RESUMEN
Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.
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Genes Homeobox , Neuronas , Humanos , Cromatina , Neurotransmisores , ProsencéfaloRESUMEN
We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.
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Reprogramación Celular , Cromatina , Cromatina/genética , Reprogramación Celular/genética , Fibroblastos , Epigénesis GenéticaRESUMEN
The recent development of spatial omics methods has enabled single-cell profiling of the transcriptome and 3D genome organization with high spatial resolution. Expanding the repertoire of spatial omics tools, a spatially resolved single-cell epigenomics method will accelerate understanding of the spatial regulation of cell and tissue functions. Here, we report a method for spatially resolved epigenomic profiling of single cells using in situ tagmentation and transcription followed by multiplexed imaging. We demonstrated the ability to profile histone modifications marking active promoters, putative enhancers, and silent promoters in individual cells, and generated high-resolution spatial atlas of hundreds of active promoters and putative enhancers in embryonic and adult mouse brains. Our results suggested putative promoter-enhancer pairs and enhancer hubs regulating developmentally important genes. We envision this approach will be generally applicable to spatial profiling of epigenetic modifications and DNA-binding proteins, advancing our understanding of how gene expression is spatiotemporally regulated by the epigenome.
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Epigenómica , Código de Histonas , Ratones , Animales , Regiones Promotoras Genéticas , Epigénesis Genética , Transcriptoma , Elementos de Facilitación Genéticos , CromatinaRESUMEN
Oligodendrocytes, the myelinating cells of the central nervous system, possess great potential for disease modeling and cell transplantation-based therapies for leukodystrophies. However, caveats to oligodendrocyte differentiation protocols ( Ehrlich et al., 2017; Wang et al., 2013; Douvaras and Fossati, 2015) from human embryonic stem and induced pluripotent stem cells (iPSCs), which include slow and inefficient differentiation, and tumorigenic potential of contaminating undifferentiated pluripotent cells, are major bottlenecks towards their translational utility. Here, we report the rapid generation of human oligodendrocytes by direct lineage conversion of human dermal fibroblasts (HDFs). We show that the combination of the four transcription factors OLIG2, SOX10, ASCL1 and NKX2.2 is sufficient to convert HDFs to induced oligodendrocyte precursor cells (iOPCs). iOPCs resemble human primary and iPSC-derived OPCs based on morphology and transcriptomic analysis. Importantly, iOPCs can differentiate into mature myelinating oligodendrocytes in vitro and in vivo. Finally, iOPCs derived from patients with Pelizaeus Merzbacher disease, a hypomyelinating leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene, showed increased cell death compared with iOPCs from healthy donors. Thus, human iOPCs generated by direct lineage conversion represent an attractive new source for human cell-based disease models and potentially myelinating cell grafts.
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Células Madre Pluripotentes Inducidas , Enfermedad de Pelizaeus-Merzbacher , Diferenciación Celular/fisiología , Fibroblastos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Oligodendroglía/metabolismo , Enfermedad de Pelizaeus-Merzbacher/genética , Enfermedad de Pelizaeus-Merzbacher/metabolismo , Enfermedad de Pelizaeus-Merzbacher/terapiaRESUMEN
Long noncoding RNAs (lncRNAs) are a diverse and pervasive class of genes. Recent studies in the mammalian brain have uncovered several novel mechanisms. LncRNA loci are often located in proximity to developmental transcriptional factors. The lncRNA product may act like a transcription factor to control distantly located genes, or in other instances, the lncRNA loci contain DNA regulatory elements that act locally on neighboring genes. Circular RNAs are covalently closed single-stranded RNAs that can control neuronal function by acting as microRNA sponges and additional mechanisms. LncRNAs can also engage in target-directed microRNA degradation to shape the pool of microRNAs and translation. Thus, diverse mechanisms allow lncRNAs to act in the nucleus and cytoplasm to control neuronal fate and function.
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Encefalopatías/patología , Encéfalo/patología , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , Animales , Encéfalo/metabolismo , Encefalopatías/genética , HumanosRESUMEN
The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteína MioD/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Linaje de la Célula/genética , Reprogramación Celular , Cromatina/química , Cromatina/metabolismo , Embrión de Mamíferos , Fibroblastos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína MioD/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Motivos de Nucleótidos , Unión Proteica , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.
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Envejecimiento/metabolismo , Reprogramación Celular , Senescencia Celular/fisiología , Fibroblastos/metabolismo , Cicatrización de Heridas , Animales , Línea Celular , Reprogramación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Mediadores de Inflamación/metabolismo , Judíos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Procesos Estocásticos , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.
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Proteínas de Unión a Calmodulina/metabolismo , Sinapsis Eléctricas/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuronas/fisiología , Células Madre Pluripotentes/fisiología , Teratoma/metabolismo , Animales , Proteínas de Unión a Calmodulina/genética , Carcinogénesis , Células Cultivadas , Reprogramación Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Neurogénesis , Transducción de Señal , Teratoma/inducido químicamente , Teratoma/patología , Ácido Valproico/toxicidadRESUMEN
Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.
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Trastorno del Espectro Autista/genética , Diferenciación Celular/genética , Mutación , Trastornos del Neurodesarrollo/genética , Neuronas/metabolismo , ARN Largo no Codificante/genética , Trastorno del Espectro Autista/patología , Niño , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 5/genética , Variaciones en el Número de Copia de ADN , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Trastornos del Neurodesarrollo/patología , Neurogénesis/genética , Neuronas/citología , Linaje , Translocación Genética/genéticaRESUMEN
Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.
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Diferenciación Celular/fisiología , Transdiferenciación Celular/fisiología , Leucocitos Mononucleares/citología , Neuronas/citología , Linfocitos T/citología , Adolescente , Adulto , Anciano , Reprogramación Celular/fisiología , Femenino , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.
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Cromatina/metabolismo , Fibroblastos/metabolismo , Neuronas/metabolismo , Reprogramación Celular , HumanosRESUMEN
Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/fisiología , Proteínas de Homeodominio/genética , Células Madre Pluripotentes/fisiología , Factores de Transcripción/genética , Animales , Ingeniería Celular , Células Cultivadas , Humanos , Ratones , Células Madre Pluripotentes/citologíaRESUMEN
Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.
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Carcinogénesis/patología , Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Carcinogénesis/metabolismo , Ciclo Celular , Cromatina/metabolismo , Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Unión Proteica , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/deficiencia , Factores de Transcripción SOXB1/genéticaRESUMEN
Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Reprogramación Celular , Células-Madre Neurales/citología , Potenciales de Acción , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Células Madre Embrionarias/citología , Fibroblastos/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Canales de Sodio/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cellular differentiation processes during normal embryonic development are guided by extracellular soluble factors such as morphogen gradients and cell contact signals, eventually resulting in induction of specific combinations of lineage-determining transcription factors. The young field of epigenetic reprogramming takes advantage of this knowledge and uses cell fate determination factors to convert one lineage into another such as the conversion of fibroblasts into pluripotent stem cells or neurons. These induced cell fate conversions open up new avenues for studying disease processes, generating cell material for therapeutic intervention such as drug screening and potentially also for cell-based therapies. However, there are still limitations that have to be overcome to fulfill these promises, centering on reprogramming efficiencies, cell identity, and maturation. In this review, we discuss the discovery of induced neuronal reprogramming, ways to improve the conversion process, and finally how to define properly the identity of those converted neuronal cells.
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Reprogramación Celular/fisiología , Neuronas/fisiología , Factores de Transcripción/fisiología , Animales , Linaje de la Célula , HumanosRESUMEN
We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O2 conditions compared to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3ß and Smad-mediated pathways. Our results showed that lowering the O2 tension at the initiation of reprogramming is a simple and efficient strategy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.