RESUMEN
We used single-cell RNA sequencing (seq) on several human induced pluripotent stem (iPS) cell-derived neural stem cell (NSC) lines and one fetal brain-derived NSC line to study inherent cell type heterogeneity at proliferating neural stem cell stage and uncovered predisposed presence of neurogenic and gliogenic progenitors. We observed heterogeneity in neurogenic progenitors that differed between the iPS cell-derived NSC lines and the fetal-derived NSC line, and we also observed differences in spontaneous differentiation potential for inhibitory and excitatory neurons between the iPS cell-derived NSC lines and the fetal-derived NSC line. In addition, using a recently published glia patterning protocol we enriched for gliogenic progenitors and generated glial cells from an iPS cell-derived NSC line.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neurogénesis , Neuroglía/citología , Línea Celular , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias Humanas/clasificación , Humanos , Células Madre Pluripotentes Inducidas/clasificación , Análisis de la Célula IndividualRESUMEN
We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.
Asunto(s)
Trastorno Autístico/patología , Proteínas de Unión al Calcio/genética , Eliminación de Gen , Células Madre Pluripotentes Inducidas/patología , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/genética , Células-Madre Neurales/patología , Análisis de la Célula Individual/métodos , Potenciales de Acción , Alelos , Trastorno Autístico/genética , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genéticaRESUMEN
Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.
RESUMEN
We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Adulto , Autofagia/fisiología , Neuronas Dopaminérgicas/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Enfermedad de Parkinson/genética , alfa-Sinucleína/genéticaRESUMEN
BACKGROUND: Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages. RESULTS: Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling. CONCLUSIONS: The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.
Asunto(s)
Encéfalo/metabolismo , Metilación de ADN/genética , Síndrome de Down/genética , Epigenómica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Adulto , Encéfalo/patología , Ensamble y Desensamble de Cromatina/genética , Islas de CpG/genética , Síndrome de Down/complicaciones , Femenino , Feto/metabolismo , Feto/patología , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Trastornos del Neurodesarrollo/etiología , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Embarazo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Trisomía/genéticaRESUMEN
CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.
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Trastorno del Espectro Autista , Discapacidad Intelectual , Trastorno del Espectro Autista/genética , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Femenino , Guanilato-Quinasas/genética , Humanos , Discapacidad Intelectual/genética , Masculino , MutaciónRESUMEN
Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the aging population, but their etiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases with known genetic cause provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein SQSTM1/p62 lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem cell (NESC) model and differentiated neurons derived from reprogrammed p62 patient cells or by CRISPR/Cas9-directed gene editing in NESCs. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings imply an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation required for normal neurodifferentiation.
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Diferenciación Celular/genética , Reprogramación Celular/genética , Metabolismo Energético/genética , Proteína Sequestosoma-1/genética , Perfilación de la Expresión Génica , Glucólisis , Humanos , Mitofagia , Modelos Biológicos , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/citología , Neuronas/metabolismo , Fosforilación Oxidativa , Estrés Oxidativo , Oxígeno/metabolismo , Proteína Sequestosoma-1/metabolismoRESUMEN
The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.
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Enfermedad de Alzheimer/metabolismo , Proteína BRCA1/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Células Cultivadas , Técnicas de Reprogramación Celular , Biología Computacional , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Humanos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuronas/patología , Fosforilación , Presenilina-1/genética , Presenilina-2/genética , Presenilina-2/metabolismo , Transducción de Señal , Transcriptoma , Fosfatasas cdc25/metabolismoRESUMEN
Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.
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Técnicas de Cultivo de Célula/métodos , Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/metabolismo , Laminina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Human induced pluripotent stem (hiPS) cell lines CTRL-9-II and CTRL-10-I were derived from healthy monozygotic twin donors using non-integrating RNA based Sendai virus reprogramming and cultured in a xeno-free chemically defined condition. The established hiPS cell lines, CTRL-9-II and CTRL-10-I, are karyotypically normal, free from reprogramming vectors, display endogenously expression of pluripotency factors at levels similar to embryonic stem cells. The generated iPS cell lines demonstrate pluripotency by passing bioinformatics assay PluriTest and by embryonic body assay.
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Reprogramación Celular , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Masculino , ARN Mensajero/metabolismo , Virus Sendai/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Gemelos MonocigóticosRESUMEN
CTL07-II is a healthy feeder-free and characterized human induced pluripotent stem (iPS) cell line. Cultured under xeno-free and defined conditions. The line is generated from healthy human fibroblasts with non-integrating Sendai virus vectors encoding the four Yamanaka factors, OCT4, SOX2, KLF4 and cMYC. The generated iPS cells are free from reprogramming vectors and their purity, karyotypic stability and pluripotent capacity is confirmed.