(D-135) Confined Migration Drives Stem Cell Epigenetics and Differentiation

Introduction:: Human mesenchymal stem cells (hMSCs) have shown potential in regenerative medicine due to their multi-lineage differentiation capacity, immunomodulatory properties, and low mutagenic potential. They can navigate complex extracellular matrix (ECM) environments, experiencing restricted migration when traversing tight spaces in interstitial tissue or gaps within blood vessel walls. While numerous studies have elucidated how different mechanical properties of the ECM can regulate hMSC behavior, the effect of confined migration on the fate of hMSCs is not well-examined, despite its prevalence in the ECM. Moreover, it is accepted that mechanical forces perceived by cells through mechanosensitive adhesions can be transmitted to the nucleus via the cytoskeleton, leading to chromatin reorganization and changes in gene expression. However, the function of nuclear mechanosensing remains elusive in the context of confined migration.


Materials and Methods:: To address these questions, we designed and fabricated a physiologically relevant, flow-free microchannel system based on polydimethylsiloxane (PDMS), which enables us to quantitatively investigate the consequences of confined migration in stem cells. Straight microchannels of constant height (10 µm) yet two different lengths (short: 25 µm, long: 150 µm) and different widths ranging from 3µm to 10 µm were used to recapitulate confined migration presenting in vivo. All microchannels were coated with collagen type I to provide cell binding sites. Cells in chips were all maintained in normal expansion media unless otherwise specified. Time-lapse microscope was used to monitor stem cell migration dynamics. Immunofluorescence staining and fluorescent microscope were employed to examine stem cell morphological changes before, under, and after confined migration, as well as epigenetic changes and the location of transcriptional factors. To study how nuclear mechanosensing is involved in confined migration, drugs inhibiting cytoskeleton contractility or organization was applied to the culture media. In addition, a dominant negative nesprin construct containing the KASH domain fused to mCherry was introduced to stem cells to disconnect the nucleus from the cytoskeleton.


Results, Conclusions, and Discussions:: We found that stem cells migrated faster under narrow confinement (3 µm wide microchannels), a phenomenon similar to cancer cells which have been shown to undergo mesenchymal-to-amoeboid transition in response to confinement. As expected, stem cells exhibited significant nuclear deformation when migrating through confinements, with an increasing nuclear aspect ratio as the degree of confinement increased. Additionally, altered cell and nuclear morphology were observed in cells exited the 3 µm wide microchannels (Figure 1a). The nuclear deformation was also found to correlate with major changes in genome regulation in stem cells that traverse narrow confinements: H3K9 acetylation was significantly upregulated post-confinement. More importantly, stem cells subjected to confined migration express more and have a higher nuclear-to-cytoplasmic ratio of Runx2, an early transcriptional factor involved in osteogenesis, than unconfined cells after 7 days (Figure 1b-c). Critically, this upregulation was found to be dependent on both the length and width of the confining microchannel, suggesting that migration through physiologically-relevant confinement is sufficient to initiate differentiation pathways in adult stem cells. The results of disrupting the cell contractility or integrity of link of nucleoskeleton and cytoskeleton suggest that nuclear mechanosensing is responsible for confinement induced changes in stem cells. Overall, these results will help to further our understanding of MSC responses to confinement and aid the development of MSC-based therapies.


Acknowledgements (Optional): : The authors would like to acknowledge the assistance of the Department of Biomedical Engineering, the Mechanobiology Institute, Microfab Core, Wet Lab Core, IT Core, and Microscopy Core. Funding support was provided by the NRF of Singapore.


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