Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes
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 (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.
Data availability
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/.
Article and author information
Author details
Funding
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.
Ethics
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).
Copyright
© 2021, Schörnig et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,029
- views
-
- 711
- downloads
-
- 43
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Developmental Biology
- Evolutionary Biology
Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genome assemblies revealed suggestive gene content differences and provided the opportunity for detailed genetic analyses. We created a single-cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined the spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting that derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how the novelties of these fish evolved.
-
- Developmental Biology
- Neuroscience
We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of ×2 and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 µm across an FOV of approximately 1.5×1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hr, visualizing the movement of over 4.0×105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.