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$$^2$$ 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.

Long-term adherence of human brain cells in vitro is enhanced by charged amine-based plasma polymer coatings

“…Other glass surface modifications for neuron cultures have focused on neurite outgrowth ( Cesca et al., 2014 ; Corey et al., 1991 ; Li et al., 2015 ; Liu et al., 2006 ) or neural stem cell differentiation ( Ananthanarayanan et al., 2010 ; Chen et al., 2018b ), but not sustained neuronal adhesion. Recently nano-structured glass was shown to facilitate PSC-derived neuron functionality ( Harberts et al., 2021 ), but again, cultures were not tested >20 days. An additional benefit of plasma polymer treatment is that the refractive index (and hence, magnitude of attractive van der Waals interactions) can be tailored to further promote the adhesion of various cell types.…”

Generation of Human iPSC‐Derived Neurons on Nanowire Arrays Featuring Varying Lengths, Pitches, and Diameters

“…Dead cells were sorted out in the pipeline by discrimination of bright nuclei (apoptotic) and medium bright nuclei (viable). [ 48 ] Viable cells were used to mask the MAP2 channel to identify MAP2‐positive cells. The NeuN/TH channels were masked by the MAP2‐positive cells to identify NeuN/TH‐positive cells.…”

“…In previous studies, we showed the general feasibility of generating human iPSC-derived neurons on NW arrays, but the utilized substrates featured only about 1 µm long NWs. [47,48] As a result, their rather basic topographies just induced a limited amount of deformational stress toward the cells. The impact of other NW array geometries-for instance, with longer NWs-on the neuronal differentiation is still unclear and thus has been addressed in this study.…”

Using 2D and 3D pluripotent stem cell models to study neurotropic viruses

“…Low rates of reproducibility and efficiency for some existing differentiation protocols may also hinder cell-type specific analyses and integration of data across different research groups. Several adaptations have emerged to overcome potential challenges of 2D models and develop more physiologically relevant networks and substrates, such as nanowire arrays ( 186 , 187 ), sandwich cultures ( 188 ), micropatterning of cell-adhesive islands ( 189 ), modulated substrates ( 190 ), and microfluidic devices ( 191 ). Few, however, have been applied to study iPSC-based models of neurotropic viruses.…”