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Embryonic stem cell-derived neurons as a model system for epigenome maturation during development

ABSTRACT: DNA methylation in neurons is directly linked to neuronal genome regulation and maturation. Unlike other tissues, vertebrate neurons accumulate high levels of atypical DNA methylation in the CH sequence context (mCH) during early postnatal brain development. Here we investigate to what extent neurons derived in vitro from both mouse and human pluripotent stem cells recapitulate in vivo DNA methylation patterns. While human ESC-derived neurons did not accumulate mCH in either 2D culture or 3D organoid models, even after prolonged culture, neurons derived from mouse ESCs in vitro acquire in vivo levels of mCH over a similar time period as both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, is preceded by expression of the post-mitotic marker Rbfox3 (NeuN), is enriched at the nuclear lamina, and negatively correlates with gene expression. We further show that CG- and CH-methylation patterning subtly differs between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional non-cell autonomous processes. As our findings show mouse ESC-derived neurons are capable of recapitulating the unique DNA methylation landscape of adult mammalian neurons in vitro over experimentally tractable timeframes, identification of such external cues, and elucidation of the writing-reading mechanisms and functional consequences of these different types and patterns of DNA methylation events, is now achievable.

ORGANISM(S): Mus musculus

PROVIDER: GSE137098 | GEO | 2023/07/25

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.

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Embryonic stem cell-derived neurons as a model system for epigenome maturation during development

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