Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies

Authors: Pei-Yu Shih (Janssen Pharmaceutica NV), Mohamed Kreir (Janssen Pharmaceutica NV), Devesh Kumar(Janssen Pharmaceutica NV), Frederik Seibt (Janssen Pharmaceutica NV), Francisco Pestana (Janssen Pharmaceutica NV), Benjamin Schmid (Bioneer A/S), Bjørn Holst (Bioneer A/S), Christian Clausen (Bioneer A/S), Rachel Steeg (Fraunhofer UK Research Ltd), Benjamin Fischer (Fraunhofer Institute for Biomedical Engineering IBMT), Juan Pita-Almenar (Janssen Pharmaceutica NV), Andreas Ebneth (Janssen Pharmaceutica NV), AlfredoCabrera-Socorro (Janssen Pharmaceutica NV)

Published in Stem Cell Research


Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a “fully-humanized” set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at

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