No abstract

Mesenchymal stromal cells (MSCs) modulate immune cells to ameliorate multiple inflammatory pathologies. Biophysical signals that regulate this process are poorly defined. By engineering hydrogels with tunable biophysical parameters relevant to bone marrow where MSCs naturally reside, we show that soft extracellular matrix maximizes the ability of MSCs to produce paracrine factors that have been implicated in monocyte production and chemotaxis upon inflammatory stimulation by tumor necrosis factor–α (TNFα). Soft matrix increases clustering of TNF receptors, thereby enhancing NF-κB activation and downstream gene expression. Actin polymerization and lipid rafts, but not myosin-II contractility, regulate mechanosensitive activation of MSCs by TNFα. We functionally demonstrate that human MSCs primed with TNFα in soft matrix enhance production of human monocytes in marrow of xenografted mice and increase trafficking of monocytes via CCL2. The results suggest the importance of biophysical signaling in tuning inflammatory activation of stromal cells to control the innate immune system.

Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

Advances in engineered hydrogels reveal how cells sense and respond to 3D biophysical cues. However, most studies rely on interfacing a population of cells in a tissue-scale bulk hydrogel, an approach that overlooks the heterogeneity of local matrix deposition around individual cells. A droplet microfluidic technique to deposit a defined amount of 3D hydrogel matrices around single cells independently of material composition, elasticity, and stress relaxation times is developed. Mesenchymal stem cells (MSCs) undergo isotropic volume expansion more rapidly in thinner gels that present an Arg-Gly-Asp integrin ligand. Mathematical modeling and experiments show that MSCs experience higher membrane tension as they expand in thinner gels. Furthermore, thinner gels facilitate osteogenic differentiation of MSCs. By modulating ion channels, it is shown that isotropic volume expansion of single cells predicts intracellular tension and stem cell fate. The results suggest the utility of precise microscale gel deposition to control single cell functions. Cells utilize tactile mechanisms to physically probe the extracellular matrix. [1] Advances in the design of engineered hydrogels have revealed that various matrix biophysical properties are sufficient to impact cellular functions independently of changes in biochemical cues, including matrix elasticity, [2,3] degradation, [4] and stress relaxation. [5] As a result, cells exert traction forces on

Extracellular vesicles (EVs) are cell-secreted particles with broad potential to treat tissue injuries by delivering cargo to program target cells. However, improving the yield of functional EVs on a per cell basis remains challenging due to an incomplete understanding of how microenvironmental cues regulate EV secretion at the nanoscale. We show that mesenchymal stromal cells (MSCs) seeded on engineered hydrogels that mimic the elasticity of soft tissues with a lower integrin ligand density secrete ∼10-fold more EVs per cell than MSCs seeded on a rigid plastic substrate, without compromising their therapeutic activity or cargo to resolve acute lung injury in mice. Mechanistically, intracellular CD63+ multivesicular bodies (MVBs) transport faster within MSCs on softer hydrogels, leading to an increased frequency of MVB fusion with the plasma membrane to secrete more EVs. Actin-related protein 2/3 complex but not myosin-II limits MVB transport and EV secretion from MSCs on hydrogels. The results provide a rational basis for biomaterial design to improve EV secretion while maintaining their functionality.