An epigenetic barrier sets the timing of human neuronal maturation

Gabriele Ciceri, Hyunwoo Cho, Meghana Kshirsagar, Arianna Baggiolini, Kelly A Aromolaran, Ryan M Walsh, Peter A Goldstein, Richard P. Koche, Christina S Leslie, Lorenz Studer

Posted on: 1 December 2022 , updated on: 2 December 2022

Preprint posted on 3 June 2022

Article now published in Nature at http://dx.doi.org/10.1038/s41586-023-06984-8

A new protocol from Ciceri and colleagues allows the differentiation of hPSC into a pure population of cortical neurons and the characterization of maturation stages in human neurons

Selected by PiCLS Dundee

Categories: cell biology, genetics, neuroscience

Prelight written by Elisenda Raga Gil and Ines Jmel-Boyer

Background

The development of the Central Nervous System (CNS) is a long and extensive process that continues after birth to reach maturity. Although there are some similarities between mammals, the brain maturation in humans is much longer. Moreover, the timing of the maturation process is key. It has been shown that human Pluripotent Stem Cell (hPSC)-derived neurons, transplanted into a mouse cortex, mature at a different pace than the surrounding neurons (they require nine months instead of one to reach maturity). These findings would suggest that human-derived neurons follow a species-specific and intrinsic program, where even in a different environment, the timing of human neuron maturation remains the same (10 times slower).

Main findings

The mechanisms controlling the pace of neural development and maturation are relatively unexplored. Understanding this better would be very useful to reach the full potential of hPSC technologies, allowing us to understand human neuronal maturation and treat brain disorders. Current protocols to differentiate hPSC towards neuronal cells, generate multiple cell lineages, that give rise to unsynchronised neurons maturing at different pace. This pre-print presents a new protocol to generate a homogeneous and synchronised population of neurons. This is achieved by first generating cortical Neural Precursor Cells (NPC) via dual SMAD and WNT signalling inhibition followed by the temporal synchronization of neurogenesis via inhibition of Notch signalling pathway. This process allowed the authors to trace the maturation state of the resulting neurons over time and to monitor key steps of maturation process based on several neuronal phenotypes.

The proposed protocol discussed in this pre-print (Ciceri et al.) allows hPSCs (OCT4+, NANOG+) to transition into NPC (FOXG1+, PAX6+, EMX2+, FEZF2+) by the 10th day of differentiation (d10). By day 20, they had a pure NPC population. The next step consisted of optimising the replating density of cells upon passaging and treating them with DAPT, an inhibitor of the Notch pathway. As a result, they obtained a synchronous neurogenesis and by day 25, NPCs exited the cell cycle and formed isochronic MAP2+ post-mitotic neurons of similar age.

Furthermore, they confirmed the functionality of the generated neurons by looking at various phenotypes such as the neurites’ length or arborisation complexity. They showed that the neurons they had generated acquired intrinsic electrophysiological properties.

Transcriptional data analysis was performed to assess the developmental stage at different timepoints during NPC-to-neuron maturation. Principal component analysis (PCA) of RNA-seq data showed which populations presented the bigger variation (and therefore which populations had the biggest difference between them): d25 to d50 presented the most pronounced changes, while for the rest of the transitions (d50 to d75 and d75 to d100) the changes were more subtle. This analysis was key to select the time points for further analysis (d25, d50 and d100).

Upregulated transcripts from the Gene Ontology (GO) analysis included, among others, components of the cytoskeleton, Ca²⁺ signalling/homeostasis and ATP biosynthesis. Some of these groups were further validated by immunofluorescent imaging. Furthermore, through ATAC-seq data analysis, the authors addressed chromatin accessibility changes and found that these aligned according to maturation stages by PCA analysis clustering.

Transcription factor (TF) motif enrichment showed that peaks with increased accessibility in young neurons were enriched with TF motifs for genes that are important during early stages of cortical development while late opening peaks were associated with activity-dependent TF motifs. Thus, the authors could show that the hPSC-derived cortical neurons differentiated using this new protocol, undergo a maturation process and are functionally active.

The authors then aimed at finding the mechanism(s) responsible for the protracted maturation of hPSC-derived neurons. They focused on genes that were monotonically downregulated during maturation. Two epigenetic-related pathways emerged: chromatin regulation (SWI/SNF, polycomb) and epigenetic related (histone demethylases and methyltransferases) pathways, suggesting an inverse correlation between the expression of these genes and neural maturation state. They knocked out 21 selected genes (18 chromatin regulators and 3 transcription factors), and found that loss of function of several of those genes induced faster neuronal maturation. Overall, Ciceri et al., have shown the existence of an epigenetic “brake” that is progressively released to ensure the slow maturation of human neurons.

By transiently treating NPCs from d12 to d20 with small molecule inhibitors against the PRC2 regulator EZH2, the histone methyltransferases EHMT1/2 and DOT1L, the authors showed that neuronal maturation was sped up, proving that EZH2, EHMT1/2 and DOT1L are key components of the epigenetic barrier at NPC stage. They further proposed that EZH2 is responsible for maintaining maturation related genes in a poised state via deposition of the H3K27me3 repressive histone mark.

Overall, the epigenetic barrier identified in this preprint has a dual mechanism: a direct one that maintains maturation genes in a poised state and an indirect one that modulates epigenetic regulators involved in the maturation process.

Future work

The authors have described a new protocol to differentiate hPSC into a pure population of cortical neurons. They focused on one type of neuron, but this method could be adapted to study other types as well and compare the epigenetic maturation signatures in each one of them, allowing us to better understand brain development and function.

In addition to having a pure population, the protocol allows researchers to have a higher number of same-age neurons, permitting the use of quantitative methods at sequential timepoints. This could unravel the gradual changes in transcription and in the epigenetic state over time. In the future, one could focus on assays requiring large numbers such as proteomics studies or 3D chromatin captures.

Using this protocol, one could also culture specific types of cells together in vitro and study their interactions and the effect this has on their development.

This new protocol will allow the study of brain injuries or developmental disorders in a more precise manner, using hPSC in order to elucidate the mechanisms underlaying these processes, but it could also have other important purposes, such as the study of the effect of drugs on development.

doi: https://doi.org/10.1242/prelights.33225

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Questions for & Answers from the authors

Gabriele Ciceri shared

Have you checked whether the fact that only one type of cell is present in this in vitro culture, has an impact on the signature and/or function of the neurons formed? Removing glia or interneurons from the in vitro environment could for instance have consequences on the function of those neurons.

In this manuscript, we studied specifically the mechanisms underlying the intrinsic timing of neuronal maturation. We are currently beginning to investigate the impact of extrinsic factors on the timing of neuronal maturation. Extrinsic factors, which include interactions among distinct neuronal and glial cell types, have been described to modulate certain neuronal properties such as pattern of neuronal functionality. Thus, we expect them to have an impact on at least some of the specific phenotypes that characterize the maturation process. As we have profiled in detail the multiple phenotypic dimensions underlying the maturation process in hPSC-derived cortical neurons, it will be interesting to test which of those aspects are specifically modulated by cell-cell interactions, for example in co-culture experiments. This set of studies could also reveal how intrinsic and extrinsic factors functionally interplay, whether they converge mechanistically, have additive or synergistic effect and/or unlock new states of neuronal maturity.

What would be the advantage of having a slow release of the “epigenetic barrier” compared to an off/on switch? What evolutionary advantage would a prolonged development confer?

In our model the slow release of the epigenetic barrier ensures that the process of neuronal maturation proceeds gradually over an extended period of time, which is one remarkable feature of human cortical development. How “slow” or “fast” cell maturation impact the emerging properties and the overall complexity of the brain is still largely unexplored experimentally. The protracted maturation characteristic of the human brain has been proposed to be linked to extended periods of neural plasticity and suggested to underly human-specific properties, such as high-order cognition, behaviours, and extended learning. Protracted timing of development in humans compared to most other species is also observed at earlier stages, such us during the specification of cell fates in the cerebral cortex and has been shown to contribute to the expansion in brain size and complexity. While protracted timing of development may have contributed to the emergence of unique human features, alterations in the timing and/or aberrant trajectories of cell maturations in extreme cases may be associated with intellectual disorders, such as autism spectrum disorders.

The small molecules and inhibitors used in this protocol are specific to neural development, but could the global strategy be applied to other types of progenitors (lung cells, for example)?

The small molecules used for the specification of the neural fate from pluripotency target signalling pathways critical for the establishment of neural identity. These pathways are also involved in the development of multiple other tissues depending on the stage. In general, these and other small molecules targeting several developmental pathways are used at specific doses and windows of treatment to guide hPSC toward specific cell fates in vitro. These methods are generally called “directed differentiations”. There are many available technologies that used similar principles (but different experimental manipulations and small molecules) to generate different types of neural and non-neural progenitor cells.

The small molecules targeting epigenetic factors to trigger precocious neuronal maturation are also general, as these pathways are involved in several cellular processes and cell types. It is possible that what we have learned about maturation from hPSC-derived cortical neurons may apply to the maturation of other human cell types as well, though we will need to test this hypothesis experimentally.

What do you think is the impact of the replating step in this protocol and how does this change from what was previously done?

The replating step of NPC at low-density contributes to promote cell cycle exit and the generation of postmitotic neurons. We think that it acts in concert with Notch inhibition, mimicking the suppression of this pathway, which normally requires cell-cell contact. In this way we can promote the generation of neurons synchronized at the time of cell cycle exit using this relatively simple but effective and scalable solution, which is one of the main advantages of this system. This aspect was critical for us to specifically track maturation related changes within a synchronized population of neurons. For example, without synchronization of neurogenesis, a subset of NPCs would persist and continue to produce new neurons over extended time periods whereby those newly born (immature neurons) intermingle with more mature, earlier born neurons. Furthermore, early versus late born neurons can have different neuronal subtype identity, each with a slightly different pace of maturation timing, which would further complicate the analysis of maturation in such non-synchronized cultures.

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An epigenetic barrier sets the timing of human neuronal maturation

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