Journal: Materials Today Bio

Article Title: Age-mimicking hydrogel stiffness recapitulates the mechanical niche of the hippocampus to regulate neural stem cell senescence

doi: 10.1016/j.mtbio.2026.102985

Figure Lengend Snippet: Age-associated hippocampal stiffening and its replication via laminin-modified hydrogels . (a) The strategy of in vivo EdU labeling and marker immunostaining for analyzing NSC proliferation and neurogenesis across various mouse age groups. ( b ) Co-staining of GFAP, EdU, and DCX in the hippocampus across different ages. Representative images showing a reduction in active radial glia-like stem cells and neuroblasts/newborn neurons with increasing age. GFAP (green), DCX (red), EdU (gray), and DAPI (blue). Scale bar, 100 μm. ( c-e ) Quantification of active radial glia-like stem cells (GFAP + EdU + ) ( c ), neuroblasts (DCX + EdU + ) ( d ), and newborn neurons (DCX + ) (e) in the SGZ area as in ( b ). n = 3 or 4 mice per group. ( f ) Schematic showing the measurement of hippocampal tissue stiffness using the Pavone nanoindenter and the design of hyaluronic acid (HA)–laminin hydrogels with tunable stiffness to mimic hippocampal mechanical properties at different postnatal ages. Soft, medium, and stiff hydrogels correspond to the mechanical characteristics of hippocampal tissues from 1-, 4-, and 12-week-old mice, respectively. ( g ) Representative images of the dentate gyrus in mouse brain slices across age groups, captured under Pavone nanoindentation microscopy. The SGZ regions measured by the nanoindentation probe are demarcated by paired colorful dashed lines. ( h ) Quantification of Young’s modulus in the hippocampal SGZ region of mice at different ages using Pavone nanoindentation. Brain slices were obtained from four mice per age group. Measurements were taken from n = 227 spots (1 week), n = 149 spots (4 weeks), n = 241 spots (8 weeks), n = 157 spots (12 weeks). (i ) Schematic illustration of the synthesis of HA@HA and HA@HA–Laminin hydrogels. Hyaluronic acid (HA) was first crosslinked with adipic dihydrazide (ADH) using EDC/HCl activation under acidic conditions (pH 3–4) to form HA@HA. Subsequently, laminin was conjugated to the HA network via CDI-mediated coupling to generate HA@HA–Laminin hydrogels. ( j ) Quantification of Young’s modulus of Soft, Medium, and Stiff HA-laminin hydrogels using the same Pavone nanoindentation used for tissue (Soft hydrogel, n = 44 spots; Medium hydrogel, n = 37 spots; Stiff hydrogel, n = 29 spots). ( k ) Immunostaining of YAP1 protein shows the subcellular localization of YAP1 in NSCs cultured on HA-laminin hydrogels of varying stiffness. Dashed lines indicate representative cells with YAP1 predominantly in the cytoplasm (indicated by arrowheads), while solid lines represent cells with YAP1 primarily in the nucleus (indicated by arrows). Scale bar, 20 μm. ( l ) Quantification of YAP1 distribution in NSCs as in ( k ) (n = 4 wells). For all quantification data, statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison tests. Data are presented as mean ± SD (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: Primary antibodies were diluted to specific concentrations in an antibody dilution buffer (5% NDS, 0.2% Triton X-100): Map2 (CST, 4542s, 1:500), GFAP (Sigma, AB5541, 1:1000), GFAP (Invitrogen, 13-0300, 1:1000), LaminB1 (Abcam, ab229025, 1:1000), Yap1 (Proteintech,13584-1-AP, 1:500), Nestin(BD Pharmingen, 611658, 1:1000), Tuj1 (Abcam, AB78078, 1:1000) and Piezo1 (Proteintech, 15939-1-AP, 1:500).

Techniques: Modification, In Vivo, Labeling, Marker, Immunostaining, Staining, Microscopy, Activation Assay, Cell Culture, Comparison