SingaporeMIT Alliance for Research & Technology 
BioSystems and Micromechanics (BioSyM) InterDisciplinary Research Group 

Macromolecular crowding directs extracellular matrix organization and

Macromolecular crowding directly alters organization of deposited extracellular matrix proteins and thus alters the orientation of the actin cytoskeleton. (A) Immunostaining of intracellular Factin (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 marrowderived 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 Factin (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 FITCconjugated rat tail typeI 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 Factin (red) after 3 days for human bone marrowderived mesenchymal stromal or stem cells cultured in basal MMC media seeded onto typeI 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). 
