Mimicking physiological stiffness or oxygen levels in vitro reorganizes mesenchymal stem cells machinery toward a more naive phenotype

Mesenchymal stem cells (MSCs) offer a promising therapeutic potential for a wide variety of pathologies. However, obtaining minimal effective doses requires an extensive in vitro expansion, which compromises their stemness and therapeutic properties. The stiffness of the umbilical cord ranges between 2 and 5kPa, and the oxygen levels fluctuate from 2.4% to 3.8%, differing from the standard in vitro culture conditions where MSCs are exposed to the stiffness of the Petri dish (2-3 GPa) and near atmospheric oxygen levels (18.5% O2). Since MSCs can sense and respond to biomechanical and chemical characteristics of the microenvironment, it was hypothesized that expanding MSCs on 3kPa platforms - mechanomodulation - or at 5% O2 levels - physioxia - could potentially impact the cellular proteome of MSCs, for long (7-10 days) or short (48h) periods. Data analysis has unveiled that culturing MSCs on soft substrates for long periods promotes the expression of various proteins related to cell redox homeostasis, such as thioredoxins and peroxiredoxins. Conversely, culturing these cells during the same period but under low oxygen levels leads to an increase in chaperone machinery proteins, such as HSP90 or TRiC. These proteins can favor the clearance of misfolded proteins and telomerase maintenance processes, possibly preventing MSCs from being driven to a senescent phenotype. Although mechanomodulation and physioxia are two distinct stimuli, both converge in downregulating the expression of histones and several ribosomal subunits, possibly decreasing translational complexity, which could hypothetically favor a more naive phenotype for MSCs. Interestingly, priming UC-MSCs (48h) leads to a differential expression of proteins of the extracellular matrix and histone subtypes. Understanding the role of these proteins in transducing environmental cues might provide insights into how conventional culture conditions significantlyalter fundamental cellular processes and support the development of a more efficient protocol to expand and empower the therapeutic potential of MSCs. In the future, employing a combination of reduced stiffness and lower oxygen levels may present a promising strategic approach. HighlightsO_LICulturing MSCs on a soft substrate (3kPa) enhances the expression of antioxidant proteins, such as thioredoxins and peroxiredoxins C_LIO_LIProtein homeostasis is remodeled in MSCs cultured under physiological levels of oxygen (5% O2) through the differential expression of the chaperone machinery C_LIO_LILowering stiffness or oxygen levels during in vitro MSCs expansion decreases histones and ribosomal subunits expression, possibly favoring a more naive phenotype C_LI