RESUMO
Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.
Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Callithrix , Técnicas de Cultura de Células , Linhagem da Célula , Células Cultivadas , Técnicas de Reprogramação Celular , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Células-Tronco Neurais/metabolismo , Pele/citologiaRESUMO
Ultrasound-targeted microbubble destruction (UTMD) is a novel means of tissue-specific gene delivery. This approach systemically infuses transgenes precoupled to gas-filled lipid microbubbles that are burst within the microvasculature of target tissues via an ultrasound signal resulting in release of DNA and transfection of neighboring cells within the tissue. Previous work has shown that adenovirus containing cDNA of UCP-1, injected into the epididymal fat pads in mice, induced localized fat depletion, improving glucose tolerance, and decreasing food intake in obese diabetic mice. Our group recently demonstrated that gene therapy by UTMD achieved beta cell regeneration in streptozotocin (STZ)-treated mice and baboons. We hypothesized that gene therapy with BMP7/PRDM16/PPARGC1A in skeletal muscle (SKM) of obese Zucker diabetic fatty (fa/fa) rats using UTMD technology would produce a brown adipose tissue (BAT) phenotype with UCP-1 overexpression. This study was designed as a proof of concept (POC) project. Obese Zucker rats were administered plasmid cDNA contructs encoding a gene cocktail with BMP7/PRDM16/PPARGC1A incorporated within microbubbles and intravenously delivered into their left thigh. Controls received UTMD with plasmids driving a DsRed reporter gene. An ultrasound transducer was directed to the thigh to disrupt the microbubbles within the microcirculation. Blood samples were drawn at baseline, and after treatment to measure glucose, insulin, and free fatty acids levels. SKM was harvested for immunohistochemistry (IHC). Our IHC results showed a reliable pattern of effective UTMD-based gene delivery in enhancing SKM overexpression of the UCP-1 gene. This clearly indicates that our plasmid DNA construct encoding the gene combination of PRDM16, PPARGC1A, and BMP7 reprogrammed adult SKM tissue into brown adipose cells in vivo. Our pilot established POC showing that the administration of the gene cocktail to SKM in this rat model of genetic obesity using UTMD gene therapy, engineered a BAT phenotype with UCP-1 over-expression. © 2017 IUBMB Life, 69(9):745-755, 2017.
Assuntos
Reprogramação Celular/genética , Diabetes Mellitus Experimental/terapia , Técnicas de Transferência de Genes , Terapia Genética , Obesidade/terapia , Tecido Adiposo Marrom/metabolismo , Animais , Proteína Morfogenética Óssea 7/genética , Diferenciação Celular/genética , Diabetes Mellitus Experimental/genética , Modelos Animais de Doenças , Humanos , Microbolhas/uso terapêutico , Músculo Esquelético/metabolismo , Músculo Esquelético/transplante , Obesidade/genética , Obesidade/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Plasmídeos/genética , Plasmídeos/uso terapêutico , Ratos , Ratos Zucker , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Braço/fisiopatologia , Isquemia Encefálica/fisiopatologia , Cognição/fisiologia , Dedos/fisiopatologia , Desempenho Psicomotor/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/fisiopatologia , Destreza Motora/fisiologia , Córtex Sensório-Motor/fisiopatologiaRESUMO
Parkinson's disease (PD) is a relentlessly progressive and currently incurable neurodegenerative disease with significant unmet medical needs. Since PD stems from the degeneration of midbrain dopaminergic (DA) neurons in a defined brain location, PD patients are considered optimal candidates for cell replacement therapy. Clinical trials for cell transplantation in PD are beginning to re-emerge worldwide with a new focus on induced pluripotent stem cells (iPSCs) as a source of DA neurons since they can be derived from adult somatic cells and produced in large quantities under current good manufacturing practices. However, for this therapeutic strategy to be realized as a viable clinical option, fundamental translational challenges need to be addressed including the manufacturing process, purity and efficacy of the cells, the method of delivery, the extent of host reinnervation and the impact of patient-centered adjunctive interventions. In this study we report on the impact of physical and cognitive training (PCT) on functional recovery in the nonhuman primate (NHP) model of PD after cell transplantation. We observed that at 6 months post-transplant, the PCT group returned to normal baseline in their daily activity measured by actigraphy, significantly improved in their sensorimotor and cognitive tasks, and showed enhanced synapse formation between grafted cells and host cells. We also describe a robust, simple, efficient, scalable, and cost-effective manufacturing process of engraftable DA neurons derived from iPSCs. This study suggests that integrating PCT with cell transplantation therapy could promote optimal graft functional integration and better outcome for patients with PD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Doença de Parkinson , Adulto , Animais , Humanos , Neurônios Dopaminérgicos/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Callithrix , Treino Cognitivo , Doença de Parkinson/cirurgia , Transplante de Células-Tronco/métodos , Diferenciação Celular/fisiologiaRESUMO
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.
Assuntos
Células-Tronco Neurais , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Doenças Neurodegenerativas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Células-Tronco Neurais/metabolismoRESUMO
Neural stem cell therapy has been galvanized by the discovery of pluripotent stem cells. The possibility to generate specialized central nervous system-specific differentiated cells using human somatic cells engineered to become induced pluripotent stem cells (iPSCs) was a game changer. This technology has broad applications in the field of regenerative medicine, in vitro disease modeling, targeted drug discovery, and precision medicine. Currently, iPSCs are one of the most promising cell sources amenable for commercialization and off-the-shelf neural stem cell therapy products. iPSCs exhibit a strong self-renewable ability that supports the development of a virtually unlimited source of neural cells for structural repair in neurological disorders. However, along with this strong proliferative capacity of iPSCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous population of human neural stem cells (hNSCs) are of paramount importance to ensure consistency in the composition of the cellular product and to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from iPSCs through the use of specific growth medium and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we will describe methods to analyze the ischemic stroke and size of grafts using magnetic resonance imaging and OsiriX software and neuroanatomical tracing procedures to study axonal remodeling after ischemic stroke and cell transplantation.
Assuntos
Isquemia Encefálica , Autorrenovação Celular , Células-Tronco Pluripotentes Induzidas , AVC Isquêmico , Células-Tronco Neurais , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Humanos , AVC Isquêmico/terapia , Modelos TeóricosRESUMO
Parkinson's disease (PD) is primarily idiopathic and a highly heterogenous neurodegenerative disease with patients experiencing a wide array of motor and non-motor symptoms. A major challenge for understanding susceptibility to PD is to determine the genetic and environmental factors that influence the mechanisms underlying the variations in disease-associated traits. The pathological hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the brain and post-mortem Lewy pathology, which leads to the loss of projecting axons innervating the striatum and to impaired motor and cognitive functions. While the cause of PD is still largely unknown, genome-wide association studies provide evidence that numerous polymorphic variants in various genes contribute to sporadic PD, and 10 to 15% of all cases are linked to some form of hereditary mutations, either autosomal dominant or recessive. Among the most common mutations observed in PD patients are in the genes LRRK2, SNCA, GBA1, PINK1, PRKN, and PARK7/DJ-1. In this review, we cover these PD-related mutations, the use of induced pluripotent stem cells as a disease in a dish model, and genetic animal models to better understand the diversity in the pathogenesis and long-term outcomes seen in PD patients.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Doença de Parkinson , Animais , Humanos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Estudo de Associação Genômica Ampla , Neurônios DopaminérgicosRESUMO
Parkinson's disease (PD) is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. The incidence of PD increases with aging and the expected burden will continue to escalate with our aging population. Since its discovery in the 1961 levodopa has remained the gold standard pharmacotherapy for PD. However, the progressive nature of the neurodegenerative process in and beyond the nigrostriatal system causes a multitude of side effects, including levodopa-induced dyskinesia within 5 years of therapy. Attenuating dyskinesia has been a significant challenge in the clinical management of PD. We report on a small molecule that eliminates the expression of levodopa-induced dyskinesia and significantly improves PD-like symptoms. The lead compound PD13R we discovered is a dopamine D3 receptor partial agonist with high affinity and selectivity, orally active and with desirable drug-like properties. Future studies are aimed at developing this lead compound for treating PD patients with dyskinesia.
Assuntos
Antiparkinsonianos/toxicidade , Dopaminérgicos/toxicidade , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Transtornos Parkinsonianos/metabolismo , Receptores de Dopamina D3/metabolismo , Animais , Callithrix , Agonistas de Dopamina/farmacologia , Agonistas de Dopamina/uso terapêutico , Discinesia Induzida por Medicamentos/prevenção & controle , Células HEK293 , Humanos , Ligantes , Transtornos Parkinsonianos/prevenção & controle , Primatas , Estrutura Secundária de Proteína , Quimpirol/farmacologia , Quimpirol/uso terapêutico , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/químicaRESUMO
BACKGROUND AND PURPOSE: Hypoxic-ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair. METHODS: The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative polymerase chain reaction. RESULTS: Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support. CONCLUSIONS: These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.
Assuntos
Axônios/fisiologia , Células-Tronco Embrionárias/transplante , Hipóxia-Isquemia Encefálica/cirurgia , Microglia/fisiologia , Neurônios/transplante , Transplante de Células-Tronco , Animais , Animais Recém-Nascidos , Humanos , Hipóxia-Isquemia Encefálica/patologia , Plasticidade Neuronal/fisiologia , Ratos , Ratos Sprague-Dawley , Transplante de Células-Tronco/métodos , TransplantesRESUMO
Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , Células-Tronco Embrionárias/citologia , Imageamento por Ressonância Magnética/métodos , Transplante de Células-Tronco/métodos , Animais , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Masculino , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
The aging of brain cells and synaptic loss are the major underlying pathophysiological processes contributing to the progressive decline in cognitive functions and Alzheimer's disease. The difference in cognitive performances observed between adult and aged subjects across species highlights the decline of brain systems with age. The inflection point in age-related cognitive decline is important for our understanding of the pathophysiology of neurodegenerative diseases and for timing therapeutic interventions. Humans and nonhuman primates share many similarities including age-dependent changes in gene expression and decline in neural and immune functions. Given these evolutionary conserved organ systems, complex human-like behavioral and age-dependent changes may be modeled and monitored longitudinally in nonhuman primates. We integrated three clinically relevant outcome measures to investigate the effect of age on cognition, motor function and diurnal activity in aged baboons. We provide evidence of a naturally-occurring age-dependent precipitous decline in movement planning, in learning novel tasks, in simple discrimination and in motivation. These results suggest that baboons aged ~20 years (equivalent to ~60 year old humans) may offer a relevant model for the prodromal phase of Alzheimer's disease and related dementias to investigate mechanisms involved in the precipitous decline in cognitive functions and to develop early therapeutic interventions.
Assuntos
Envelhecimento/fisiologia , Doença de Alzheimer/fisiopatologia , Disfunção Cognitiva/fisiopatologia , Actigrafia , Adulto , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico , Animais , Técnicas de Observação do Comportamento , Comportamento Animal/fisiologia , Encéfalo , Ritmo Circadiano/fisiologia , Cognição/fisiologia , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/etiologia , Modelos Animais de Doenças , Feminino , Humanos , Aprendizagem/fisiologia , Masculino , Pessoa de Meia-Idade , Motivação/fisiologia , Movimento/fisiologia , Testes Neuropsicológicos , PapioRESUMO
Neural stem cells (NSCs) are defined by three necessary but not sufficient criteria: (1) self-renewable, (2) ability to generate a large number of progeny, and (3) ability to differentiate into the principal central nervous system (CNS) cell types, neurons, astrocytes, and oligodendrocytes. There are various approaches to derive neural lineages from pluripotent stem cells. It is well recognized that the chosen method of NSC derivation is critical to answering the basic biology question under investigation, to the success rate in drug discovery and to the efficacy of the therapeutic cells intended for repairing the CNS. There are three critical attributes of NSCs: (1) well-defined and stable cellular composition, (2) consistent process of perpetuation that avoids drift in composition, and (3) stable phenotype or therapeutic activity of the NSCs or their differentiated progeny. Over the past decades, we have been continuously developing consistent processes for generating stable, multipotent self-renewable NSCs from various sources. In this chapter, we report a method to generate NSCs from induced pluripotent stem cells.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Diferenciação Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Meios de Cultura , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacosRESUMO
Dopaminergic (DA) neurons are involved in many critical functions within the central nervous system (CNS), and dopamine neurotransmission impairment underlies a wide range of disorders from motor control deficiencies, such as Parkinson's disease (PD), to psychiatric disorders, such as alcoholism, drug addictions, bipolar disorders, schizophrenia and depression. Neural stem cell-based technology has potential to play an important role in developing efficacious biological and small molecule therapeutic products for disorders with dopamine dysregulation. Various methods of differentiating DA neurons from pluripotent stem cells have been reported. In this chapter, we describe a simple technique using dopamine-inducing factors (DIFs) to differentiate neural stem cells (NSCs), isolated from induced pluripotent stem cells (iPSCs) into DA neurons.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Animais , Biomarcadores , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismoRESUMO
Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Expressão Gênica , Genes Reporter , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Edição de Genes , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismo , RNA Guia de Cinetoplastídeos , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP beads.
Assuntos
Biblioteca Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise de Célula Única , Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma , Fluxo de TrabalhoRESUMO
This chapter describes a pipeline for basic bioinformatics analysis of single-cell sequencing data (see Chap. 10 : Single-Cell Library Preparation). Starting with raw sequencing data, we describe how to quality check samples, to create an index from a reference genome, to align the sequences to an index, and to quantify transcript abundances. The curated data sets will enable differential expression analysis, population analysis, and pathway analysis.
Assuntos
Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise de Sequência de RNA , Biologia Computacional/métodos , SoftwareRESUMO
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of the midbrain dopaminergic neurons, which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease, however about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) with high prevalence in Ashkenazi Jewish patients and in North African Berber and Arab patients. It is still not fully understood how this mutation leads to PD pathology. In this study, we derived induced pluripotent stem cells (iPSCs) from an Ashkenazi Jewish patient with G2019S LRRK2 mutation to isolate self-renewable multipotent neural stem cells (NSCs) and to model this form of PD in vitro. To investigate the cellular diversity and disease pathology in the NSCs, we used single cell RNA-seq transcriptomic profiling. The evidence suggests there are three subpopulations within the NSCs: a committed neuronal population, intermediate stage population and undifferentiated stage population. Unbiased single-cell transcriptomic analysis revealed differential expression and dysregulation of genes involved in PD pathology. The significantly affected genes were involved in mitochondrial function, DNA repair, protein degradation, oxidative stress, lysosome biogenesis, ubiquitination, endosome function, autophagy and mitochondrial quality control. The results suggest that G2019S LRRK2 mutation may affect multiple cell types in a non-cell autonomous mechanism of PD pathology and that unbiased single-cell transcriptomics holds promise for personalized medicine.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Judeus , Mutação , Células-Tronco Neurais/metabolismo , Doença de Parkinson/etnologia , Análise de Célula ÚnicaRESUMO
Rapid and reliable assessment of mitochondrial bioenergetics is a vital tool in drug discovery studies aimed at reversing or improving mitochondrial dysfunction. Induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) carry and replicate the donor disease pathology and can be an ideal cellular model for phenotypic screening of compounds. Herein we describe the use of Seahorse XFe96 analyzer to assess mitochondrial functions in iPSC-derived NSCs for drug screening.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Bioensaio , Diferenciação Celular , Células Cultivadas , Análise de Dados , Matriz Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Consumo de OxigênioRESUMO
Neural stem cells (NSCs) are multipotent and self-renewing precursor cells that give rise to all cell types of the central nervous system (CNS). They can be used for modeling CNS in vitro, for developmental studies and for cell replacement therapies. NSCs can be derived from pluripotent stem cells through differentiation using specific growth factors. Nonhuman primates (NHP) are critical preclinical models for translational research. Induced pluripotent stem cells (iPSCs) can be generated from NHP for the purposes of allogenic or autologous cell replacement studies. Here, we describe the derivation of NSCs from NHP iPSCs.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Separação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Callithrix , Técnicas de Cultura de Células , Separação Celular/métodosRESUMO
Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.