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

Abstract

We generated induced excitatory sensory 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 than the human cells. In accordance, during the first two 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. 4Here, we use a direct conversion system in which chimpanzee, bonobo and human iPSCs are converted into neurons using an NGN2 overexpression system [23,24].Unlike what has been previously reported, we obtain a neuronal cell population containing a majority of sensory neurons. We describe their temporal differentiation in terms of gene expression, electrophysiology and dendritic arborization. 5 RESULTS Maturation of human and ape induced neurons in vitroWe generated iNeurons from three chimpanzee (SandraA, JoC, ciPS01), one bonobo (BmRNA) and three human (409B2, SC102A, HmRNA) iPSC lines and from one human ESC line (H9) using forced expression of the pan-neurogenic transcription factor neurogenin 2 (NGN 2; Figure 1A) [23,24]. We followed their differentiation using molecular, electrophysiological and morphological approaches for up to 8 weeks, as indicated in the ( Figure 1A, B). Single cell RNA sequencing (scRNAseq) was performed for one chimpanzee (SandraA) and three human (409B2, SC102A1 and H9) cell lines, electrophysiology was performed using all eight cell lines and morphological analyses were done with two chimpanzee (SandraA and JoC), the bonobo (BmRNA) and three human (409B2, SC102A1 and H9) cell lines. We will refer to ape iNs for results where we combined chimpanzee and bonobo iNs for analysis.Differentiation was characterized by a downregulation of the stem cell markers NANOG, OCT4 and SOX2 and of the neural progenitor's markers ID1 and ID3 in both chimpanzee and human cells ( Supplementary Figure 1), by a change in cellular morphology and by the extension of neurites ( Figure 1C). Chimpanzee, bonobo and human iNs showed a neuron-like morphology at day 7 (d7) of differentiation and formed a dense network by d14. Neurites were positive for TUJI (beta-III-tubulin, a neuronal marker) starting from d3 of differentiation in apes and humans ( Supplementary Figure 2).By the end of the differentiation at d35, both ape and human cells formed networks that were positive for MAP2 (microtubule associated protein-2, marker for mature

From stem and progenitor cells to neurons in the developing neocortex: key differences among hominids

“…Intriguingly, similar developmental/maturation timing differences have also been found among hominid neocortical neurons [73,74] (Figure 1). This suggests that a comprehensive characterisation of neural cell differences among hominids requires taking into account the timing of each characteristic process throughout development.…”

“…Also, once fully mature, human IPSC-derived neurons tended to be electrophysiologically more active than chimpanzee ones, although they matured at a slower pace [73]. A slower maturation rate of human neurons was also observed in a recent preprint when using a direct conversion approach, in which induced cortical and sensory neurons were obtained via forced expression of Ngn2 in iPSCs, bypassing the neural progenitor stage [74]. Induced human neurons were found to mature more slowly in terms of both morphology and function.…”

“…As to the latter, this was found to be the case especially regarding the sEPSCs (spontaneous excitatory post-synaptic currents), a measure of spontaneous synaptic activity. Interestingly, the delayed synaptic communication was paralleled by a delayed expression of genes encoding synaptic proteins [74]. This experimental pipeline allowed to uncouple the generation of neurons from NPCs (developmental process) from the neuronal maturation processes per se, minimized the effects of the local environment, and suggested that neoteny is an intrinsic feature of human excitatory neurons and is not limited to pyramidal neurons.…”

Harnessing pluripotent stem cells as models to decipher human evolution

“…It is to this question, therefore, that inter-species iPSC models have been applied most frequently (see Mostajo-Radj et al [125] for an extensive review of this field). In the absence of primary tissue, which is hard to obtain and stage, several studies have used iPSCderived brain cell types using 2D culture differentiation protocols from different developmental time points in an attempt to explore differences that can be linked to human-specific cognition [122,[126][127][128][129][130][131][132][133]. For example, Otani et al made elegant use of the staged nature of a 2D cortical development protocol [134] to examine cortical cell proliferation across humans, chimpanzees and macaques, finding differences in the amount of time cortical progenitor cells remained proliferative across the three species [129] that could potentially be linked to differences in brain cell numbers between them.…”

Learning about cell lineage, cellular diversity and evolution of the human brain through stem cell models

“…Similarly, excitatory sensory neurons differentiated from human stem cells showed delayed neuronal maturation and expression of genes involved in dendrite and synapse development when compared to counterparts derived from chimpanzee or bonobo [75]. The molecular network that control neoteny in human start to be understood.…”