Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

  1. Maria Schörnig
  2. Xiangchun Ju
  3. Luise Fast
  4. Sebastian Ebert
  5. Anne Weigert
  6. Sabina Kanton
  7. Theresa Schaffer
  8. Nael Nadif Kasri
  9. Barbara Treutlein
  10. Benjamin Marco Peter
  11. Wulf Hevers
  12. Elena Taverna

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Abstract

We generated induced excitatory neurons (iNeurons, iNs) from chimpanzee, bonobo, and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single-cell RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee and bonobo than the human cells. In accordance, during the first 2 weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo, and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs, and this difference in timing likely depends on functions downstream of NGN2.

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  1. Version published to 10.7554/elife.59323 on eLife
  2. eLife

    ###Reviewer #3:

    The study by Taverna et al. uses NGN2-induction in human, chimpanzee, and bonobo pluripotent stem cells to attempt to decouple the process of neuronal maturation from the cell cycle in order to study species-specific differences in neuronal maturation. Using single cell RNA sequencing, analysis of neuronal morphology, and electrophysiological recordings, the study argues that neuronal maturation is delayed in human compared to chimpanzee and bonobo among a heterogeneous class of sensory neurons and that this delay is cell-intrinsic. However, the current data are incompletely analyzed and do not provide strong support for this conclusion.

    Major comments:

    The dramatic differences in cell type composition of the induced neurons across species, revealed by single cell sequencing in Figure 2A, pose significant problems for the interpretation of the rest of the results. Specifically, if the chimpanzee cells are biased to making different sensory neuron cell types than the human cells, then differences in maturation rates between cell types rather than between species could drive the results. The authors must take into account the influence of cell type, individual, and species in order to support their claims of species differences.

    First, the number of individuals (only one chimpanzee individual) used for single-cell analysis is inadequate. There could be individual differences in timing and neuronal composition between lines that are independent of species and are not accounted for. At least 3-5 individuals per species should be used to enable statistical analysis of species differences. Ideally, the same lines should be used for single-cell analysis and morphological/physiological analyses. Staining for the cluster markers discovered from the current single cell analysis could also be applied to the remaining individuals to understand whether induced neurons have a similar composition across all the individuals from the three species.

    If the single chimpanzee individual shown in the single cell data is really representative of the three chimpanzee lines used elsewhere in the manuscript, the dramatic differences in neuronal types across species must be taken into account in subsequent analyses. For example, gene expression in Figure 3 could be analyzed on a cluster by cluster basis rather than grouping all neuronal clusters together. As shown, the differences across species could just be due to cell-type specific differences (for example, cluster 4 appears to be made up of entirely chimpanzee neurons while cluster 5 has more equal species representation). For physiology and morphology experiments, post hoc marker staining could ensure that neurons of the same type are compared across species, or if not registered to individual cells, it could still reveal the similarities and differences in composition between plates.

    Does NGN2 induction make a valid cell type? The authors should compare their expression data to previous work utilizing NGN2 induction (Zhang et al 2013) as well as to data from mouse and human tissue samples. It would be helpful to clarify whether the differences with previous work (i.e. induction of sensory neurons compared to cortical neurons) are due to incomplete characterization previously or to a different outcome here. And most importantly, it would be helpful to more clearly identify the endogenous cell types modeled in this data, perhaps by integration with primary sensory and cortical neurons single cell datasets.

    Do the BRN2 and CUX1-positive cells show co-expression with other cortical markers, like FOXG1 and EMX2, to support the statement that some of these cells may be cortical, or are these genes also expressed in some sensory neurons, or are these simply cells of mixed identify that lack in vivo counterparts?

    Please provide more detail about the NGN2 expression system as utilized across species.

    For each species, was the corresponding NGN2 gene used? If so, are there sequence differences between species that could influence differentiation?

    Is the time course of NGN2 expression the same across species?

    What are the dynamics of NGN2 induction in this system compared to normal differentiation - does persistent NGN2 expression after differentiation ultimately keep neurons in a more immature state?

    Does the NGN2 system entirely de-couple differentiation from cell cycle as the authors claim or do a few cell cycles still occur post-induction, and does this number differ between species? The focus in the introduction on cognition and the role of cortical differences between humans and non-human primates is puzzling in light of the claim that most of the neurons generated in this study are sensory neurons. If the authors' conclusions are valid, then it seems that this finding should be framed differently. Are there known species differences in sensory neurons? Do these results suggest that delayed maturation is a more general phenomenon and not restricted to brain regions involved in cognition?

    The following sentence in the discussion attempts to address this point: "Of note, sensory neurons are interesting from an evolutionary point of view, as the development and evolution of working memory in humans is linked to a higher integration of sensory functions in the human prefrontal cortex." However, this statement and the references cited instead support the view that species differences might be found in the prefrontal cortex rather than in sensory neurons.

  3. eLife

    ###Reviewer #2:

    This is a well written MS looking at comparing the rate/tempo of maturation of Chimpanzee, Bonobo and human neurons. The work is well done and easy to follow. The core findings are that human neurons, developed in vitro via a well-established directed differentiation protocol mature slower than the NHP neurons.

    Several groups have previously used both in vivo and in vitro models (similar to the one used here) to define cross-species maturation features. These earlier studies have shown that indeed human cells develop more slowly than other species (like mice or Chimpanzees). The authors recognize this work in their introduction. While the finding of slower human neuron maturation is not completely novel, the current work furthers these earlier studies by adding additional characterization of electrophysiological and molecular properties of the neurons made. It also highlights an underappreciated presence of sensory neurons in these cultures.

    Things to consider:

    1. Definitive characterization of the neurons produced by Ngn2 overexpression. Prior work defined the neurons mostly as pyramidal, of cortical origin. Here, the authors claim both mix identity (very probable) and the presence of large numbers of sensory neurons. One is left wondering whether this is a slightly different differentiation protocol, whether the interpretation of the data is different, or whether variability is high. If the authors classify the single cell RNA data from prior studies with this same protocol, would they still conclude that these are sensory neurons? If the authors could prove that the protocol produces bona fide sensory neurons, that would be an advance for the field. That may require direct comparison to endogenous sensory neurons (beyond a small number of markers) and classification based on electrophysiological properties (which the authors do have). Are these sensory neurons based on physiology?

    2. Could one use the system to point at mechanisms that may mediate the observed differences in maturation rates? This would move the field forward in a powerful way.

  4. eLife

    ###Reviewer #1:

    The results are somewhat underdeveloped and there are several aspects of the study that can be improved by deeper analyses:

    1. The rigor of the experiments and statistical analysis is not clear. Although the use of several lines of iPSCs from each species is a strength, there are no details of how many batches of differentiation/induction were done or how many replicates were used for analysis. This is especially important for structural and functional analysis that can vary between lines and batches.

    2. The identity of the induced neurons as sensory neurons is interesting but is based solely on gene expression (scRNAseq). It would be more compelling if the authors would show other characteristics that identified this population of neurons. It is possible that some neurons express these sensory neuron genes, but do not express the proteins and/or do not differentiate into functional sensory neurons.

    3. The proportions of cells in each cluster of the scRNAseq would be informative to 1) identify changes as the neurons mature and compare between species, and 2) identify differences between species, as the authors state (page 9) that same populations were found in different proportions.

    4. Given the valuable time course scRNA seq data, the analysis of neuron maturation over time is somewhat limited. More sophisticated analysis of gene expression changes/coexpression would strengthen the overall impact of the data.

    5. Similarly, the discussion is superficial and focused on consequences but not causes of differences in neuron maturation time. The discussion does not build on the rich and extensive transcriptomic data to provide any mechanistic hypotheses of the causes of the differences.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    The manuscript by Schörnig presents an elegant comparison of structural and functional maturation of cortical neurons from different primate species that is of broad interest to researchers interested in evolutionary neuroscience and those who are interested in the unique qualities of the human cortex. The authors use an induced neuron approach to generate cortical-like neurons from iPSCs from different species and compare the structure, function and gene expression of the different neurons over time in culture. This strategy bypasses development and provides much more heterogeneous cultures for analysis. While the results are largely descriptive, they provide very interesting resource data providing insight into both primate neural development and human-specific attributes.

  6. Version published to 10.1101/2020.05.21.094912v1 on bioRxiv