophic actions of the novel AMPA receptor potentiator, LY404187, in rodent models of Parkinson's disease.

Recent developments in the molecular biology and pharmacology of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has led to the discovery of selective, potent and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and evidence suggests that they play important roles in plasticity and cognitive processes. Activation of AMPA receptors also increases neuronal activation and activity-dependent signalling, which may increase brain-derived neurotrophic factor (BDNF) expression and enhance cell proliferation in the brain. We therefore hypothesised that an AMPA receptor potentiator may provide neurotrophic effects in rodent models of Parkinson's disease. In the present studies we report that the potent and selective AMPA receptor potentiator, R,S-N-2-(4-(4-Cyanophenyl)phenyl)propyl 2-propanesulfonamide (LY404187), provides both functional, neurochemical and histological protection against unilateral infusion of 6-hydroxydopamine into the substantia nigra or striatum of rats. The compound also reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in mice. Interestingly, we were also able to observe large functional and histological effects when we delayed treatment until after cell death had occurred (3 or 6 days after 6-hydroxydopamine infusion), supporting a neurotrophic mechanism of action. In addition, LY404187 provided a dose-dependent increase in growth-associated protein-43 expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new therapy to halt the progression and perhaps repair the degeneration in Parkinson's disease.

The neuronal nitric oxide synthase inhibitor, TRIM, as a neuroprotective agent: effects in models of cerebral ischaemia using histological and magnetic resonance imaging techniques.

Most neuroprotective compounds that appear promising in the pre-clinical phase of testing are subsequently dismissed as relatively ineffective when entered into large-scale clinical trials. Many pre-clinical studies of potential neuroprotective candidates evaluate efficacy in only one or possibly two different models of ischaemia. In this study we examined the effects of 1,2-trifluoromethylphenyl imidazole (TRIM), a novel neuronal nitric oxide synthase (nNOS) inhibitor, in three models of cerebral ischaemia (global gerbil, global rat and focal rat). In addition, to follow the progression of the pathology, we also compared traditional histology methods with more advanced magnetic resonance imaging (MRI) as endpoint measures for neurological damage and neuroprotection. TRIM (50 mg/kg i.p.) prevented ischaemia-induced hippocampal damage following global ischaemia in gerbils when administered before or immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. Further studies indicated that the compound (administered at 50 mg/kg, i.p., immediately after occlusion) also protected in a rat four-vessel occlusion (4-VO) model using both histological and diffusion-weighted (DW) imaging techniques. In a final study, TRIM (50 mg/kg i.p. 30 min after occlusion) provided a significant reduction in infarct volume at 4 and 24 h as measured using diffusion-weighted (DW) and proton density (PD)-weighted magnetic resonance imaging (MRI). This was confirmed using histological techniques. These studies confirm that nNOS inhibitors may have utility in stroke and provide evidence that combined magnetic resonance and histological methods can provide a powerful method of assessing neuronal damage in rodent models of cerebral ischaemia.

LY503430, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor potentiator with functional, neuroprotective and neurotrophic effects in rodent models of Parkinson's disease.

Glutamate is the major excitatory transmitter in the brain. Recent developments in the molecular biology and pharmacology of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors have led to the discovery of selective, potent, and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes. In the present study, we first characterized a novel AMPA receptor potentiator, (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430), on recombinant human GLUA1-4 and native preparations in vitro and then evaluated the potential neuroprotective effects of the molecule in rodent models of Parkinson's disease. Results indicated that submicromolar concentrations of LY503430 selectively enhanced glutamate-induced calcium influx into human embryonic kidney 293 cells transfected with human GLUA1, GLUA2, GLUA3, or GLUA4 AMPA receptors. The molecule also potentiated AMPA-mediated responses in native cortical, hippocampal, and substantia nigra neurons. We also report here that LY503430 provided dose-dependent functional and histological protection in animal models of Parkinson's disease. The neurotoxicity after unilateral infusion of 6-hydroxydopamine into either the substantia nigra or the striatum of rats and that after systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice were reduced. Interestingly, LY503430 also had neurotrophic actions on functional and histological outcomes when treatment was delayed until well after (6 or 14 days) the lesion was established. LY503430 also produced some increase in brain-derived neurotrophic factor in the substantia nigra and a dose-dependent increases in growth associated protein-43 (GAP-43) expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new disease modifying therapy for Parkinson's disease.

An AMPA receptor potentiator modulates hippocampal expression of BDNF: an in vivo study.

AMPA receptor activation has been demonstrated to increase the neuronal expression of brain derived neurotrophic factor (BDNF). In the present study, we investigated the effect of a novel AMPA receptor potentiator (LY404187) and its active isomer (LY451646) on the expression of BDNF protein and mRNA, as well as TrkB mRNA in rat hippocampus. LY404187 administered for 7 days (1 mg/kg) significantly increased the number of BDNF immunopositive cells in the dentate gyrus, but not other hippocampal subfields. Chronic treatment (7 days) with LY451646 (0.5 mg/kg, comparable to 1 mg/kg of LY404187) increased the level of both BDNF and TrkB mRNA expression in the dentate gyrus, CA3 and CA4 of the hippocampus. However, chronic treatment with lower doses of LY451646 (0.125 and 0.25 mg/kg) decreased the level of BDNF and TrkB mRNA in hippocampus, whilst the highest used dose of LY451646 (1 mg/kg) had no effect on BDNF and TrkB mRNA in hippocampus. In contrast, acute treatment with LY451646 produced an increase in BDNF mRNA levels at doses of 0.125 and 0.25 mg/kg in the hippocampus (CA4, CA3 and dentate gyrus, but not in CA1). LY451646 at 0.5 mg/kg had no effect, but at 1.0 mg/kg decreased the level of BDNF mRNA in hippocampus. Acute treatment with LY451646 did not affect the TrkB receptor mRNA levels in hippocampus. Our results demonstrate that biarylpropylsulfonamide AMPA receptor potentiators are capable of modulating the expression of BDNF and TrkB mRNA in a dose- and time-dependent manner. The increase in both BDNF protein and mRNA expression in the dentate gyrus but not in CA1 indicates a specific role of AMPA receptors in the regulation of BDNF expression in this hippocampal subfield. The regulation of BDNF expression by biarylpropylsulfonamids such as LY451646 may have important therapeutical implications for this class of molecule in the treatment of depression and other CNS disorders.