Geraldo, M. | bioRxiv Science

bioRxiv2024

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 Whartons jelly niche are accommodated to the stiffness and oxygen level found at the umbilical cord (UC), which are 2 to 5kPa (Youngs modulus) and 2.4% to 3.8% O2, respectively. However in vitro culture conditions (2-3 GPa and 18.5% O2) 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% O2) (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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/609692v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16350corg.highwire.dtl.DTLVardef@1f4c18borg.highwire.dtl.DTLVardef@1ce9c0aorg.highwire.dtl.DTLVardef@1666d0b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

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

cell biology↗