Gyeong Eon Chang

Graphene Oxide Hierarchical Patterns for the Derivation of Electrophysiologically Functional Neuron-like Cells from Human Neural Stem Cells

Graphene has shown great potential for biomedical engineering applications due to its electrical conductivity, mechanical strength, flexibility, and biocompatibility. Topographical cues of culture substrates or tissue-engineering scaffolds regulate the behaviors and fate of stem cells. In this study, we developed a graphene oxide (GO)-based patterned substrate (GPS) with hierarchical structures capable of generating synergistic topographical stimulation to enhance integrin clustering, focal adhesion, and neuronal differentiation in human neural stem cells (hNSCs). The hierarchical structures of the GPS were composed of microgrooves (groove size: 5, 10, and 20 μm), ridges (height: 100-200 nm), and nanoroughness surfaces (height: ∼10 nm). hNSCs grown on the GPS exhibited highly elongated, aligned neurite extension along the ridge of the GPS and focal adhesion development that was enhanced compared to that of cells grown on GO-free flat substrates and GO substrates without the hierarchical structures. In particular, GPS with a groove width of 5 μm was found to be the most effective in activating focal adhesion signaling, such as the phosphorylation of focal adhesion kinase and paxillin, thereby improving neuronal lineage commitment. More importantly, electrophysiologically functional neuron-like cells exhibiting sodium channel currents and action potentials could be derived from hNSCs differentiated on the GPS even in the absence of any of the chemical agents typically required for neurogenesis. Our study demonstrates that GPS could be an effective culture platform for the generation of functional neuron-like cells from hNSCs, providing potent therapeutics for treating neurodegenerative diseases and neuronal disorders.

The Ca2+ -activated chloride channel anoctamin-2 mediates spike-frequency adaptation and regulates sensory transmission in thalamocortical neurons

Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+-activated Cl− channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission.

Suppression of Sin3A activity promotes differentiation of pluripotent cells into functional neurons

Sin3 is a transcriptional corepressor for REST silencing machinery that represses multiple neuronal genes in non-neuronal cells. However, functions of Sin3 (Sin3A and Sin3B) in suppression of neuronal phenotypes are not well characterized. Herein we show that Sin3A knockdown impedes the repressive activity of REST and enhances differentiation of pluripotent P19 cells into electrophysiologically active neurons without inducing astrogenesis. It is also found that silencing Sin3B induces neurogenesis of P19 cells with a lower efficiency than Sin3A knockdown. The results suggest that Sin3A has a more profound effect on REST repressive machinery for silencing neuronal genes in P19 cells than Sin3B. Furthermore, we show that a peptide inhibitor of Sin3A-REST interactions promotes differentiation of P19 cells into functional neurons. Observations made in studies using genetic deletion and a synthetic inhibitor suggests that Sin3A plays an important role in the repression of neuronal genes by the REST regulatory mechanism.

A potent and selective small molecule inhibitor of sirtuin 1 promotes differentiation of pluripotent P19 cells into functional neurons

Sirtuin 1 (SIRT1) is known to suppress differentiation of pluripotent/multipotent cells and neural progenitor cells into neurons by blocking activation of transcription factors critical for neurogenesis. EX-527 is a highly selective and potent inhibitor against SIRT1 and has been used as a chemical probe that modulates SIRT1-associated biological processes. However, the effect of EX-527 on neuronal differentiation in pluripotent cells has not been well elucidated. Here, we report an examination of EX-527 effects on neurogenesis of pluripotent P19 cells. The results showed that EX-527 greatly accelerated differentiation of P19 cells into neurons without generation of cardiac cells and astrocytes. Importantly, neurons derived from P19 cells treated with EX-527 generated voltage-dependent sodium currents and depolarization-induced action potentials. The findings indicate that the differentiated cells have electrophysiological properties. The present study suggests that the selective SIRT1 inhibitor could have the potential of being employed as a chemical inducer to generate functionally active neurons.

Combining Suppression of Stemness with Lineage-Specific Induction Leads to Conversion of Pluripotent Cells into Functional Neurons.

Sox2 is a key player in the maintenance of pluripotency and stemness, and thus inhibition of its function would abrogate the stemness of pluripotent cells and induce differentiation into several types of cells. Herein we describe a strategy that relies on a combination of Sox2 inhibition with lineage-specific induction to promote efficient and selective differentiation of pluripotent P19 cells into neurons. When P19 cells transduced with Skp protein, an inhibitor of Sox2, are incubated with a neurogenesis inducer, the cells are selectively converted into neurons that generate depolarization-induced sodium currents and action potentials. This finding indicates that the differentiated neurons are electrophysiologically active. Signaling pathway studies lead us to conclude that a combination of Skp with the neurogenesis inducer enhances neurogenesis in P19 cells by activating Wnt and Notch pathways. The present differentiation protocol could be valuable to selectively generate functionally active neurons from pluripotent cells.

A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans

Animals use pheromones as a conspecific chemical language to respond appropriately to environmental changes. The soil nematode Caenorhabditis elegans secretes ascaroside pheromones throughout the lifecycle, which influences entry into dauer phase in early larvae, in addition to sexual attraction and aggregation. In adult hermaphrodites, pheromone sensory signals perceived by worms usually elicit repulsion as an initial behavioral signature. However, the molecular mechanisms underlying neuronal pheromone sensory process from perception to repulsion in adult hermaphrodites remain poorly understood. Here, we show that pheromone signals perceived by GPA-3 is conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role by controlling glutaminase gene expression. We further provide evidence that this modulatory role for DAF-16/FoxO seems to be conserved evolutionarily by electro-physiological study in mouse primary hippocampal neurons that are responsible for glutamatergic neurotransmission. These findings provide the basis for understanding the nematode pheromone signaling, which seems crucial for adaptation of adult hermaphrodites to changes in environmental condition for survival.

Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons

Biophysical cues can improve the direct reprogramming of fibroblasts into neurons that can be used for therapeutic purposes. However, the effects of a three-dimensional (3D) environment on direct neuronal reprogramming remain unexplored. Here, we show that brain extracellular matrix (BEM) decellularized from human brain tissue facilitates the plasmid-transfection-based direct conversion of primary mouse embryonic fibroblasts into induced neuronal (iN) cells. We first show that two-dimensional (2D) surfaces modified with BEM significantly increase the generation efficiency of iN cells and enhance neuronal transdifferentiation and maturation. Moreover, in an animal model of ischaemic stroke, iN cells generated on the BEM substrates and transplanted into the brain led to significant improvements in locomotive behaviours. We also show that compared with the 2D BEM substrates, 3D BEM hydrogels recapitulating brain-like microenvironments further promote neuronal conversion and potentiate the functional recovery of the animals. Our findings suggest that 3D microenvironments can boost nonviral direct reprogramming for the generation of therapeutic neuronal cells.Hydrogels made from decellularized human brain tissue facilitate the direct conversion of primary mouse embryonic fibroblasts into induced neuronal cells that lead to therapeutic outcomes after transplantation in an animal model of ischaemic stroke.

Photoactive poly(3-hexylthiophene) nanoweb for optoelectrical stimulation to enhance neurogenesis of human stem cells

Optoelectrical manipulation has recently gained attention for cellular engineering; however, few material platforms can be used to efficiently regulate stem cell behaviors via optoelectrical stimulation. In this study, we developed nanoweb substrates composed of photoactive polymer poly(3-hexylthiophene) (P3HT) to enhance the neurogenesis of human fetal neural stem cells (hfNSCs) through photo-induced electrical stimulation. Methods: The photoactive nanoweb substrates were fabricated by self-assembled one-dimensional (1D) P3HT nanostructures (nanofibrils and nanorods). The hfNSCs cultured on the P3HT nanoweb substrates were optically stimulated with a green light (539 nm) and then differentiation of hfNSCs on the substrates with light stimulation was examined. The utility of the nanoweb substrates for optogenetic application was tested with photo-responsive hfNSCs engineered by polymer nanoparticle-mediated transfection of an engineered chimeric opsin variant (C1V1)-encoding gene. Results: The nanoweb substrates provided not only topographical stimulation for activating focal adhesion signaling of hfNSCs, but also generated optoelectrical stimulation via photochemical and charge-transfer reactions upon exposure to 539 nm wavelength light, leading to significantly enhanced neuronal differentiation of hfNSCs. The optoelectrically stimulated hfNSCs exhibited mature neuronal phenotypes with highly extended neurite formation and functional neuron-like electrophysiological features of sodium currents and action potentials. Optoelectrical stimulation with 539 nm light simultaneously activated both C1V1-modified hfNSCs and nanoweb substrates, which upregulated the expression and activation of voltage-gated ion channels in hfNSCs and further increased the effect of photoactive substrates on neuronal differentiation of hfNSCs. Conclusion: The photoactive nanoweb substrates developed in this study may serve as platforms for producing stem cell therapeutics with enhanced neurogenesis and neuromodulation via optoelectrical control of stem cells.

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule–induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.