abstract
Mesenchymal stem cells (MSC) are essential for tissue regeneration, although their biological variability (e.g. interdonor differences) limits their therapeutic potential. New strategies to accurately monitor/predict MSC osteogenic capacity are needed for selecting the most suitable donors/cells. Here, intra- and extracellular metabolic variations accompanying human adipose-derived MSC (hAMSC) osteodifferentiation are characterized for 3 donors over 21 days using untargeted nuclear magnetic resonance (NMR) metabolomics. A donor-independent 9-endometabolite signature (choline, ethanolamine, UDP-GlcNAc, UDP-GalNAc, methylguanidine, phosphocreatine, phosphocholine, ADP and one unknown) predicts osteodifferentiation from day 7, with > 95% sensitivity, specificity, and classification rates, achieving 100% predictive scores by day 14. In addition, osteogenic-specific donor-independent patterns are identified in 17 exometabolites: i) reduced uptake of glutamine, branched-chain amino acids, 2-hydroxyisobutyrate, pyruvate and glucose; ii) increased secretion of 3-hydroxybutyrate, ornithine, pyroglutamate and lactate; iii) decreased secretion of 3-hydroxyisobutyrate, _-ketoglutarate and citrate; and iv) consistent final levels of alanine, choline and histidine (regardless of their variations). This 17-exometabolite signature enables non-invasive detection of osteodifferentiation with 100% sensitivity, specificity, and classification after day 4. These findings show that differentiating cells efficiently manage metabolic resources by reconfiguring key pathways, e.g. partially shifting from glycolysis/OxPhos to the PCr-Cr axis for ATP and phosphate production, activating antioxidative mechanisms, and adapting protein glycosylation and membrane metabolism. These donor-independent signatures constitute a robust tool to accurately assess/predict osteogenic potential, enabling effective and early MSC donor selection.
authors
Daniela S.C. Bispo, João A. Rodrigues, Inês Graça, Catarina S.H. Jesus, Marlene Correia, Iola F. Duarte, Brian J. Goodfellow, Mariana B. Oliveira, João F. Mano, Ana M. Gil
our authors
Ana Maria Pissarra Coelho Gil
Associate Professor with Aggregation
Brian James Goodfellow
Assistant Professor
Catarina Sofia Henriques de Jesus
Junior Researcher
Daniela Bispo
PhD Student
Inês C. R. Graça
Research Fellowship
Iola Melissa Fernandes Duarte
Principal Researcher
João A. Rodrigues
Junior Researcher
João Mano
Full professor
Mariana Braga de Oliveira
Assistant Researcher
Marlene Correia
PhD StudentProjects
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)
Metabolite-activated 3D stem cell differentiation into bone (BetterBone)
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).