Stem cell footprinting during osteogenic differentiation

• Autor(a) principal: Correia, Marlene Cardoso
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/37152
• topic: Metabolomics; Metabonomics; Metabolic footprinting; Exometabolome; Nuclear magnetic resonance (NMR) spectroscopy; Stem cells (SCs); Mesenchymal stem cells (MSCs); Osteogenic differentiation

Metabolomics has been employed in stem cell research to unveil fundamental metabolic information about their characteristics and functions. In particular, metabolic footprinting studies the exometabolome of cells, providing information about the uptake and secretion of metabolites from culture media, and thus offering a non-destructive way to monitor cell metabolism over time. However, the study of intracellular metabolism has been favoured over the years, and there is a need to now articulate both types of information to characterize cell metabolism as a whole. This comprehensive approach was applied to the study of 2D osteogenic differentiation of human adipose-derived mesenchymal stem cells, using metabolomics by nuclear magnetic resonance (NMR) spectroscopy. Osteogenic media samples were collected over three independent experiments using distinct cell donors (to characterize donor variability), and control media samples of cells in proliferation were also used and compared (to identify markers of both proliferation and osteogenesis). A data correction was devised to account for the effect of media exchanges during periods of 21-28 days, thus allowing a continuous analysis of cell proliferation and differentiation over time. Metabolic features were identified for cell differentiation in relation to proliferation, for each donor independently; then donor variability was addressed. Cell proliferation metabolic changes reflected cell requirements for glucose and glutamine (for energy supply), and marked secretion of most amino acids, often after day 7 of cell culture. A set of donor-independent markers of cell proliferation were proposed to comprise glucose, glutamine, citrate, threonine and 3-hydroxybutyrate. In addition, osteogenic-specific metabolic features included less extensive amino acid secretion and marked lactate production. In particular, variations in isoleucine, glutamine and lactate could be proposed as osteogenic-specific donor-independent markers. A preliminary 3D cell culture experiment was performed, and results attested the need to define an adequate number of cells for metabolomic studies. As time did not allow, 2D ¹H NMR spectra of cell media culture were not explored in this work to confirm putative assignments of a few metabolites and to attempt to identify unknown peaks, which is a recognized limitation of this work.