RESUMEN

Patient-specific stem cells derived from somatic cell nuclear transfer (SCNT) embryos or from induced pluripotent stem cells (iPSCs) could be used to treat various diseases with minimal immune rejection. Many studies using these cells have been conducted in rats and mice; however, there exist numerous dissimilarities between the rodents and humans limiting the clinical predictive power and experimental utility of rodent experiments alone. Nonhuman primates (NHPs) share greater homology to human than rodents in all respects, including genomics, physiology, biochemistry, and the immune system. Thus, experimental data obtained from monkey studies would be more predictive for designing an effective cell replacement therapy in humans. Unfortunately, there are few iPSC lines and even fewer SCNT lines that have been derived in NHPs, hampering broader studies in regenerative medicine. One promising potential therapy would be the replacement of dopamine neurons that are lost in Parkinson's disease. After dopamine depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the African green monkey (Chlorocebus sabaeus) shows the most complete model of Parkinsonism compared with other species and brain pathology and behavioral changes are almost identical to those in humans after accidental exposure to MPTP. Therefore, we have developed a SCNT procedure to generate multiple pluripotent stem cell lines in this species for studies of possible treatment of Parkinsonism and for comparing with cells derived from iPSCs. Using 24 female monkeys as egg donors and 7 somatic cell donor monkeys, we have derived 11 SCNT embryonic stem cell lines that expressed typical stemness genes and formed all three germ layer derivatives. We also derived two iPSC lines using an episome-mediated reprogramming factor delivery system. This report describes the process for deriving these cell lines and proving their pluripotency for differentiation into various potentially therapeutic cells.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Técnicas de Transferencia Nuclear , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Bandeo Cromosómico , Clonación de Organismos , Medios de Cultivo , Análisis Citogenético , ADN/genética , Neuronas Dopaminérgicas/metabolismo , Desarrollo Embrionario , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Genotipo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Ovario/fisiología , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem cells (PS cells) and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. Here, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could surpass the limitations of current monkey models of neurological and psychiatric disease, but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Asunto(s)

Discusiones Bioéticas , Modelos Animales de Enfermedad , Ética en Investigación , Trasplante de Células Madre/ética , Quimera por Trasplante , Animales , Haplorrinos , Humanos

RESUMEN

Generating human organs inside interspecies chimeras might one day produce patient-specific organs for clinical applications, but further advances in identifying human chimera-competent pluripotent stem (PS) cells are needed. Moreover, the potential for human PS cells to contribute to the brains in human-animal chimeras raises ethical questions. The use of non-human primate (NHP) chimera-competent PS cells would allow one to test interspecies organogenesis strategies while also bypassing such ethical concerns. Here, we provide the first evidence for a putative chimera-competent pluripotent state in NHPs. Using histone deacetylase (HDAC) and selective kinase inhibition, we converted the PS cells of an Old World monkey, the African Green monkey (aGM), to an ERK-independent cellular state that can be propagated in culture conditions similar to those that sustain chimera-competency in rodent cells. The obtained stem cell lines indefinitely self-renew in MEK inhibitor-containing culture media lacking serum replacement and FGF. Compared to conventional PS cells, the novel stem cells express elevated levels of KLF4, exhibit more intense nuclear staining for TFE3, and manifest increased mitochondrial membrane depolarization. These data are preliminary but indicate that the key to deriving primate chimera-competent PS cells is to shield cells from the activation of ERK, PKC, and WNT signaling. Because of the similarity of aGMs to humans, the more ethically palatable use of NHP cells, and the more similar gestation length between aGMs and large animals such as sheep, the aGM cell lines described herein will serve as a useful tool for evaluating the efficacy and safety of interspecies organogenesis strategies. Future studies will examine chimera-competency and generalizability to human cells.

Asunto(s)

Quimera/embriología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Células Madre Pluripotentes/citología , Animales , Bioética , Células Cultivadas , Chlorocebus aethiops , Humanos , Factor 4 Similar a Kruppel , Organogénesis

RESUMEN

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem (PS) cells and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. In this study, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could not only surpass the limitations of current monkey models of neurological and psychiatric disease but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Asunto(s)

Modelos Animales de Enfermedad , Haplorrinos/genética , Trastornos Mentales/genética , Enfermedades Neurodegenerativas/genética , Quimera por Trasplante/genética , Animales , Investigaciones con Embriones/ética , Haplorrinos/fisiología , Humanos , Trastornos Mentales/patología , Enfermedades Neurodegenerativas/patología , Trasplante de Células Madre/ética , Trasplante de Células Madre/métodos , Trasplante de Células Madre/normas , Quimera por Trasplante/fisiología

RESUMEN

The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.

Asunto(s)

Células Madre Pluripotentes/citología , Animales , Diferenciación Celular/fisiología , Humanos , Trasplante de Órganos

RESUMEN

Clinical trials testing the hypothesis that fetal dopamine grafts would provide antiparkinsonian benefit in patients who had already developed side effects from their long-term use of L-dopa revealed, in some cases, the presence of dyskinesias even in the absence of L-dopa. The form, intensity, and frequency of these dyskinesias were quite variable, but their manifestation slowed the clinical development of cell replacement therapies. Rodent models of graft-induced dyskinesias (GIDs) have been proposed, but their accuracy in modeling GIDs has been questioned because they usually require amphetamine for their presentation. The present study attempted to model GIDs in parkinsonian monkeys and, for the first time, to test the effect of grafts on previously dyskinetic monkeys. Toward this end, monkeys were rendered parkinsonian with n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and dyskinetic with levodopa. They then received intraputamenal grafts of fetal dopaminergic cells, control cerebellar cells, or vehicle bilaterally and were studied for 18 months. Dopaminergic cells were grafted in a manner designed to produce either "hot spot" or "widespread" striatal innervation. Although levodopa-induced dyskinesias could be elicited postoperatively, GIDs were never observed in any animal at any time after grafting. Grafted monkeys were also challenged with levodopa but did not show any greater responses to these challenges than before grafting. These studies support the development of future dopamine neuron cell transplantation therapy-based approaches, indicating that in relevant primate models with appropriate cell preparation methodology, with successful graft survival and putamenal dopamine innervation, there is no evidence of graft-induced dyskinesias. J. Comp. Neurol. 525:498-512, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Cerebelo/trasplante , Neuronas Dopaminérgicas/trasplante , Discinesia Inducida por Medicamentos/fisiopatología , Trasplante de Tejido Fetal , Intoxicación por MPTP/terapia , Mesencéfalo/trasplante , Neuronas/trasplante , Animales , Antiparkinsonianos/toxicidad , Calbindinas/metabolismo , Núcleo Caudado/patología , Núcleo Caudado/fisiopatología , Cerebelo/metabolismo , Chlorocebus aethiops , Dopamina/administración & dosificación , Dopamina/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Levodopa/toxicidad , Intoxicación por MPTP/patología , Intoxicación por MPTP/fisiopatología , Masculino , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Microglía/metabolismo , Microglía/patología , Neuronas/metabolismo , Neuronas/patología , Putamen/patología , Putamen/fisiopatología , Putamen/cirugía , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Cell therapy has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying potentially viable human embryos and can be used to generate an unlimited supply of neural cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine (DA) levels in rodent and nonhuman primate models of PD. Here we report the results of a 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms. The hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys. Transplantation of hpNSCs was safe and well tolerated by the animals with no dyskinesia, tumors, ectopic tissue formation, or other test article-related serious adverse events. We observed that hpNSCs promoted behavioral recovery; increased striatal DA concentration, fiber innervation, and number of dopaminergic neurons; and induced the expression of genes and pathways downregulated in PD compared to vehicle control animals. These results provide further evidence for the clinical translation of hpNSCs and support the approval of the world's first pluripotent stem cell-based phase I/IIa study for the treatment of PD (Clinical Trial Identifier NCT02452723).

Asunto(s)

Intoxicación por MPTP/terapia , Células-Madre Neurales/trasplante , Recuperación de la Función/fisiología , Animales , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Diferenciación Celular , Células Cultivadas , Chlorocebus aethiops , Análisis por Conglomerados , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Inmunohistoquímica , Cariotipo , Intoxicación por MPTP/inducido químicamente , Intoxicación por MPTP/patología , Masculino , Células-Madre Neurales/citología , Partenogénesis

RESUMEN

BACKGROUND AND PURPOSE: Ischemic stroke is the leading cause of upper extremity motor impairments. Although several well-characterized experimental stroke models exist, modeling of upper extremity motor impairments, which are unique to primates, is not well established. Cortical representation of dexterous movements in nonhuman primates is functionally and topographically similar to that in humans. In this study, we characterize the African green monkey model of focal ischemia reperfusion with a defined syndrome, impaired dexterous movements. METHODS: Cerebral ischemia was induced by transient occlusion of the M3 segment of the left middle cerebral artery. Motor and cognitive functions after stroke were evaluated using the object retrieval task with barrier-detour. Postmortem magnetic resonance imaging and histopathology were performed to map and characterize the infarct. RESULTS: The middle cerebral artery occlusion consistently produced a necrotic infarct localized in the sensorimotor cortex in the middle cerebral artery territory. The infarction was reproducible and resulted in significant loss of fine motor function characterized by impaired dexterity. No significant cognitive impairment was detected. Magnetic resonance imaging and histopathology demonstrated consistent and significant loss of tissue on the left parietal cortex by the central sulcus covering the sensorimotor area. The results suggest that this species has less collateralization, which closely resembles humans. CONCLUSIONS: The reported nonhuman primate model produces a defined and reproducible syndrome relevant to our understanding of ischemic stroke, cortical representation, and sensorimotor integration controlling dexterous movements. This model will be useful in basic and translational research addressing loss of arm function and dexterity.

Asunto(s)

Brazo/fisiopatología , Isquemia Encefálica/fisiopatología , Cognición/fisiología , Dedos/fisiopatología , Desempeño Psicomotor/fisiología , Accidente Cerebrovascular/fisiopatología , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/fisiopatología , Destreza Motora/fisiología , Corteza Sensoriomotora/fisiopatología

RESUMEN

Accumulation of alpha-synuclein (α-syn) in Lewy bodies and neurites of midbrain dopamine neurons is diagnostic for Parkinson's disease (PD), leading to the proposal that PD is a toxic gain-of-function synucleinopathy. Here we discuss the alternative viewpoint that α-syn displacement from synapses by misfolding and aggregation results in a toxic loss-of-function. In support of this hypothesis we provide evidence from our pilot study demonstrating that knockdown of endogenous α-syn in dopamine neurons of non-human primates reproduces the pattern of nigrostriatal degeneration characteristic of PD.

RESUMEN

The possibility of enhancing endogenous brain repair following neurological disorders, such as Parkinson's disease (PD), is of considerable recent interest. One such mechanism may exist in the striatum as an upregulated population of tyrosine hydroxylase (TH)-immunoreactive neurons that appear after 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) lesions in nonhuman primates as well as in humans with PD. An intriguing possibility is that these endogenous neurons reflect a compensatory mechanism to mitigate the loss of striatal DA due to progressive destruction of the nigrostriatal pathway. The possibility of enhancing the number and function of this population is attractive; however, it is crucial to gain further information about these cells in order to comprehend more fully their possible therapeutic potential. The current research was designed to investigate the fate of this endogenous population in African green monkeys rendered parkinsonian by MPTP lesions. Specifically, we assessed changes in the numbers of striatal neurons expressing TH at differing stages of the toxin-induced behavioral disability and discovered a close relationship with symptom severity and striatal DA neuron numbers. Increased numbers of striatal TH-positive neurons were associated with MPTP treatment that produced parkinsonian symptoms compared to numbers of these neurons in MPTP-treated asymptomatic animals and untreated controls. Expression of striatal DA neurons peaked at the manifestation of symptoms in mild/moderate animals and remained stable in animals that were severely parkinsonian. Furthermore, in severely debilitated animals that improved after fetal dopaminergic grafts, we discovered a return to control levels of the endogenous population. Taken together, our results further support the concept that this population of DA neurons responds to variations in striatal DA tone and may serve as a compensatory mechanism to restore striatal DA levels in the context of significant depletion. Artificially manipulating this endogenous population could prove beneficial for PD treatment, especially for individuals in early disease stages.

Asunto(s)

Neuronas Dopaminérgicas/metabolismo , Intoxicación por MPTP/patología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Núcleo Caudado/metabolismo , Chlorocebus aethiops , Modelos Animales de Enfermedad , Intoxicación por MPTP/metabolismo , Masculino , Índice de Severidad de la Enfermedad

RESUMEN

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

Asunto(s)

Intoxicación por MPTP/terapia , Células-Madre Neurales/trasplante , Óvulo/citología , Enfermedad de Parkinson Secundaria/terapia , Animales , Encéfalo/metabolismo , Encéfalo/patología , Chlorocebus aethiops , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Dopamina/análisis , Dopamina/metabolismo , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Células-Madre Neurales/citología , Oxidopamina/toxicidad , Enfermedad de Parkinson Secundaria/inducido químicamente , Ratas , Ratas Sprague-Dawley , Distribución Tisular , Trasplante Heterólogo

RESUMEN

BACKGROUND: Cognitive deficits are a core symptom of schizophrenia, yet they remain particularly resistant to treatment. The model provided by repeatedly exposing adult nonhuman primates to phencyclidine has generated important insights into the neurobiology of these deficits, but it remains possible that administration of this psychotomimetic agent during the pre-adult period, when the dorsolateral prefrontal cortex in human and nonhuman primates is still undergoing significant maturation, may provide a greater understanding of schizophrenia-related cognitive deficits. METHODS: The effects of repeated phencyclidine treatment on spine synapse number, dopamine turnover and BDNF expression in dorsolateral prefrontal cortex, and working memory accuracy were examined in pre-adult monkeys. RESULTS: One week following phencyclidine treatment, juvenile and adolescent male monkeys demonstrated a greater loss of spine synapses in dorsolateral prefrontal cortex than adult male monkeys. Further studies indicated that in juvenile males, a cognitive deficit existed at 4 weeks following phencyclidine treatment, and this impairment was associated with decreased dopamine turnover, decreased brain derived neurotrophic factor messenger RNA, and a loss of dendritic spine synapses in dorsolateral prefrontal cortex. In contrast, female juvenile monkeys displayed no cognitive deficit at 4 weeks after phencyclidine treatment and no alteration in dopamine turnover or brain derived neurotrophic factor messenger RNA or spine synapse number in dorsolateral prefrontal cortex. In the combined group of male and female juvenile monkeys, significant linear correlations were detected between dopamine turnover, spine synapse number, and cognitive performance. CONCLUSIONS: As the incidence of schizophrenia is greater in males than females, these findings support the validity of the juvenile primate phencyclidine model and highlight its potential usefulness in understanding the deficits in dorsolateral prefrontal cortex in schizophrenia and developing novel treatments for the cognitive deficits associated with schizophrenia.

Asunto(s)

Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cognición , Dopamina/metabolismo , Fenciclidina , Corteza Prefrontal/metabolismo , Esquizofrenia/metabolismo , Psicología del Esquizofrénico , Médula Espinal/metabolismo , Sinapsis/metabolismo , Factores de Edad , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Masculino , Memoria a Corto Plazo , Corteza Prefrontal/fisiopatología , Esquizofrenia/inducido químicamente , Esquizofrenia/patología , Esquizofrenia/fisiopatología , Factores Sexuales , Médula Espinal/fisiopatología , Médula Espinal/ultraestructura , Sinapsis/ultraestructura , Factores de Tiempo

RESUMEN

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Asunto(s)

Terapia Genética/métodos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Intoxicación por MPTP/terapia , Mesencéfalo/cirugía , Células-Madre Neurales/trasplante , Neuritas/trasplante , Neurogénesis , Medicina Regenerativa/métodos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Línea Celular , Linaje de la Célula , Forma de la Célula , Supervivencia Celular , Chlorocebus aethiops , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Humanos , Intoxicación por MPTP/inducido químicamente , Intoxicación por MPTP/genética , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Mesencéfalo/metabolismo , Mesencéfalo/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neuritas/metabolismo , Neuritas/patología , Nicho de Células Madre , Factores de Tiempo , Transducción Genética , Transfección , Regulación hacia Arriba

RESUMEN

The potential for "replacement cells" to restore function in Parkinson's disease has been widely reported over the past 3 decades, rejuvenating the central nervous system rather than just relieving symptoms. Most such experiments have used fetal or embryonic sources that may induce immunological rejection and generate ethical concerns. Autologous sources, in which the cells to be implanted are derived from recipients' own cells after reprogramming to stem cells, direct genetic modifications, or epigenetic modifications in culture, could eliminate many of these problems. In a previous study on autologous brain cell transplantation, we demonstrated that adult monkey brain cells, obtained from cortical biopsies and kept in culture for 7 weeks, exhibited potential as a method of brain repair after low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused dopaminergic cell death. The present study exposed monkeys to higher MPTP doses to produce significant parkinsonism and behavioral impairments. Cerebral cortical cells were biopsied from the animals, held in culture for 7 weeks to create an autologous neural cell "ecosystem" and reimplanted bilaterally into the striatum of the same six donor monkeys. These cells expressed neuroectodermal and progenitor markers such as nestin, doublecortin, GFAP, neurofilament, and vimentin. Five to six months after reimplantation, histological analysis with the dye PKH67 and unbiased stereology showed that reimplanted cells survived, migrated bilaterally throughout the striatum, and seemed to exert a neurorestorative effect. More tyrosine hydroxylase-immunoreactive neurons and significant behavioral improvement followed reimplantation of cultured autologous neural cells as a result of unknown trophic factors released by the grafts.

Asunto(s)

Encéfalo/citología , Intoxicación por MPTP/complicaciones , Intoxicación por MPTP/cirugía , Trastornos Mentales/etiología , Neuronas/trasplante , Recuperación de la Función/fisiología , Animales , Biopsia , Recuento de Células , Chlorocebus aethiops , Modelos Animales de Enfermedad , Modelos Lineales , Masculino , Trastornos Mentales/terapia , Necrosis/etiología , Compuestos Orgánicos , Factores de Tiempo , Trasplante Autólogo/métodos , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

A human embryonic stem cell (HESC) line, H1, was studied after differentiation to a dopaminergic phenotype in vitro in order to carry out in vivo studies in Parkinsonian monkeys. To identify morphological characteristics of transplanted donor cells, HESCs were transfected with a GFP lentiviral vector. Gene expression studies were performed at each step of a neural rosette-based dopaminergic differentiation protocol by RT-PCR. In vitro immunofluorescence revealed that >90% of the differentiated cells exhibited a neuronal phenotype by ß-III-tubulin immunocytochemistry, with 17% of the cells coexpressing tyrosine hydroxylase prior to implantation. Biochemical analyses demonstrated dopamine release in culture in response to potassium chloride-induced membrane depolarization, suggesting that the cells synthesized and released dopamine. These characterized, HESC-derived neurons were then implanted into the striatum and midbrain of MPTP (1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine)-exposed monkeys that were triple immunosuppressed. Here we demonstrate robust survival of transplanted HESC-derived neurons after 6 weeks, as well as morphological features consistent with polarization, organization, and extension of processes that integrated into the host striatum. Expression of the dopaminergic marker tyrosine hydroxylase was not maintained in HESC-derived neural grafts in either the striatum or substantia nigra, despite a neuronal morphology and expression of ß-III-tubulin. These results suggest that dopamine neuronal cells derived from neuroectoderm in vitro will not maintain the correct midbrain phenotype in vivo in nonhuman primates, contrasted with recent studies showing dopamine neuronal survival using an alternative floorplate method.

Asunto(s)

1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , Neuronas Dopaminérgicas/citología , Células Madre Embrionarias/citología , Neuronas/citología , Enfermedad de Parkinson/terapia , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/fisiología , Chlorocebus aethiops , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Células Madre Embrionarias/metabolismo , Humanos , Masculino , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología

RESUMEN

We combined viral vector delivery of human glial-derived neurotrophic factor (GDNF) with the grafting of dopamine (DA) precursor cells from fetal ventral mesencephalon (VM) to determine whether these strategies would improve the anti-Parkinson's effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, an animal model for Parkinson's disease (PD). Both strategies have been reported as individually beneficial in animal models of PD, leading to clinical studies. GDNF delivery has also been reported to augment VM tissue implants, but no combined studies have been done in monkeys. Monkeys were treated with MPTP and placed into four balanced treatment groups receiving only recombinant adeno-associated virus serotype 5 (rAAV5)/hu-GDNF, only fetal DA precursor cells, both together, or a buffered saline solution (control). The combination of fetal precursors with rAAV5/hu-GDNF showed significantly higher striatal DA concentrations compared with the other treatments, but did not lead to greater functional improvement in this study. For the first time under identical conditions in primates, we show that all three treatments lead to improvement compared with control animals.

Asunto(s)

Dependovirus/genética , Dopamina/metabolismo , Trasplante de Tejido Fetal , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Intoxicación por MPTP/terapia , Mesencéfalo/trasplante , Enfermedad de Parkinson/terapia , Animales , Conducta Animal , Trasplante de Tejido Encefálico , Chlorocebus aethiops , Terapia Combinada , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Terapia Genética , Vectores Genéticos , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Virus de la Anemia Infecciosa Equina/genética , Intoxicación por MPTP/fisiopatología , Intoxicación por MPTP/psicología , Masculino , Mesencéfalo/citología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/psicología

RESUMEN

Several addictive or neurotoxic drugs are dependent on the dopamine transporter (DAT) and/or vesicular monoamine transporter (VMAT2) to exert their detrimental effects on dopamine neurons. For example, methamphetamine and MPTP are substrates for both DAT and VMAT2, with the ratio of DAT to VMAT2 in striatum being a determinant of the degree of toxicity inflicted by these drugs on dopamine neurons. Thus, the susceptibility of dopamine neurons to agents whose pharmacology involves DAT and VMAT2 may vary during development if the ontogeny of DAT and VMAT2 differs, and this is relevant as exposure of dopamine neurons to toxic agents during development is hypothesized to underlie some neurological or psychiatric disorders. However, the relative expression of DAT and VMAT2 has not been studied in either primate or nonprimate fetal brain, and this was addressed in the present study by measuring the binding of specific radioligands of DAT and VMAT2 to striatal membranes from nonhuman primates at mid-gestation, late-gestation, and the postnatal and adult periods. Dopamine concentration was also determined in striatal tissue from the same brains. These data indicate that in striatum of primates, unlike rodents, there is a sharp increase in DAT and VMAT2 expression after mid-gestation, with adult levels being attained at the time of birth. In addition, this study demonstrated that there is a coordinated expression of DAT and VMAT2 from the time of mid-gestation to adulthood.

Asunto(s)

Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Animales , Chlorocebus aethiops , Cuerpo Estriado/embriología , Cuerpo Estriado/crecimiento & desarrollo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Ensayo de Unión Radioligante , Proteínas de Transporte Vesicular de Monoaminas/genética

RESUMEN

Prevalent use of bisphenol-A (BPA) in the manufacture of resins, plastics and paper products has led to frequent exposure of most people to this endocrine disruptor. Some rodent studies have suggested that BPA can exert detrimental effects on brain development. However as rodent models cannot be relied on to predict consequences of human exposure to BPA during development, it is important to investigate the effects of BPA on non-human primate brain development. Previous research suggests that BPA preferentially targets dopamine neurons in ventral mesencephalon and glutamatergic neurons in hippocampus, so the present work examined the susceptibility of these systems to low dose BPA exposure at the fetal and juvenile stages of development in non-human primates. Exposure of pregnant rhesus monkeys to relatively low levels of BPA during the final 2 months of gestation, induced abnormalities in fetal ventral mesencephalon and hippocampus. Specifically, light microscopy revealed a decrease in tyrosine hydroxylase-expressing (dopamine) neurons in the midbrain of BPA-exposed fetuses and electron microscopy identified a reduction in spine synapses in the CA1 region of hippocampus. In contrast, administration of BPA to juvenile vervet monkeys (14-18 months of age) was without effect on these indices, or on dopamine and serotonin concentrations in striatum and prefrontal cortex, or on performance of a cognitive task that tests working memory capacity. These data indicate that BPA exerts an age-dependent detrimental impact on primate brain development, at blood levels within the range measured in humans having only environmental contact with BPA.

Asunto(s)

Compuestos de Bencidrilo/toxicidad , Región CA1 Hipocampal/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Mesencéfalo/efectos de los fármacos , Fenoles/toxicidad , Efectos Tardíos de la Exposición Prenatal , Sinapsis/efectos de los fármacos , Factores de Edad , Animales , Conducta Animal/efectos de los fármacos , Biomarcadores/metabolismo , Región CA1 Hipocampal/embriología , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/fisiología , Chlorocebus aethiops , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Edad Gestacional , Macaca mulatta , Masculino , Exposición Materna/efectos adversos , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Mesencéfalo/patología , Embarazo , Serotonina/metabolismo , Sinapsis/metabolismo , Sinapsis/patología , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Parkinson's disease is usually characterized as a movement disorder; however, cognitive abilities that are dependent on the prefrontal cortex decline at an early stage of the disease in most patients. The changes that underlie cognitive deficits in Parkinson's disease are not well understood. We hypothesize that reduced dopamine signalling in the prefrontal cortex in Parkinson's disease is a harbinger of detrimental synaptic changes in pyramidal neurons in the prefrontal cortex, whose function is necessary for normal cognition. Our previous data showed that monkeys exposed to the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but not exhibiting overt motor deficits (motor-asymptomatic), displayed cognitive deficits in prefrontal cortex-dependent tasks. The present results demonstrate that motor-asymptomatic MPTP-treated monkeys have a reduced dopamine concentration and a substantially lower number (50%) of asymmetric (excitatory) spine synapses in layer II/III, but not layer V, of the dorsolateral prefrontal cortex, compared to controls. In contrast, neither dopamine concentration nor asymmetric synapse number was altered in the entorhinal cortex of MPTP-treated monkeys. Together, these findings suggest that the number of asymmetric spine synapses on dendrites in the prefrontal cortex is dopamine-dependent and that the loss of synapses may be a morphological substrate of the cognitive deficits induced by a reduction in dopamine neurotransmission in this region.

Asunto(s)

Trastornos del Conocimiento/metabolismo , Espinas Dendríticas/metabolismo , Dopamina/metabolismo , Trastornos Parkinsonianos/metabolismo , Corteza Prefrontal/metabolismo , Sinapsis/metabolismo , Animales , Chlorocebus aethiops , Trastornos del Conocimiento/patología , Espinas Dendríticas/patología , Masculino , Trastornos Parkinsonianos/patología , Corteza Prefrontal/patología , Sinapsis/patología

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by the unremitting degeneration of motor neurons. Multiple processes involving motor neurons and other cell types have been implicated in its pathogenesis. Neural stem cells (NSCs) perform multiple actions within the nervous system to fulfill their functions of organogenesis and homeostasis. We test the hypothesis that transplanted, undifferentiated multipotent migratory NSCs may help to ameliorate an array of pathological mechanisms in the SOD1(G93A) transgenic mouse model of ALS. On the basis of a meta-analysis of 11 independent studies performed by a consortium of ALS investigators, we propose that transplanted NSCs (both mouse and human) can slow both the onset and the progression of clinical signs and prolong survival in ALS mice, particularly if regions sustaining vital functions such as respiration are rendered chimeric. The beneficial effects of transplanted NSCs seem to be mediated by a number of actions including their ability to produce trophic factors, preserve neuromuscular function, and reduce astrogliosis and inflammation. We conclude that the widespread, pleiotropic, modulatory actions exerted by transplanted NSCs may represent an accessible therapeutic application of stem cells for treating ALS and other untreatable degenerative diseases.

Asunto(s)

Esclerosis Amiotrófica Lateral/patología , Células-Madre Neurales/citología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Superóxido Dismutasa/genética , Superóxido Dismutasa-1