Summary Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 ( NGN2 ) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2 -forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2 -iN heterogeneity can confound results that are sensitive to neuron type.

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 (scRNAseq) 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 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.

We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Human neurons engineered from induced pluripotent stem cells (iPSCs) through Neurogenin 2 (Ngn2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here we used single-cell transcriptomics to dissect the cell states that emerge during Ngn2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to Ngn2 expression level and the duration of Ngn2 forced expression. Our data reveals that Ngn2 dosage can regulate neuron fate acquisition, and that Ngn2-iN heterogeneity can confound results that are sensitive to neuron type.

The cellular, molecular and functional comparison of neurons from closely related species is crucial in evolutionary neurobiology. The access to living tissue and post-mortem brains of humans and non-human primates is limited and the state of the tissue might not allow recapitulating important species-specific differences. A valid alternative is offered by neurons derived from induced pluripotent stem cells (iPSCs) obtained from humans and non-human apes and primates. We will review herein the contribution of iPSCs-derived neuronal models to the field of evolutionary neurobiology, focusing on species-specific aspects of neuron’s cell biology and timing of maturation. In addition, we will discuss the use of iPSCs for the study of ancient human traits.