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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, cortical neurons derived from mouse ESCs acquired in vivo levels of mCH over a similar time period in both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition was coincident with a transient increase in Dnmt3a, preceded by the postmitotic marker Rbfox3 (NeuN), was enriched at the nuclear lamina, and negatively correlated with gene expression. We further found that methylation patterning subtly differed between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional noncell autonomous processes. Our findings show that mouse ESC-derived neurons, in contrast to those of humans, can recapitulate the unique DNA methylation landscape of adult neurons in vitro over experimentally tractable timeframes, which allows their use as a model system to study epigenome maturation over development.

SUBMITTER: Martin S

PROVIDER: S-EPMC10218269 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

Martin Sally S Poppe Daniel D Olova Nelly N O'Leary Conor C Ivanova Elena E Pflueger Jahnvi J Dechka Jennifer J Simmons Rebecca K RK Cooper Helen M HM Reik Wolf W Lister Ryan R Wolvetang Ernst J EJ

Genes 20230422 5

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 or ...[more]

PMID: 37239317

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Project description:IntroductionProlactin (PRL) is a pleiotropic hormone implicated in various physiological processes; however, its contribution to neurodevelopment, particularly early corticogenesis, remains insufficiently characterized. In this study, we investigate PRL's regulatory influence on the initial stages of cortical development, with an emphasis on its effects on neuronal and astrocytic differentiation.MethodsWe employed a standardized in vitro differentiation protocol to generate cortical neurons from mouse embryonic stem cells (mESCs). Prolactin receptor (PRLr) expression was evaluated in pluripotent stem cells, neural stem cells (NSCs), immature neurons, and mature neurons using both PCR and immunofluorescence. These analyses revealed dynamic changes in PRLr expression throughout the differentiation process. Additionally, cells were treated with varying concentrations of PRL during early and late differentiation phases, enabling assessment of its impact on neuronal phenotypic distribution and morphological complexity.ResultsEarly PRL administration significantly enhanced the population of β-tubulin III + immature neurons, promoting neuronal survival without altering NSC proliferation. Furthermore, PRL treatment increased the abundance of Tbr1 + and NeuN + neurons, augmented dendritic complexity, and accelerated neuronal maturation. In contrast, PRL exposure at later stages of neural differentiation did not yield comparable effects. Notably, PRL delayed the maturation of protoplasmic astrocytes, although the total astrocyte population was not affected.DiscussionThese findings highlight PRL's pivotal role as a regulator of early corticogenesis by modulating neuronal survival, dendritic development, and astrocyte maturation. PRL thus emerges as a potential key factor in neurodevelopment, underscoring its importance in the hormonal regulation of neural differentiation and maturation. These insights may have broader implications for understanding the molecular and cellular mechanisms underlying normal and pathological neurodevelopment.

Project description:Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Dataset Information

Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

Publications

Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

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