Readaptation of mesenchymal stem cells to high stiffness and oxygen environments modulate the extracellular matrix

The therapeutic potential of mesenchymal stem cells (MSCs) has been explored over the past decades due to their ability to modulate the microenvironment through paracrine signaling. Consequently, the secretome of MCSs has emerged as a cell-free therapy rather than a cell therapy, offering the advantages of being readily commercialized as an off-the-shelf product without immunogenicity compatibility issues. As a result, strategies to manipulate and enhance the secretory profile of MSCs’ secretome are emerging. MSCs from the Wharton’s jelly niche are accommodated to the stiffness and oxygen level found at the umbilical cord (UC), which are 2 to 5kPa (Young’s modulus) and 2.4% to 3.8% O ₂ , respectively. However in vitro culture conditions (2-3 GPa and 18.5% O ₂ ) are largely different from the one observed in vivo. Here, we present a proteomic characterization of the secretome of MSCs primed (48h) or readapted (7-10 days) to soft (3kPa) (mechanomodulated) or low oxygen levels (5% O ₂ ) (physioxia). Maintaining MSCs on soft platforms for long periods increased the secretion of proteins associated with cell redox homeostasis, such as protein disulfide isomerases and mitochondrial proteins, while physioxia enhanced the secretion of immunomodulatory proteins. The high secretion of these proteins might confer a therapeutical advantage by favoring a regenerative environment at the injury site. Interestingly, lowering the stiffness or oxygen converged on the downregulation of several extracellular matrix proteins (ECM), particularly collagen fibrils, on primed and readapted cells. These results suggest that a massive reorganization of the extracellular space occurs upon culturing MSCs on conventional culture conditions, which may affect not only matrix stiffness but also several signaling pathways initiated at the cell membrane, such as PDGF signaling pathways (e.g., PI3K-AKT), consequently biasing stem cell fate. In conclusion, mimicking physiological culture conditions in vitro modulates secretome composition, which may empower its therapeutical properties by enriching proteins that promote cell survival.