Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation

Abstract

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

An Intracellular Metabolic Signature as a Potential Donor-Independent Marker of the Osteogenic Differentiation of Adipose Tissue Mesenchymal Stem Cells

“…For technical reasons, only the osteogenesis assay was performed for donor 3, while a slightly different protocol was followed (namely, different extraction solvent volumes [ 36 ], passage 7 compared to 5 and 6 for donors a and 2) and cell proliferation could not be adequately assessed. The cryopreserved hAMSCs from each donor were thawed, plated in culture flasks (T175), expanded and passaged as described before [ 36 , 37 ].…”

“…The medium was exchanged twice a week for 21 days. On days 0, 1, 4, 7, 14, and 21, cells were trypsinized (as previously described [ 37 ]) and collected in triplicate (specifically for donor 1, samples were also collected for day 28). For donor 2, two of the three samples of day 21 were discarded due to microbial contamination.…”

“…pNP absorbance at 450 nm was measured (microplate reader, Synergie HTX, BioTek Instruments, Winooski, VT, USA) and ALP activity was calculated using a pNP calibration curve (0.0−0.5 mM). For calcium quantification and determination of osteocalcin (OCN, a late marker of osteogenic differentiation [ 51 ]) protocols were followed as described previously [ 36 , 37 ]. ALP activity, calcium concentrations and OCN secretion were normalized by total dsDNA.…”

“…To the best of our knowledge, only a few metabolomic studies of MSCs heterogeneity have been carried out [ 23 , 27 , 28 , 29 , 30 ], employing both targeted and untargeted approaches and mostly using mass spectrometry (MS)-based methods. Metabolomics may be carried out using either MS or nuclear magnetic resonance (NMR) spectroscopy, techniques which provide complementary information [ 31 ] and which have proved valuable in determining the detailed metabolic adaptations during cell differentiation [ 32 , 33 ], for instance, to find metabolites with inductive roles in differentiation [ 34 , 35 , 36 , 37 ] or aid in the comparison between different MSC donors [ 23 , 28 , 29 , 30 ]. For example, an MS-based untargeted study unveiled that human adipose tissue MSCs (hAMSCs) from obese individuals secreted higher amounts of metabolites associated with glycolysis, tricarboxylic acid cycle (TCA) cycle, pentose phosphate pathway (PPP) and polyol pathway while showing decreased proliferation, migration and differentiation abilities [ 23 ].…”

“…To achieve this, the detailed polar endometabolome adaptations of hAMSCs harvested from two randomly chosen donors, found to differ in proliferating and differentiation extensions, were measured at several timepoints throughout 21 days, under osteogenic and basal (control) conditions. The detailed and holistic nature of NMR metabolomics [ 33 , 36 , 37 ] enabled different families of hydrophilic metabolites to be identified simultaneously and their time course changes to be determined. The metabolite trajectories observed in common for the two donors, either descriptive of cell proliferation or of osteogenic differentiation, were proposed as potential donor-independent markers of each biological process.…”