Human mesenchymal stem cells (hMSCs), during in vitro expansion, gradually lose their distinct spindle morphology, self-renewal ability, multi-lineage differentiation potential and enter replicative senescence. This loss of cellular function is a major roadblock for clinical applications which demand cells in large numbers. Here, we demonstrate a novel role of substrate stiffness in the maintenance of hMSCs over long-term expansion. When serially passaged for 45 days from passage 3 to passage 18 on polyacrylamide gel of Young's modulus E =5 kPa, hMSCs maintained their proliferation rate and showed nine times higher population doubling in comparison to their counterparts cultured on plastic Petri-plates. They did not express markers of senescence, maintained their morphology and other mechanical properties such as cell stiffness and cellular traction, and were significantly superior in adipogenic differentiation potential. These results were demonstrated in hMSCs from two different sources, umbilical cord and bone marrow. In summary, our result shows that a soft gel is a suitable substrate to maintain the stemness of mesenchymal stem cells. As preparation of polyacrylamide gel is a well-established, and well-standardized protocol, we propose that this novel system of cell expansion will be useful in therapeutic and research applications of hMSCs.

Cell–cell interaction via substrate deformation in turn modifies cellular response to substrate rigidity.

Efficient homing of human mesenchymal stem cells (hMSCs) is likely to be dictated by a combination of physical and chemical factors present in the microenvironment. However, crosstalk between the physical and chemical cues remains incompletely understood. Here, we address this question by probing the efficiency of epidermal growth factor (EGF)-induced hMSC chemotaxis on substrates of varying stiffness (3, 30 and 600 kPa) inside a polydimethylsiloxane (PDMS) microfluidic device. Chemotactic speed was found to be the sum of a stiffness-dependent component and a chemokine concentration-dependent component. While the stiffness-dependent component scaled inversely with stiffness, the chemotactic component was independent of stiffness. Faster chemotaxis on the softest 3 kPa substrates is attributed to a combination of weaker adhesions and higher protrusion rate. While chemotaxis was mildly sensitive to contractility inhibitors, suppression of chemotaxis upon actin depolymerization demonstrates the role of actin-mediated protrusions in driving chemotaxis. In addition to highlighting the collective influence of physical and chemical cues in chemotactic migration, our results suggest that hMSC homing is more efficient on softer substrates.

Microbial lipids play a critical role in the pathogenesis of infectious diseases by modulating the host cell membrane properties, including lipid/protein diffusion and membrane organization. Mycobacterium tuberculosis (Mtb) synthesizes various chemically distinct lipids that are exposed on its outer membrane and interact with host cell membranes. However, the effects of the structurally diverse Mtb lipids on the host cell membrane properties to fine-tune the host cellular response remain unknown. In this study, we employed membrane biophysics and cell biology to assess the effects of different Mtb lipids on cell membrane mechanics, lipid diffusion, and the cytoskeleton of THP-1 macrophages. We found that Mtb lipids modulate macrophage membrane properties, actin cytoskeleton, and biochemical processes, such as protein phosphorylation and lipid peroxidation, in a virulence lipid-selective manner. These results emphasize that Mtb can fine-tune its interactions with the host cells governed by modulating the lipid profile on its surface. These observations provide a novel lipid-centric paradigm of Mtb pathogenesis that is amenable to pharmacological inhibition and could promote the development of robust biomarkers of Mtb infection and pathogenesis.

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.