Journal: bioRxiv

Article Title: Mechanical environment afforded by engineered polymer hydrogel critically regulates survival of neural stem cells transplanted in the injured spinal cord via Piezo1-mediated mechanotransduction

doi: 10.1101/2025.04.15.648586

Figure Lengend Snippet: (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 kPa 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 kPa to 0.2 kPa stiffness for 24 hrs. 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 kPa 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

Article Snippet: Cell culture vessels coated with polyacrylamide (PAA) hydrogel, featuring different elastic modulus levels, were purchased from Matrigen ( http://matrigene.com ).

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