Journal: Advanced Science

Article Title: Mechanical Environment Afforded by Engineered Hydrogel Critically Regulates Survival of Neural Stem Cells Transplanted in the Injured Spinal Cord via Piezo1‐Mediated Mechanotransduction

doi: 10.1002/advs.202507160

Figure Lengend Snippet: Influence of the substrate stiffness on the adhesive properties and the viability of cultured NSCs. A) A diagram depicting the in vitro culture system providing polymer matrix with varying degree of mechanical stiffness. PAA = polyacrylamide. B) Representative images of NSCs obtained during adhesion assay. NSCs expressing GFP were grown on substrates with mechanical stiffness ranging from 25 to 0.2 kPa. Upper panel: Phase‐contrast images. Middle panel: NSCs were visualized using GFP fluorescence. Boxed regions show magnified cell morphologies below. Scale bars = 20 µm. C–E) Quantitative graphs comparing the number of cells adhered (C), areas of cell spreading (D), and the perimeter of cell boundary (E). Each dot represents an independent culture replicate, with each replicate being an average value from three coverslips. Error bars represent SEM. ** and *** indicates p < 0.05 and p < 0.001 by one‐way ANOVA followed by Tukey's post hoc analysis. F,G) Representative images of NSCs for cell survival assay. NSCs were cultured on hydrogel substrates ranging from 25 to 0.2 kPa stiffness for 24 h. Subsequently, cell survival assay was performed. Live cells are labeled by Calcein‐AM (green), and dead cells are labeled by Ethidium Homodimer‐1 (red). Quantitative graph depicting the percentage of dead cells (G). Each dot represents an independent culture replicate, with each replicate being an average value from three coverslips. Error bars indicate SEM. Scale bars = 100 µm. H,I) Representative images of phospho‐ERK immunostaining (H). Arrows indicate GFP positive cells colocalized with phospho‐ERK (pERK, magenta) expression. The total number of NSCs was divided by pERK positive cells (I). Error bars represent SEM. ** indicates p < 0.01 by unpaired t ‐test. Each dot represents an independent culture replicate, with each replicate being an average value from three coverslips. Scale bar = 50 µm. J,K) Representative images of NSCs for cell survival assay with sodium arsenite treatment. Sodium arsenite (NaAsO 2 ) was used to induce cellular stress for 24 h (J). Quantitative graph of percentage of dead cells (K). N = 6 and 4 for 25 and 0.2 kPa groups, respectively. Error bars represent SEM. * indicates p < 0.05 by unpaired t ‐test. Error bars represent the SEM. Scale bars = 100 µm. (L) Graphical illustration of the 3D hydrogel live/dead cell survival assay methods. (M) Representative confocal microscopic projection images of NSCs cultured in 3D hydrogels with different concentrations. Scale bar = 100 µm. (N) Quantification of dead cell percentages in 3D hydrogel cultures. *** indicates p < 0.001 by unpaired t ‐test. N = 4 independent 3D cultures for both 10% and 16% hydrogel concentrations. Each dot represents an independent 3D culture replicate. Error bars represent SEM.

Article Snippet: For spontaneous intracellular Ca 2+ imaging, glass bottom coverslips or hydrogel‐bound coverslips in a confocal dish (Matrigen, CA, #SV3520‐EC) were pre‐coated with Poly‐D‐Lysine for 18 h in a 37 °C incubator.

Techniques: Adhesive, Cell Culture, In Vitro, Polymer, Cell Adhesion Assay, Expressing, Fluorescence, Clonogenic Cell Survival Assay, Labeling, Immunostaining

Journal: Advanced Science

Article Title: Mechanical Environment Afforded by Engineered Hydrogel Critically Regulates Survival of Neural Stem Cells Transplanted in the Injured Spinal Cord via Piezo1‐Mediated Mechanotransduction

doi: 10.1002/advs.202507160

Figure Lengend Snippet: Actin polymerization determines the stiffness‐dependent cellular elasticity and intracellular calcium dynamics in NSCs. A) Nanoindentation analysis of the elastic modulus of NSCs cultured on hydrogel substrates with stiffness of 100, 25, and 12 kPa. Atomic force microscopy (AFM) was used to measure cellular membrane stiffness. Whiskers indicate the range of the minimum and maximum values. The lines within the boxes indicate the median values. *** indicates p < 0.001 compared to 100 kPa, and # indicates p < 0.05 compared to 25 kPa group by one‐way ANOVA followed by Tukey's post hoc analysis. N = 20, 25, and 20 cells for 100, 25, and 12 kPa, respectively. B,C) Representative images of filamentous actin (F‐actin) visualization in NSCs grown on 100, 25, 12, 2, and 0.2 kPa hydrogel substrates (B). F‐actin was stained using Alexa Fluor 594‐Phalloidin co‐labeled with DAPI. Quantification graph of actin staining intensity (C). Each dot represents a measurement of one or two coverslips from an independent culture. Error bars represent SEM. *** and * indicate p < 0.001 and p < 0.05, respectively, by one‐way ANOVA followed by Tukey's post hoc analysis. Error bars represent SEM. Scale bar = 5 µm. D) Changes in NSC plasma membrane elastic modulus measured by AFM following treatment with 20 µM of cytochalasine D (CytoD), actin destabilizer. N = 20 and 30 cells for 100 and 12 kPa (DMSO) groups, respectively, and N = 20 for both 100 and 12 kPa (CytoD) groups. Whiskers indicate the range of the minimum and maximum values. The lines within the boxes indicate the median values. *** indicates p < 0.001 and ** indicates p < 0.01 by two‐way ANOVA followed by Bonferroni's post hoc analysis. E) Intracellular calcium oscillations in NSCs grown on 25 kPa and 0.2 kPa hydrogel substrates. Left: representative calcium intensity maps (red: high level of Ca2+, blue: low level of Ca2+). Right: representative plots of calcium oscillations over a 60 s period. Each color represents an individual cell. F) Quantitative graphs of intracellular calcium oscillation parameters: responding cells (%), frequency (events s −1 ), and amplitude (F max /F 0 ). ** indicates p < 0.01 by unpaired t ‐test. N = 200 and 190 cells from 4 independent cultures for 0.2 and 25 kPa groups. Each dot represents an average value from an independent culture. G) Intracellular calcium oscillation after cytochalasin D (CytoD) treatment. H) Quantification graphs of intracellular calcium oscillation parameters: responding cells (%), frequency (events s −1 ), and amplitude (F max /F 0 ). * indicates p < 0.05 by unpaired t ‐test. N = 150 cells for 0.2 kPa + DMSO; N = 130 cells for 0.2 kPa + cytoD; N = 150 cells for 25 kPa + DMSO; and N = 150 cells for 25 kPa + cytoD group. Three independent cultures were conducted. Error bars represent SEM. Each dot represents an average value from an independent culture.

Article Snippet: For spontaneous intracellular Ca 2+ imaging, glass bottom coverslips or hydrogel‐bound coverslips in a confocal dish (Matrigen, CA, #SV3520‐EC) were pre‐coated with Poly‐D‐Lysine for 18 h in a 37 °C incubator.

Techniques: Cell Culture, Microscopy, Membrane, Staining, Labeling, Clinical Proteomics