Robust neuronal differentiation of human iPSC-derived neural progenitor cells cultured on densely-spaced spiky silicon nanowire arrays

Abstract

Nanostructured cell culture substrates featuring nanowire (NW) arrays have been applied to a variety of basic cell lines and rodent neurons to investigate cellular behavior or to stimulate cell responses. However, patient-derived human neurons—a prerequisite for studying e.g. neurodegenerative diseases efficiently—are rarely employed due to sensitive cell culture protocols and usually long culturing periods. Here, we present human patient induced pluripotent stem cell-derived neurons cultured on densely-spaced spiky silicon NW arrays (600 NWs/ 100 µm^2 with NW lengths of 1 µm) which show mature electrophysiological characteristics after only 20 days of culturing. Exemplary neuronal growth and network formation on the NW arrays are demonstrated using scanning electron microscopy and immunofluorescence microscopy. The cells and neurites rest in a fakir-like settling state on the NWs only in contact with the very NW tips shown by cross-sectional imaging of the cell/NW interface using focused ion beam milling and confocal laser scanning microscopy. Furthermore, the NW arrays promote the cell culture by slightly increasing the share of differentiated neurons determined by the quantification of immunofluorescence microscopy images. The electrophysiological functionality of the neurons is confirmed with patch-clamp recordings showing the excellent capability to fire action potentials. We believe that the short culturing time to obtain functional human neurons generated from patient-derived neural progenitor cells and the robustness of this differentiation protocol to produce these neurons on densely-spaced spiky nanowire arrays open up new pathways for stem cell characterization and neurodegenerative disease studies.