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Peer-reviewed journal article

An epigenetic barrier sets the timing of human neuronal maturation

Gabriele CiceriiD, Arianna BaggioliniiD, Hyunwoo ChoiD, Meghana KshirsagariD, Silvia K. Benito-KwiecinskiiD, Ryan M. WalshiD, Kelly A. Aromolaran, Alberto J. Gonzalez-HernandeziD, Hermany MungubaiD, So Yeon KooiD, Nan XuiD, Kaylin J. Sevilla, Peter A. GoldsteiniD, Joshua LevitziD, Christina S. LeslieiD, Richard P. KocheiD, Lorenz StuderiD

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Abstract

The pace of human brain development is highly protracted compared to most other species. The maturation of cortical neurons is particularly slow, extending over months to years to reach adult-like functions. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cell (hPSC) during in vitro differentiation or upon transplantation into the murine brain in vivo. Those findings suggest the presence of a cell intrinsic clock that sets the pace of neuronal maturation, though the molecular nature of such a clock has remained elusive. Here, we identify an epigenetic developmental program which sets the timing of human neuronal maturation. First, we developed a human PSC-based approach to synchronize the birth of cortical neurons in vitro which allowed us to define a detailed atlas of progressive morphological, functional, and molecular maturation in human cortical neurons. Interestingly, we observed a slow, temporal unfolding of maturation programs that is limited by the retention of a specific set of epigenetic factors. Loss-of-function studies for several of those factors in cortical neurons enables precocious molecular and physiological maturation. Remarkably, transient inhibition of EZH2, EHMT1/2 or DOT1L, at the progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Therefore, our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an "epigenetic barrier" in progenitor cells. Mechanistically, this barrier acts by holding transcriptional maturation programs in a poised state that gets gradually released during neuronal differentiation to ensure the prolonged timeline characteristic of human cortical neuron maturation.

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Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (3 Jun 2022) · CC BY-NC-ND 4.0

Chromatin regulators including EZH2, EHMT1/2, and DOT1L establish an epigenetic barrier in human neural progenitors that poises maturation genes and sets the protracted pace of cortical neuron maturation.

Human cortical neurons require months to years to reach functional maturity both in vivo and during pluripotent stem cell differentiation, hindering in vitro disease modeling, and the molecular clock governing this protracted schedule has been unknown. Using a synchronized human cortical neurogenesis platform, this study demonstrates that neuronal maturation speed is pre-programmed prior to neurogenesis by an epigenetic barrier in neural progenitor cells.

Key results

  1. ShownSynchronized differentiation of human pluripotent stem cells generates homogeneous, coetaneous TBR1+ cortical neurons that progressively mature over 100 days without ongoing neurogenesis.

    Dual-SMAD and WNT inhibition followed by low-density replating and Notch inhibition (10 uM DAPT) at day 20 drove cell-cycle exit of Ki67+ progenitors into MAP2+/TBR1+ post-mitotic neurons by day 25, which across days 25, 50, 75, and 100 increased neurite length, action potential amplitude/kinetics, and mEPSCs, transitioning to synchronous network calcium firing by day 60. · A hPSC-based platform to study human neuronal…; hPSC-derived neurons follow a gradual functional…; Figure legends

  2. ShownTranscriptomic and chromatin accessibility profiling across neuronal maturation reveals progressive activation of synaptic and activity-dependent enhancers alongside monotonic downregulation of epigenetic regulators.

    RNA-seq and ATAC-seq at days 25, 50, 75, and 100 identified ~20,000 differentially accessible ATAC-seq peaks grouped into 9 clusters (with late-opening clusters 2 and 3 enriched for MEF2 and AP-1/FOSL2 motifs and synaptic ontologies) and monotonic downregulation (FDR <= 5%) of chromatin regulators from PRC1/2, NuRD, BAF, and histone methyltransferase/demethylase complexes. · hPSC-derived neurons follow a gradual functional…; An epigenetic switch drives neuronal maturation; RNAseq and analysis; Figure legends

  3. ShownCRISPR/Cas9 knockout of monotonically downregulated chromatin regulators in post-mitotic cortical neurons accelerates molecular and electrophysiological maturation.

    In an arrayed CRISPR/Cas9 screen of 21 downregulated genes (18 chromatin regulators and 3 TFs; 2 gRNAs/gene) in day 25 GPI::Cas9 neurons, knockout of roughly half of the chromatin regulators (including CBX2, RNF2, EPC1, EPC2, EZH2, MTF2, CHD3, KMT5B, SMARCA4, SMARCAD1, and BRD1) increased NEFH and STX1A protein at day 35 and elevated spontaneous calcium spike amplitude. · An epigenetic switch drives neuronal maturation; Figure legends

  4. ShownTransient small-molecule inhibition of EZH2, EHMT1/2, or DOT1L in dividing neural progenitor cells primes newborn neurons for rapid functional and transcriptional maturation.

    Treating cortical NPCs from day 12 to day 20 (before washout and neurogenesis) with inhibitors of EZH2 (GSK343), EHMT1/2 (UNC00638), or DOT1L (EPZ004777) at 2 or 4 uM did not alter PAX6, FOXG1, or Ki67/MAP2 ratios at day 20, but induced elevated NEFH and STX1A protein and synaptic/ion-transport transcripts at day 35 and significantly increased calcium spike amplitude, frequency, and network synchronicity at day 40. · An epigenetic barrier in NPCs sets the rate of…; EdU labelling and small molecule treatments; Figure legends

  5. ShownEZH2-mediated H3K27me3 maintains neuronal maturation effector genes and competing chromatin regulators in a bivalent poised state in neural progenitor cells.

    CUT&RUN profiling of H3K27ac, H3K4me3, H3K27me3, and H3K9me3 identified 8 differential peak clusters between NPCs and neurons, revealing that cluster 1 and 3 maturation loci (including FOS, FOSB, NPAS4, BDNF, NEFH, TUBA4A, and chromatin regulators JADE2 and CHD5) carry bivalent H3K27me3/H3K4me3 marks in NPCs that lose H3K27me3 during maturation and are dose-dependently derepressed by transient NPC EZH2 inhibition. · An epigenetic barrier in NPCs sets the rate of…; Figure legends

Methods, data and reproducibility

Synchronized cortical excitatory neurogenesis from WA09 (H9) and GPI::Cas9 hPSCs using dual-SMAD/WNT inhibition (days 0-9) and low-density passaging with DAPT (days 20-30), tracking maturation at days 25-100 via Imaris, patch-clamp, GCaMP6m, RNA-seq, ATAC-seq, and CUT&RUN. Tested barrier genes via arrayed lentiviral CRISPR/Cas9 knockouts at day 25 and transient epigenetic inhibitor treatment in days 12-20 NPCs.

  • Synchronized WA09 (H9) and GPI::Cas9 hPSC-derived cortical NPCs and neurons profiled by bulk RNA-seq, ATAC-seq, CUT&RUN, patch-clamp, and calcium imaging across days 25, 50, 75, and 100 · human · n = n = 3 runs (RNA-seq), n = 2 (ATAC-seq, CUT&RUN), n = 25-43 neurons/timepoint
  • BrainSpan Atlas of the Developing Human Brain reference gene expression · human · https://www.brainspan.org
  • Published single-cell RNA-seq dataset of developing mouse neocortex (Di Bella et al., 2021) · mouse · PMID: 34163074
  • ENCODE hg19 blacklisted genomic regions for CUT&RUN peak filtering · human · https://www.encodeproject.org/annotations/ENCSR636HFF
Methods & tools
FastQC, STAR, HTSeq, DESeq2, DAVID, URD, Trimmomatic, MACS2, HOMER, MEME Suite (FIMO), Bowtie2, Picard Tools
Data & accessions
https://www.brainspan.org/https://www.encodeproject.org/annotations/ENCSR636HFF
Reagents & environment
Trimmomatic version 0.36, TrimGalore version 0.4.5, cutadapt version 1.15, FastQC version 0.11.5, bowtie2 version 2.3.4.1, Picard Tools version 2.16.0, featureCounts version 1.6.1, Homer v4.11
Reproducibility gaps
All newly generated genomic datasets (RNA-seq, ATAC-seq, CUT&RUN) are reported as deposited at GEO with accession number pending; custom MATLAB scripts for calcium imaging are not linked; GPI::Cas9 hPSC line requires a Material Transfer Agreement.
Limitations (3)
  • The synchronized neuronal monoculture is largely devoid of glial cells such as astrocytes, leaving the role of neuron-glia interactions and extrinsic circuit cues in modulating maturation unexamined. · stated by the authors
  • Experiments focused on early-born lower-layer TBR1+ cortical excitatory neurons, so whether identical epigenetic barriers govern upper-layer cortical or non-cortical neuronal lineages remains to be tested. · stated by the authors
  • Perturbation screens tested 21 selected candidates and did not exhaustively evaluate all monotonically downregulated epigenetic factors or other enriched transcriptional pathways. · stated by the authors

Who should read:Read this if you differentiate human pluripotent stem cells into neurons or study developmental timing and want protocols to synchronize neurogenesis and accelerate functional maturation epigenetically.

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