RESUMEN
BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.
Asunto(s)
Citometría de Flujo , Células Madre Multipotentes/citología , Células-Madre Neurales/citología , Línea Celular , HumanosRESUMEN
The transcriptional programs that establish neuronal identity evolved to produce the rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression of certain transcription factors can also endow non-neural cells with neuronal properties. The relationship between reprogramming factors and the transcriptional networks that produce neuronal identity and diversity remains largely unknown. Here, from a screen of 598 pairs of transcription factors, we identify 76 pairs of transcription factors that induce mouse fibroblasts to differentiate into cells with neuronal features. By comparing the transcriptomes of these induced neuronal cells (iN cells) with those of endogenous neurons, we define a 'core' cell-autonomous neuronal signature. The iN cells also exhibit diversity; each transcription factor pair produces iN cells with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct transcription factor input 'codes' to defined transcriptional outputs, this study delineates cell-autonomous features of neuronal identity and diversity and expands the reprogramming toolbox to facilitate engineering of induced neurons with desired patterns of gene expression and related functional properties.
Asunto(s)
Reprogramación Celular/genética , Neuronas/citología , Neuronas/metabolismo , Animales , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/efectos de los fármacos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor â¼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0-2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development.
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Clonación Molecular , Mutación/genética , Neuronas/fisiología , Análisis de Secuencia de ADN , Factores de Edad , Animales , Animales Recién Nacidos , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Cadherinas/metabolismo , División Celular/genética , Elementos Transponibles de ADN/genética , Embrión de Mamíferos , Femenino , Humanos , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Repeticiones de Microsatélite/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Transferencia Nuclear , Bulbo Olfatorio/citología , Bulbo Olfatorio/embriología , Bulbo Olfatorio/crecimiento & desarrollo , Oocitos/fisiologíaRESUMEN
The nervous system is comprised of a vast diversity of distinct neural cell types. Differences between neuronal subtypes drive the assembly of neuronal circuits and underlie the subtype specificity of many neurological diseases. Yet, because neurons are irreversibly post-mitotic and not readily available from patients, it has not been feasible to study specific subtypes of human neurons in larger numbers. A powerful means to study neuronal diversity and neurological disease is to establish methods to produce desired neuronal subtypes in vitro. Traditionally this has been accomplished by treating pluripotent or neural stem cells with growth factors and morphogens that recapitulate exogenous developmental signals. These approaches often require extended periods of culture, which can limit their utility. However, more recently, it has become possible to produce neurons directly from fibroblasts using transcription factors and/or microRNAs. This technique referred to as direct reprogramming or transdifferentiation has proven to be a rapid, robust, and reproducible method to generate mature neurons of many different subtypes from multiple cell sources. Here, we highlight recent advances in generating neurons of specific subtypes using direct reprogramming and outline various scenarios in which induced neurons may be applied to studies of neuronal function and neurological disease.
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Ingeniería Celular/métodos , Reprogramación Celular , Fibroblastos , MicroARNs , Células-Madre Neurales , Neuronas , Factores de Transcripción , Animales , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/terapia , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.
Asunto(s)
Fibroblastos/fisiología , Sistema Nervioso Periférico/citología , Células Receptoras Sensoriales/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Femenino , Fibroblastos/ultraestructura , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Nociceptores/ultraestructura , Técnicas de Placa-Clamp , Sistema Nervioso Periférico/ultraestructura , Embarazo , Receptor trkC/genética , Células Receptoras Sensoriales/ultraestructura , Factor de Transcripción Brn-3A/genética , Factor de Transcripción Brn-3A/fisiologíaRESUMEN
Aberrant cell-cycle activity and DNA damage are emerging as important pathological components in various neurodegenerative conditions. However, their underlying mechanisms are poorly understood. Here, we show that deregulation of histone deacetylase 1 (HDAC1) activity by p25/Cdk5 induces aberrant cell-cycle activity and double-strand DNA breaks leading to neurotoxicity. In a transgenic model for neurodegeneration, p25/Cdk5 activity elicited cell-cycle activity and double-strand DNA breaks that preceded neuronal death. Inhibition of HDAC1 activity by p25/Cdk5 was identified as an underlying mechanism for these events, and HDAC1 gain of function provided potent protection against DNA damage and neurotoxicity in cultured neurons and an in vivo model for ischemia. Our findings outline a pathological signaling pathway illustrating the importance of maintaining HDAC1 activity in the adult neuron. This pathway constitutes a molecular link between aberrant cell-cycle activity and DNA damage and is a potential target for therapeutics against diseases and conditions involving neuronal death.