NMR Metabolomics Assessment of Osteogenic Differentiation of Adipose-Tissue-Derived Mesenchymal Stem Cells

Abstract

This Article presents, for the first time to our knowledge, an untargeted nuclear magnetic resonance (NMR) metabolomic characterization of the polar intracellular metabolic adaptations of human adipose-derived mesenchymal stem cells during osteogenic differentiation. The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts, and untargeted metabolomics may unveil novel metabolic information on the osteogenic differentiation of MSCs, allowing their behavior to be understood and monitored/guided toward effective therapies. Our results unveiled statistically relevant changes in the levels of just over 30 identified metabolites, illustrating a highly dynamic process with significant variations throughout the whole 21-day period of osteogenic differentiation, mainly involving amino acid metabolism and protein synthesis; energy metabolism and the roles of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation; cell membrane metabolism; nucleotide metabolism (including the specific involvement of O-glycosylation intermediates and NAD + ); and metabolic players in protective antioxidative mechanisms (such as glutathione and specific amino acids). Different metabolic stages are proposed and are supported by putative biochemical explanations for the metabolite changes observed. This work lays the groundwork for the use of untargeted NMR metabolomics to find potential metabolic markers of osteogenic differentiation efficacy.

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

“…The hAMSCs from donor 3 (a 52-year-old Caucasian female, Lot 63557082) were obtained from a liposuction procedure and purchased from ATCC. 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 ].…”

“…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.…”

1H NMR Metabolite Monitoring during the Differentiation of Human Induced Pluripotent Stem Cells Provides New Insights into the Molecular Events That Regulate Embryonic Chondrogenesis

“…Employing 1 H NMR metabolomics affords a semi-targeted assay whereby the simultaneous measurement of multiple metabolites within the 1 H metabolome can be monitored during biological changes [ 41 , 42 ]. The combination of metabolite fingerprinting (intracellular) and footprinting (extracellular/secreted) enables a holistic study of cell function so that we can tease apart responses to highly complex stimuli, such as those that occur during embryogenesis and the differentiation of pluripotent stem cells [ 43 , 44 , 45 ].…”