Influence of Altered Local Effective Concentration on Cytoskeletal and Ecm Structure in Human Mesenchymal Stem Cells

Abstract

Mesenchymal stromal or stem cells (MSCs) are a type of pluripotent precursor cell found in bone marrow, which can be coaxed in vitro to express certain characteristics of differentiated mesenchymal tissue lineages. Many therapeutic applications of MSCs require isolation from a given patient, expansion to useful cell numbers in culture, and reimplantation. However, the in vivo and in vitro culture conditions remain in stark contrast, and confound interpretation of experiments and development of therapeutic uses for MSCs. For example, in vivo, the extracellular microenvironment is highly crowded with proteins; in vitro, the extracellular culture media is decidedly dilute. Here, we use multiple methods to consider whether this differential crowding can directly alter MSC structure and function. We consider a series of macromolecular crowders within the culture media of MSCs, expanded under basal (non-differentiating) conditions. We then quantify changes in population-level proliferation and migration, and in extracellular and intracellular protein structure and cortical cytoskeletal stiffness via immunocytochemistry, optical microscopy, and atomic force microscopy-enabled imaging and nanoindentation of living MSCs. We find significant effects of “crowded” culture conditions on cell architecture, extracellular matrix structure, and attendant cell properties. These results motivate the development of more physiologically relevant environments for both in vitro studies and biodevices, to ultimately answer fundamental questions of physiological and pathological behavior for MSCs.