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.
Sequencing data for single cells have been deposited in ArrayExpress under the accession code E-MTAB-9233 and under Mendeley Data with doi: 10.17632/y3s4hnyvg6. To make our scRNAseq data accessible to the neuroscience community, we provide a ShinyApp-based web browser for data exploration, called iNeuronExplorer. https://bioinf.eva.mpg.de/shiny/iNeuronExplorer/ Morphological data for neurons and a custom made script for analysis have been deposited in GitHub under the URL: https://github.com/BenjaminPeter/schornig_ineuron.
scRNAseq datasetArrayExpress, E-MTAB-9233.
Scripts for Schoernig et al. 2020BenjaminPeter / schornig_ineuron.
iNeuronExplorerMPI EVA webbrowser, shiny/iNeuronExplorer/.
scRNAseq datasetMendeley Data, doi: 10.17632/y3s4hnyvg6.
This work was supported by the Max Planck Society.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Human subjects: For this study we used three human (hiPS409-B2, SC102A1, HmRNA), three chimpanzee (SandraA, JoC, ciPS01) and one bonobo (BmRNA) iPS cell lines and one additional human ES cell line (H9). The human iPSC lines hiPS409-B2 and SC102A1 were purchased from the Riken BRC Cellbank and System Biosciences, respectively. The human iPSCs line HmRNA (generated in this study) was reprogrammed from human dermal fibroblasts using the StemMACS mRNA transfection kit. The cell line was validated for pluripotency markers by immunohistochemical staining using the Human Pluripotent Stem Cell 3-Colour Immunohistochemistry Kit and were differentiated into the three different germ layers using the Human Pluripotent Stem Cell Functional Identification kit and StemMACS Trilineage Differentiation Kit. Karyotyping was carried out using Giemsa banding at the Stem Cell Engineering facility, a core facility of CMCB at Technische Universität Dresden. Karyotypes were found to be normal. The human ES cell line H9 was purchased from WiCell. The chimpanzee iPSC lines SandraA and JoC as well as the bonobo iPSCs line BmRNA were generated in a previous study (Kanton et al., Nature, 2019). The chimpanzee iPSCs ciPS01 line was provided by the Max-Delbrück-Centrum für Molekulare Medizin, Berlin.The rtT A/Ngn2-positive iPSCs/ESCs hiPS409-B2_Ngn2, SandraA_Ngn2, BmRNA_Ngn2, H9_Ngn2, SC102A1_Ngn2, HmRNA_Ngn2, ciPS01_Ngn2 and JoC_Ngn2 were generated using lentiviral vectors to stably integrate the transgenes into the genome of the stem cells and differentiate the stem cells into neurons as previously described by Frega et al., Jove, 2017.Our cultures were regularly controlled for mycoplasma.Permission to work with human and non-human primate iPSC lines and Ngn2-inducible cell lines was obtained through the Sächsisches Staatsministerium für Umwelt und Landwirtschaft (Az.: 55-8811.72/26, Az.: 55-8811.72/26/350). The use of human ESCs was approved by the ethics committee of the Robert Koch Institut (https://www.rki.de/DE/Content/Gesund/Stammzellen/Register/reg-20161027-Paeaebo.html).
- Anita Bhattacharyya, University of Wisconsin, Madison, United States
© 2021, Schörnig et al.
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