abstract
Mesenchymal stem cells (MSCs) are promising tools in regenerative medicine, particularly in bone tissue engineering, owing to their ability to differentiate into osteogenic lineages and secrete bioactive factors essential for tissue repair. However, their potential is strongly influenced by donor-dependent variability. Proteomics offers a powerful strategy to elucidate osteogenic differentiation mechanisms, providing insights to guide MSC behavior toward improved commitment. While intracellular proteomic changes during osteodifferentiation are well documented, the exoproteome remains less characterized, despite its central role in matrix remodeling and intercellular signaling. Here, we characterized the exoproteome of human adipose-derived MSCs (hAMSCs) from three donors cultured under basal and osteogenic conditions for 21 days. Conditioned media were collected at days 0, 7, 14, and 21, and proteins analyzed by mass spectrometry.Proteomic profiling revealed inter-donor variability and a conserved set of exoproteins altered during osteogenic differentiation. These included proteins linked to cytoskeletal regulation (tubulins, talin-1), adhesion (CCN5, fibulin-5), extracellular matrix remodeling (progranulin, mimecan, clusterin) and bone-related pathways (periostin, osteonectin, prothrombin), reinforcing their potential as extracellular markers of lineage commitment. GO enrichment analysis showed significant overrepresentation of extracellular and collagen-containing matrix, as well as lysosomal/secretory pathways, reflecting active remodeling of the extracellular environment. Our findings support the exoproteome as a valuable window into the molecular dynamics of MSC osteogenic differentiation while revealing donor variability as a determinant of outcomes. The identification of conserved and donor-specific signatures provides a framework for defining early and late extracellular markers of differentiation, with implications for donor stratification, personalized regenerative medicine and biomaterial design.
authors
Rodrigues JEA; Maurício T; Graça I; Bispo DSC; Oliveira MB; Mano JF; Domingues P; Gil AM
our authors
Ana Maria Pissarra Coelho Gil
Associate Professor with Aggregation
Daniela Bispo
PhD Student
Inês C. R. Graça
Research Fellowship
João A. Rodrigues
Junior Researcher
João Mano
Full professor
Mariana Braga de Oliveira
Assistant ResearcherProjects
Metabolite-activated 3D stem cell differentiation into bone (BetterBone)
A Metabolomics-guided Bioreactor for Improved Engineered Bone Implants (BioImplant)
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
acknowledgements
BetterBone (2022.04286.PTDC, doi: 10.54499/2022.04286.PTDC) & BIOIMPLANT (PTDC/BTM-ORG/28835/2017), through COMPETE2020/FEDER (POCI-01-0145-FEDER-028835). CICECO-Aveiro Institute of Materials UIDB/50011/2020 (doi: 10.54499/UIDB/50011/2020), UIDP/50011/2020 (doi: 10.54499/UIDP/50011/2020) & LA/P/0006/2020 (doi: 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC). SFRH/BD/150655/2020 (doi: 10.54499/SFRH/BD/150655/2020, DSB PhD grant). Portuguese National NMR Network (Project Nº022161) through FEDER (COMPETE 2020/POCI/PORL/FCT (PIDDAC).