Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE266359 | GEO | 2024/09/13
REPOSITORIES: GEO
Similar Datasets
Project description:Human neurons engineered from induced pluripotent stem cells (iPSCs) through Neurogenin 2 (Ngn2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here we used single-cell transcriptomics to dissect the cell states that emerge during Ngn2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to Ngn2 expression level and the duration of Ngn2 forced expression. Our data reveals that Ngn2 dosage can regulate neuron fate acquisition, and that Ngn2-iN heterogeneity can confound results that are sensitive to neuron type.
2021-08-05 | E-MTAB-10632 | biostudies-arrayexpress
Project description:Direct conversion of iPSCs into neurons by NGN2 over-expression is a popular method for disease modeling. Here we report a standardized method to generate functional excitatory cortical neurons using clonal, targeted engineered iPSC lines and defined reagents. We demonstrate that our protocol generates consistent excitatory cortical neurons at scale. These cultures can be maintained healthily for at least 150 days and expresses the majority of the GWAS genes found in autism, Parkinson’s disease, and Alzheimer’s disease. Comprehensive temporal omics, functional, and morphological profiling demonstrated continued progression of neuronal maturation. Deep functional characterization through transcriptomic, imaging, and functional assays revealed the coordinated action of multiple pathways in neuronal maturation. Our method and profiling data can serve as reference for developing human in vitro neuronal models for disease modeling.
2024-09-15 | GSE244368 | GEO
Project description:Direct conversion of iPSCs into neurons by NGN2 over-expression is a popular method for disease modeling. Here we report a standardized method to generate functional excitatory cortical neurons using clonal, targeted engineered iPSC lines and defined reagents. We demonstrate that our protocol generates consistent excitatory cortical neurons at scale. These cultures can be maintained healthily for at least 150 days and expresses the majority of the GWAS genes found in autism, Parkinson’s disease, and Alzheimer’s disease. Comprehensive temporal omics, functional, and morphological profiling demonstrated continued progression of neuronal maturation. Deep functional characterization through transcriptomic, imaging, and functional assays revealed the coordinated action of multiple pathways in neuronal maturation. Our method and profiling data can serve as reference for developing human in vitro neuronal models for disease modeling.
2024-09-15 | GSE244370 | GEO
Project description:Direct conversion of iPSCs into neurons by NGN2 over-expression is a popular method for disease modeling. Here we report a standardized method to generate functional excitatory cortical neurons using clonal, targeted engineered iPSC lines and defined reagents. We demonstrate that our protocol generates consistent excitatory cortical neurons at scale. These cultures can be maintained healthily for at least 150 days and expresses the majority of the GWAS genes found in autism, Parkinson’s disease, and Alzheimer’s disease. Comprehensive temporal omics, functional, and morphological profiling demonstrated continued progression of neuronal maturation. Deep functional characterization through transcriptomic, imaging, and functional assays revealed the coordinated action of multiple pathways in neuronal maturation. Our method and profiling data can serve as reference for developing human in vitro neuronal models for disease modeling.
2024-09-15 | GSE244369 | GEO
Project description:Ngn2 induces diverse neuronal lineages from human pluripotency
| PRJEB46177 | ENA
Project description:Two closely related proneural bHLH transcription factors, Ascl1 and Ngn2, both can induce mouse embryonic stem cells to differentiate into induced neurons. Although Ascl1 and Ngn2 induced transcriptional programs partially overlap, it is not known whether the differences translate into the distinct mechanisms. We found that both transcription factors induce mutually exclusive side populations by binding and inducing different lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we revealed differential genetic dependencies between Ascl1 and Ngn2. Specifically, Ascl1 is more dependent on factors including Tcf7l1, that regulate pluripotency and cell cycle. In the absence of the pluripotency network repressor Tcf7l1 Ascl1 is still able to repress the core pluripotency genes, however failed to exit cell cycle. Overexpression of Cdkn1c induced cell cycle exit and restored the generation of neurons. This study illuminates two different mechanistic approaches to convert ESC to induced neurons: rapid shutdown of the initial state while installing a terminal cell identity versus repurposing the initial gene regulatory network to allow generation of neurons via intermediate states. Understanding the precise mechanism and the variety of cell states induced is essential to design efficient cell replacement therapeutic applications or precise tools for disease modelling.
2023-07-17 | GSE208199 | GEO
Project description:Two closely related proneural bHLH transcription factors, Ascl1 and Ngn2, both can induce mouse embryonic stem cells to differentiate into induced neurons. Although Ascl1 and Ngn2 induced transcriptional programs partially overlap, it is not known whether the differences translate into the distinct mechanisms. We found that both transcription factors induce mutually exclusive side populations by binding and inducing different lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we revealed differential genetic dependencies between Ascl1 and Ngn2. Specifically, Ascl1 is more dependent on factors including Tcf7l1, that regulate pluripotency and cell cycle. In the absence of the pluripotency network repressor Tcf7l1 Ascl1 is still able to repress the core pluripotency genes, however failed to exit cell cycle. Overexpression of Cdkn1c induced cell cycle exit and restored the generation of neurons. This study illuminates two different mechanistic approaches to convert ESC to induced neurons: rapid shutdown of the initial state while installing a terminal cell identity versus repurposing the initial gene regulatory network to allow generation of neurons via intermediate states. Understanding the precise mechanism and the variety of cell states induced is essential to design efficient cell replacement therapeutic applications or precise tools for disease modelling.
2023-07-17 | GSE206871 | GEO
Project description:Two closely related proneural bHLH transcription factors, Ascl1 and Ngn2, both can induce mouse embryonic stem cells to differentiate into induced neurons. Although Ascl1 and Ngn2 induced transcriptional programs partially overlap, it is not known whether the differences translate into the distinct mechanisms. We found that both transcription factors induce mutually exclusive side populations by binding and inducing different lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we revealed differential genetic dependencies between Ascl1 and Ngn2. Specifically, Ascl1 is more dependent on factors including Tcf7l1, that regulate pluripotency and cell cycle. In the absence of the pluripotency network repressor Tcf7l1 Ascl1 is still able to repress the core pluripotency genes, however failed to exit cell cycle. Overexpression of Cdkn1c induced cell cycle exit and restored the generation of neurons. This study illuminates two different mechanistic approaches to convert ESC to induced neurons: rapid shutdown of the initial state while installing a terminal cell identity versus repurposing the initial gene regulatory network to allow generation of neurons via intermediate states. Understanding the precise mechanism and the variety of cell states induced is essential to design efficient cell replacement therapeutic applications or precise tools for disease modelling.
2023-07-17 | GSE206870 | GEO
Project description:Two closely related proneural bHLH transcription factors, Ascl1 and Ngn2, both can induce mouse embryonic stem cells to differentiate into induced neurons. Although Ascl1 and Ngn2 induced transcriptional programs partially overlap, it is not known whether the differences translate into the distinct mechanisms. We found that both transcription factors induce mutually exclusive side populations by binding and inducing different lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we revealed differential genetic dependencies between Ascl1 and Ngn2. Specifically, Ascl1 is more dependent on factors including Tcf7l1, that regulate pluripotency and cell cycle. In the absence of the pluripotency network repressor Tcf7l1 Ascl1 is still able to repress the core pluripotency genes, however failed to exit cell cycle. Overexpression of Cdkn1c induced cell cycle exit and restored the generation of neurons. This study illuminates two different mechanistic approaches to convert ESC to induced neurons: rapid shutdown of the initial state while installing a terminal cell identity versus repurposing the initial gene regulatory network to allow generation of neurons via intermediate states. Understanding the precise mechanism and the variety of cell states induced is essential to design efficient cell replacement therapeutic applications or precise tools for disease modelling.
2023-07-17 | GSE206869 | GEO
Project description:Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. Here, we combine single-cell multiomics with genome-wide profiling of 3D nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodelling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identify Yy1, a transcriptional co-factor, recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes, and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.
2024-04-30 | GSE208742 | GEO