Abstract
Cells can deform their local niche in three dimensions via whole-cell movements such as spreading, migration or volume expansion. These behaviours, occurring over hours to days, influence long-term cell fates including differentiation. Here we report a whole-cell movement that occurs in sliding hydrogels at the minutes timescale, termed cell tumbling, characterized by three-dimensional cell dynamics and hydrogel deformation elicited by heightened seconds-to-minutes-scale cytoskeletal and nuclear activity. Studies inhibiting or promoting the cell tumbling of mesenchymal stem cells show that this behaviour enhances differentiation into chondrocytes. Further, it is associated with a decrease in global chromatin accessibility, which is required for enhanced differentiation. Cell tumbling also occurs during differentiation into other lineages and its differentiation-enhancing effects are validated in various hydrogel platforms. Our results establish that cell tumbling is an additional regulator of stem cell differentiation, mediated by rapid niche deformation and nuclear mechanotransduction.
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Data availability
The data supporting the findings of the study are available in the Article and its Supplementary Information. The ATAC-seq experiment data are available in GEO accession GSE239277 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). Raw imaging data are available from the corresponding author upon request owing to large file sizes and numbers. Source data are provided with this paper.
Code availability
All new code written for the paper can be accessed via GitHub at https://github.com/Stanford-Fan-Yang-lab/CellTumbling.
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Acknowledgements
We would like to acknowledge NIH grant nos. R01DE024772 (F.Y.) and R01AR074502 (F.Y.) and the Stanford Bio-X Interdisciplinary Initiative Program (F.Y.) for grant support. M.A. would like to thank Bio-X Stanford Interdisciplinary Graduate Fellowship for support. S.S. would like to thank the NIH F31 predoctoral fellowship (no. 5F31CA246972-02) and Stanford NIH Biotechnology training program for support. We would like to thank N. Su, J. Lee and S. Kim for their helpful discussions, T. T. Susanto for help with the plasmid midiprep and the Chaudhuri Lab at Stanford University for kindly allowing the use of their laboratory’s rheometer. We would like to acknowledge the Stanford Gene Vector and Virus Core (GVVC) for plasmid lentivirus packaging.
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Contributions
M.A., X.T. and F.Y. conceived the study design. M.A., G.M., X.T. and S.S. performed the experiments. G.M. wrote all the new codes for image analysis with input from M.A. M.A., E.L.-F. and A.S.-C. conducted the ATAC-seq experiment design and data analysis. M.A., P.C.C., A.S. and S.C.H. conducted the microrheology characterization and data analysis. X.T., H.-P.L. and M.A. performed the AFM experiment and analyses. S.J. and X.T. conducted the polymer synthesis and characterization. A.J.M. participated in the experimental design of characterizing the chromatin state and accessibility. F.Y. supervised the study. M.A. and F.Y. wrote the manuscript with inputs from all authors.
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Extended data
Extended Data Fig. 1 Quantification of cell tumbling and nuclear movements upon cytoskeleton inhibition and LINC complex inhibition.
(a) (Left) Centroid speed and (Right) Major axis angular speed upon cytoskeletal inhibition. N ≥ 15 across 3 gels per condition (b) Nuclear movement speed upon cytoskeleton inhibition. N ≥ 20 across 3 gels per condition. (c) (Left) Centroid speed and (Right) Major axis angular speed upon LINC complex inhibition. N ≥ 15 across 3 gels per condition. (d) Nuclear movement speed (5-minute intervals for a total period of 16 hours) upon LINC complex inhibition. N ≥ 20 across 3 gels per condition. ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test.
Extended Data Fig. 2 Characterization of focal adhesions and the role of adhesive ligands in cell tumbling.
(a) Representative immunofluorescence images for focal adhesion markers in SG (top) and CG (bottom) after 24 hours of chondrogenic induction and Fibronectin after 12 hours of chondrogenic induction as observed from 3 independent experiments. Marker of interest- green, F-Actin- red, and DAPI- blue. Scale- 10μm. (b) Centroid speed and (c) Major axis angular speed upon perturbation of adhesive ligands. N ≥ 15 across 3 gels per condition (d) Nuclear movement speed upon perturbation of adhesive ligands. N ≥ 20 across 3 gels per condition. ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test.
Extended Data Fig. 3 Characterization of Ezrin in cell tumbling.
Note- NSC is the Ezrin inhibitor NSC668394. (a) Representative western blotting images and (b) Quantifications of western blotting images after 24 hours of chondrogenic induction. Protein expression for each marker was normalized to GAPDH. N = 3 biological replicates per condition. Data reported represent mean value ± s.d. (c) Representative immunofluorescence images for Ezrin (green), F-Actin (red) and DAPI (blue) in SG (top), SG with ezrin inhibitor (middle), and CG (bottom) after 24 hours of chondrogenic induction. Scale- 10μm. (d) Quantification of interquartile range of Ezrin intensity at the cell cortex highlighting ezrin expression heterogeneity. N ≥ 15 across 3 gels per condition (e) Cortical Actin, (f) Centroid speed, (g) Major axis angular speed, and (h) nuclear movement speed upon Ezrin inhibition. N ≥ 15 across 3 gels per condition ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test.
Extended Data Fig. 4 Role of cytoskeleton in early-stage tumbling-enhanced chondrogenesis.
(a–c) Gene expression of chondrogenic markers after 3 days of chondrogenic induction with cytoskeletal inhibition. N = 3 gels per condition. (a) SOX9 (b) ACAN (c) COL2A1 (d) (Top) Treatment regimen of different cytoskeleton inhibitors for the first 3 days in the 21-day chondrogenic induction period. (Bottom) Representative Safranin-O histology on cryo-sectioned samples for sGAG deposition after 21 days of chondrogenic induction with different cytoskeleton inhibitors as observed from 3 independent experiments. Scale- 200 μm (shared between all images). ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD for Extended Data Fig. 4a–c.
Extended Data Fig. 5 Role of LINC complex in early-stage tumbling-enhanced chondrogenesis.
(a–c) Gene expression of chondrogenic markers after 3 days of chondrogenic induction with LINC complex inhibition. N = 3 gels per condition. (a) SOX9 (b) ACAN (c) COL2A1 (d) (Top) Treatment regimen of LINC complex inhibition for the first 3 days in the 21-day chondrogenic induction period. (Bottom) Representative Safranin-O histology on cryo-sectioned samples for sGAG deposition after 21 days of chondrogenic induction with LINC complex inhibition as observed from 2 independent experiments. Scale- 200 μm (shared between all images). ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD for Extended Data Fig. 5a–c.
Extended Data Fig. 6 Role of adhesive ligands in early-stage tumbling-enhanced chondrogenesis.
(a–c) Gene expression of chondrogenic markers after 3 days of chondrogenic induction with adhesive ligand perturbations. N = 3 gels per condition. (a) SOX9 (b) ACAN (c) COL2A1 (d) (Top) Treatment regimen of different adhesive ligand perturbations for the first 3 days in the 21-day chondrogenic induction period. (Bottom) Representative Safranin-O histology on cryo-sectioned samples for sGAG deposition after 21 days of chondrogenic induction with different adhesive ligand perturbations as observed from 3 independent experiments. Scale- 200 μm (shared between all images). ns, not significant; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. P value, one–way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD for Extended Data Fig. 6a–c.
Supplementary information
Supplementary Information
Supplementary Notes 1–8, captions for Supplementary Videos 1–10, Figs. 1–10, Table 1, Methods and references.
Supplementary Video 1
Representative videos of cell tumbling (single cells) in bright field in SG and CG.
Supplementary Video 2
Representative videos of cell tumbling (wide field) in bright field in SG and CG.
Supplementary Video 3
Representative video showing the centroid and angular speed tracking during cell tumbling for quantifications. The first cell is in SG, and the second cell is in CG.
Supplementary Video 4
Representative videos of seconds-scale F-actin dynamics (time interval, 5 s), minutes-scale nuclear movement (time interval, 5 min) and seconds-scale nuclear deformations (time interval, 3 s).
Supplementary Video 5
Representative videos of cell tumbling and nuclear movement with cytoskeleton inhibitors. Scale bar, 10 μm.
Supplementary Video 6
Representative videos of cell tumbling and nuclear movement with LINC complex inhibition. Green, DN-KASH-mCherry; red, NucSpot Live 650. Scale bar, 10 μm.
Supplementary Video 7
Representative videos of cell tumbling with adhesive ligand perturbations. Scale bar, 10 μm.
Supplementary Video 8
Representative video showing cell tumbling during MSC osteogenesis and adipogenesis in SG.
Supplementary Video 9
Representative video showing cell tumbling during MSC chondrogenesis in degradable and viscoelastic CG.
Supplementary Video 10
Representative video showing cell tumbling in other cell types in SG.
Supplementary Data 1
Data for Supplementary Figs. 1–5,7–10.
Supplementary Data 2
Statistical test with exact P values for each comparison for Figs. 1–6, Extended Data Figs. 1–6 and Supplementary Figs. 1–10.
Source data
Source Data Fig. 1
Statistical source data for Fig. 1.
Source Data Fig. 2
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Source Data Fig. 4
Statistical source data for Fig. 4.
Source Data Fig. 5
Statistical source data for Fig. 5.
Source Data Fig. 6
Statistical source data for Fig. 6.
Source Data Figs. 4 and 5 and Extended Data Fig. 3
Full-length unprocessed western blots.
Source Data Extended Data Fig. 1
Statistical source data for Extended Data Fig. 1.
Source Data Extended Data Fig. 2
Statistical source data for Extended Data Fig. 2.
Source Data Extended Data Fig. 3
Statistical source data for Extended Data Fig. 3.
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Statistical source data for Extended Data Fig. 4.
Source Data Extended Data Fig. 5
Statistical source data for Extended Data Fig. 5.
Source Data Extended Data Fig. 6
Statistical source data for Extended Data Fig. 6.
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Ayushman, M., Mikos, G., Tong, X. et al. Cell tumbling enhances stem cell differentiation in hydrogels via nuclear mechanotransduction. Nat. Mater. 24, 312–322 (2025). https://doi.org/10.1038/s41563-024-02038-0
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DOI: https://doi.org/10.1038/s41563-024-02038-0
