Manipulating neural stem cell (NSC) fate is of great importance for improving the therapeutic efficacy of NSCs to treat neurodegenerative disorders. Biophysical cues, in addition to biochemical factors, regulate NSC phenotype and function. In this study, we assessed the extent to which surface nanotopography of culture substrates modulates human NSC (hNSC) differentiation. Fibronectin-coated polymer substrates with diverse nanoscale shapes (groove and pillar) and dimensions (ranging from 300 to 1500 nm groove width and pillar gap) were used to investigate the effects of topographical cues on hNSC morphology, alignment, focal adhesion, and differentiation. The majority of nanopatterned substrates induced substantial changes in cellular morphology and alignment along the patterned shapes, leading to alterations in focal adhesion and F-actin reorganization. Certain types of nanopatterned substrates, in particular the ones with small nanostructures (e.g., 300-300 nm groove ridges and 300-300 nm pillar diameter gaps), were found to effectively enhance focal adhesion complex development. Consequently, these substrates enhanced hNSC differentiation toward neurons and astrocytes. Nanotopographical-induced formation of focal adhesions in hNSCs activates integrin-mediated mechanotransduction and intracellular signaling pathways such as MEK-ERK, which may ultimately promote gene expression related to NSC differentiation. This strategy of manipulating matrix surface topography could be applied to develop culture substrates and tissue engineered scaffolds that improve the efficacy of NSC therapeutics.

Schwann cells (SCs) constitute a crucial element of the peripheral nervous system, by structurally supporting the formation of myelin and conveying vital trophic factors to the nervous system. However, the functions of SCs in developmental and regenerative stages remain unclear. Here, we investigated how optogenetic stimulation (OS) of SCs regulates their development. In SC monoculture, OS substantially enhanced SC proliferation and the number of BrdU + -S100ß + -SCs over time. In addition, OS also markedly promoted the expression of both Krox20 and myelin basic protein (MBP) in SC culture medium containing dBcAMP/NRG1, which induced differentiation. We found that the effects of OS are dependent on the intracellular Ca 2+ level. OS induces elevated intracellular Ca 2+ levels through the T-type voltage-gated calcium channel (VGCC) and mobilization of Ca 2+ from both inositol 1,4,5-trisphosphate (IP 3 )-sensitive stores and caffeine/ryanodine-sensitive stores. Furthermore, we confirmed that OS significantly increased expression levels of both Krox20 and MBP in SC-motor neuron (MN) coculture, which was notably prevented by pharmacological intervention with Ca 2+ . Taken together, our results demonstrate that OS of SCs increases the intracellular Ca 2+ level and can regulate proliferation, differentiation, and myelination, suggesting that OS of SCs may offer a new approach to the treatment of neurodegenerative disorders.

The schwann cells of the peripheral nervous system are indispensable for the formation, maintenance, and modulation of synapses over the life cycle. They not only recognize neuron-glia signaling molecules, but also secrete gliotransmitters. Through these processes, they regulate neuronal excitability and thus the release of neurotransmitters from the nerve terminal at the neuromuscular junction. Gliotransmitters strongly affect nerve communication, and their secretion is mainly triggered by synchronized Ca signaling, implicating Ca waves in synapse function. Reciprocally, neurotransmitters released during synaptic activity can evoke increases in intracellular Ca levels. A reconsideration of the interplay between the two main types of cells in the nervous system is due, as the concept of nervous system activity comprising only neuron-neuron and neuron-muscle action has become untenable. A more precise understanding of the roles of schwann cells in nerve-muscle signaling is required.

Increasing evidence demonstrates that optogenetics contributes to the regulation of brain behavior, cognition, and physiology, particularly during myelination, potentially allowing for the bidirectional modulation of specific cell lines with spatiotemporal accuracy. However, the type of cell to be targeted, namely, glia vs neurons, and the degree to which optogenetically induced cell activity can regulate myelination during the development of the peripheral nervous system (PNS) are still underexplored. Herein, we report the comparison of optogenetic stimulation (OS) of Schwann cells (SCs) and motor neurons (MNs) for activation of myelination in the PNS. Capitalizing on these optogenetic tools, we confirmed that the formation of the myelin sheath was initially promoted more by OS of calcium translocating channelrhodopsin (CatCh)-transfected SCs than by OS of transfected MNs at 7 days in vitro (DIV). Additionally, the level of myelination was substantially enhanced even until 14 DIV. Surprisingly, after OS of SCs, > 91.1% ± 5.9% of cells expressed myelin basic protein, while that of MNs was 67.8% ± 6.1%. The potent effect of OS of SCs was revealed by the increased thickness of the myelin sheath at 14 DIV. Thus, the OS of SCs could highly accelerate myelination, while the OS of MNs only somewhat promoted myelination, indicating a clear direction for the optogenetic application of unique cell types for initiating and promoting myelination. Together, our findings support the importance of precise cell type selection for use in optogenetics, which in turn can be broadly applied to overcome the limitations of optogenetics after injury.