Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings

Abstract

Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

Engineering circuits of human iPSC-derived neurons and rat primary glia

“…Polydimethylsiloxane (PDMS) microstructures were based on a previously published design and prepared as described in Girardin et al (2022). PDMS microstructures consist of a micropatterned two-layer 120 µm thin membrane fabricated by Wunderlichips (Switzerland).…”

“…Electrophysiological experiments were performed using 60-electrode microelectrode arrays (60MEA500/30iR-Ti-gr, Multi Channel Systems). Substrates were prepared according to the protocol detailed in Girardin et al (2022), which includes a Poly-D-lysine (PDL, P6407, Sigma Aldrich) coating of substrates and alignment of the PDMS microstructures on the substrates (MEA or glass-bottom dish), as shown in Figure 1B. For MEAs, a single PFPA-PVP coated PDMS microstructure was used and aligned, whereas for glass-bottom dishes, four PFPA-PVP PDMS microstructures were used, without the need for alignment.…”

“…. iPSC di erentiation Exactly as also described in Girardin et al (2022), human iPSCs were generated following a previously published protocol (Giorgetti et al, 2019) and transfected with a doxycycline-inducible Neurogenin-2 (NGN2) gene. Differentiation into neurons was induced by a 3-day exposure to doxycycline as reported in Russell et al (2018).…”

“…Bottom-up neuroscience focuses on building and studying elementary circuits of neurons to infer the principles of more complex assemblies, with the main goal of getting a better understanding of the mechanisms behind information processing in the brain (Aebersold et al, 2016). Bottom-up neuroscience tools allow to build multiple, topologically-controlled circuits of tens to hundreds of neurons, which can be electrically stimulated and recorded from in parallel using microelectrode arrays (MEA) (Forró et al, 2018;Duru et al, 2022;Girardin et al, 2022;.…”

“…However, compared to primary neurons of animal origin, human iPSC-derived neurons often suffer from poor survival after differentiation (Chen et al, 2021). In spite of this challenge, human NGN2 iNeurons have been successfully used with bottom-up neuroscience tools to build in vitro circuits composed of very few cells (Girardin et al, 2022). Human NGN2 iNeurons are routinely co-cultured with mice or rat primary astrocytes to support their maturation and function (Zhang et al, 2013;Frega et al, 2017;Fernandopulle et al, 2018;Rhee et al, 2019).…”