Brooke R. Snyder

Towards a transgenic model of Huntington’s disease in a non-human primate

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10–15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.

Putative Dental Pulp-Derived Stem/Stromal Cells Promote Proliferation and Differentiation of Endogenous Neural Cells in the Hippocampus of Mice

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune‐suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells, which express Nanog, Rex‐1, Oct‐4, and various cell surface antigens, and have multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre‐existing Nestin+ neural progenitor cells (NPCs) and β‐tubulin‐III+ mature neurons to the site of the graft. Additionally, many cells born during the first 7 days after implantation proliferated, forming NPCs and neurons, and, to a lesser extent, underwent astrogliosis, forming astrocytes and microglia, by 30 days after implantation. Although the DPSC graft itself was short term, it had long‐term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of ciliary neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor for up to 30 days after implantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the central nervous system. DPSCs would be a preferable cell source for therapy due to the possibility of a “personalized” stem cell, avoiding the problems associated with host immune rejection.

Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.

Gene therapy for motor neuron diseases requires efficient gene delivery to motor neurons (MNs) throughout the spinal cord and brainstem. The present study compared adeno-associated viral (AAV) vector serotypes 1, 6, 8, and 9 for spinal cord delivery in adult mice, by the intraparenchymal or intrathecal route of administration. Whereas intraparenchymal injections resulted in local transduction of the lumbar segment of the spinal cord, intrathecal injections led to a broader distribution, transducing cells along the sacral, lumbar, and lower thoracic spinal cord. Overall, AAV6 and AAV9 performed better than the other serotypes. Dramatic differences in cell-specific expression patterns could be observed when constructs bearing the chicken β-actin (Cba) versus cytomegalovirus (CMV) promoter were compared. In summary, intrathecal delivery of AAV6 or AAV9 vectors containing the CMV promoter yielded the strongest levels of biodistribution and MN transduction in the spinal cord.

Human Multipotent Stromal Cells (MSCs) Increase Neurogenesis and Decrease Atrophy of the Striatum in a Transgenic Mouse Model for Huntington's Disease

Background Implantation of human multipotent stromal cells from bone marrow (hMSCs) into the dentate gyrus of the hippocampus of mice was previously shown to stimulate proliferation, migration and neural differentiation of endogenous neural stem cells. We hypothesized that hMSCs would be beneficial in a mouse model of Huntington disease (HD) due to these neurogenic effects. Results We implanted hMSCs into the striatum of transgenic mice (N171-82Q) that are a model for HD. The implanted hMSCs rapidly disappeared over 3 to 15 days. However, they increased proliferation and neural differentiation of endogenous neural stem cells for up to 30 days. They also increased neurotrophic signaling and decreased atrophy of the striatum in 3-month old HD mice implanted with hMSCs one month earlier. Conclusions The results therefore suggested that neural implantation of hMSCs may be of benefit in HD but a number of parameters of dose, treatment schedule, and route of administration need to be optimized.

Cervical multilevel intraspinal stem cell therapy: assessment of surgical risks in Gottingen minipigs.

Study Design. Assessment of long-term surgical risks from multiple intraspinal cell injections. Objective. To prove that multilevel-targeted cell injection to the spinal cord can be a feasible and safe procedure. Summary of Background Data. Neural cell transplantation has been proposed as a treatment for a variety of neurologic disorders, including degenerative, ischemic, autoimmune, and traumatic etiologies. Among these diseases, the lack of effective treatment for amyotrophic lateral sclerosis has prompted the search for cell-based neuroprotection or motor neuron-replacement therapies. Methods. Fifteen female minipigs, divided into 3 experimental groups, underwent either 5 or 10 unilateral injections of neural stem cells or 10 vehicle injections into the C3–C5 segments of the spinal cord, using a device and technique developed for safe and accurate injection into the human spinal cord. All animals received intravenous Tacrolimus (0.025 mg/kg) BID during the course of the study. Sensory and motor functions as well as general morbidity were assessed for 28 days. Full necropsy was performed and spinal cords were analyzed for graft survival. This study was performed under Good Laboratory Practice conditions. Results. Neither mortality nor permanent surgical complications were observed within the 28-day study period. All animals returned to preoperative baseline showing full motor function recovery. Graft survival was demonstrated by immunohistochemistry. Conclusion. Clinically acceptable neural progenitor survival, distribution, and density were achieved using the number of injections and surgical techniques specifically developed for this purpose.

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

BackgroundBackgroundChimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.ResultsChDPSCs were isolated from an incisor and a canine of a forty-seven year old female chimpanzee. A homogenous population of ChDPSCs was established in early culture at a high proliferation rate and verified by the expression pattern of thirteen cell surface markers. The ChDPSCs are multipotent and were capable of differentiating into osteogenic, adipogenic and chondrogenic lineages under appropriate in vitro culture conditions. ChDPSCs also express stem cell (Sox-2, Nanog, Rex-1, Oct-4) and osteogenic (Osteonectin, osteocalcin, osteopontin) markers, which is comparable to reported results of rhesus monkey BMSCs (rBMSCs), hBMSCs and hDPSCs. Although ChDPSCs vigorously proliferated during the initial phase and gradually decreased in subsequent passages, the telomere length indicated that telomerase activity was not significantly reduced.ConclusionThese results demonstrate that ChDPSCs can be efficiently isolated from post-mortem teeth of adult chimpanzees and are multipotent. Due to the almost identical genome composition of humans and chimpanzees, there is an emergent need for defining the new role of chimpanzee modeling in comparative medicine. Teeth are easy to recover at necropsy and easy to preserve prior to the retrieval of dental pulp for stem/stromal cells isolation. Therefore, the establishment of ChDPSCs would preserve and maximize the applications of such a unique and invaluable animal model, and could advance the understanding of cellular functions and differentiation control of adult stem cells in higher primates.

Intramuscular administration of a VEGF zinc finger transcription factor activator (VEGF-ZFP-TF) improves functional outcomes in SOD1 rats.

Abstract Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron loss leading to paralysis and death. Vascular endothelial growth factor (VEGF) has angiogenic, neurotrophic, and neuroprotective properties, and has preserved neuromuscular function and protected motor neurons in rats engineered to overexpress the human gene coding the mutated G93A form of the superoxide dismutase-1 (SOD1). We assessed the effects of intramuscular administration of a plasmid that encodes a zinc finger protein transcription factor (ZFP-TF) engineered to induce VEGF expression in the SOD1 rat model of ALS. Weekly injections of the plasmid preserved ipsilateral hindlimb grip strength and markedly improved rotarod performance in SOD1 rats compared to the vehicle-treated group. The number of motor neurons and the proportion of innervated neuromuscular junctions were similar in both groups. In conclusion, our data suggest that administration of the VEGF-ZFP-TF may be neuroprotective and has potential as a safe and practical approach for the management of motor disability in ALS.

Viral vector-mediated gene transfer for CNS disease.

Importance of the field: Gene therapy is a promising strategy for the treatment of many neurological disorders that currently lack effective treatment. Recent improvements in vectorology and vector engineering have improved overall safety and delivery of viral vectors. Areas covered in this review: This review discusses the current state of viral vector development and clinical use, as well as routes of delivery, and clinical trials for neurological disorders. What the reader will gain: Viral vectors may be delivered directly or remotely to the CNS, largely depending on the nature of the disease and the tropism of the vector. Nonetheless, delivery remains one of the major limitations of successful gene transfer to the CNS. Take home message: Although the majority of clinical trials have centered on gene replacement and neuroprotection approaches, the field is advancing in the direction of neuromodulation, gene silencing and other newer strategies.

Monkey hybrid stem cells develop cellular features of Huntington's disease.

BackgroundPluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntingtons disease. Additionally, a Huntingtons cell model that develops robust pathological features of Huntingtons disease would be valuable for drug discovery research.ResultsTo test this hypothesis, a pluripotent Huntingtons disease monkey hybrid cell line (TrES1) was established from a tetraploid Huntingtons disease monkey blastocyst generated by the fusion of transgenic Huntingtons monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntingtons disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID) mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntingtons disease cellular features is influenced by neural developmental events.ConclusionsHuntingtons disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntingtons disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntingtons disease pathogenesis.

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

BackgroundDental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntingtons disease (HD), have not been fully investigated.ResultsTo examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile.ConclusionsOur results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.