Singapore-MIT Alliance for Research & Technology

BioSystems and Micromechanics (BioSyM) Inter-Disciplinary Research Group

Macromolecular crowding directs extracellular matrix organization and
mesenchymal stem cell behavior

(MIT News release)

SMART-BioSyM, MIT and NUS researchers demonstrate effects of macromolecular crowding on the cell and matrix materials. In the work soon to be published in PLoS One, they (Adam S. Zeiger (MIT/SMART), Felicia C. Loe (SMART), Ran Li (MIT), Michael Raghunath (NUS), and Krystyn J. Van Vliet (MIT/SMART)) describe how when one introduces small molecules (called crowders) into the fluids that stem cells (and all tissue cells) live in under typical lab conditions, this crowding induces dramatic alignment of the protein networks (called matrices or scaffolds) outside the cell.

Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

Macromolecular crowding directly alters organization of deposited extracellular matrix proteins and thus alters the orientation of the actin cytoskeleton. (A) Immunostaining of intracellular F-actin (red), intracellular vinculin (green) as a focal adhesion protein involved in the linking of integrin to actin cytoskeleton, and nucleus (blue, DAPI) of human bone marrow-derived mesenchymal stromal or stem cells (MSCs) after 3 days of cell culture in media containing macromolecular crowders (+MMC media) and (B) -MMC media. Scale bars = 30 μm. (C) Quantification of average angular standard deviation for F-actin (N=10 +MMC, N=10 –MMC, p=0.0223) where lower values indicate a higher degree of alignment. (D) Effective Young’s elastic modulus in kPa measured by atomic force microscopy enabled nanoindentation of MSCs +/- MMC suggests a stiffening of the cortical cytoskeleton +MMC. (E) Average angular standard deviation of FITC-conjugated rat tail type-I collagen network deposited on plasma treated glass coverslips, (F) in media absent of macromolecular crowders (- MMC) and, (G) +MMC. Scale bars = 25 μm. (H) Immunostaining of F-actin (red) after 3 days for human bone marrow-derived mesenchymal stromal or stem cells cultured in basal -MMC media seeded onto type-I collagen networks formed under –MMC, or (I) +MMC conditions (N=13 +MMC, N=13 –MMC, p=0.0001). Scale bars = 25 μm. (J) Average angular standard deviation of actin fibers for H and I. Values are reported as mean +/- standard error of measurement. * indicates statistical significance (p < 0.05). ** indicates statistical significance (p = 0.0001).