291 THE USE OF THE DYNAMIC IMPACT APPROACH AND DESORPTION ELECTROSPRAY IONIZATION–MASS SPECTROSCOPY TO ANALYZE ADIPOGENESIS IN PORCINE ADIPOSE-DERIVED STEM CELLS

Abstract

The measurement of end products of metabolism in combination with the dynamic measurement of the transcriptome can be a valuable approach to study complex processes, such as adipogenesis. For this purpose, we have combined the analysis of metabolic pathways using transcriptomics data with in situ lipid imaging obtained by desorption electrospray ionization–mass spectroscopy (DESI-MS). The transcriptome data was obtained from a previous experiment where adipose-derived stem cells (ASC) from 3 individual pigs were induced to differentiate into adipocytes for 21 days. The metabolic pathway analysis of transcriptomics data was performed using the dynamic impact approach (DIA) to determine the directional impact of pathways and gene categories. For the DESI-MS, porcine ASC were induced towards adipogenic differentiation (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33) for 2 weeks in 24-well plates over round coverslips. Direct lipid profile of fatty acids and phospholipids in 3 different locations in undifferentiated and differentiated ASC was performed. Relative values of ion counts in each location were used for statistical analysis by means of a two-tailed t-test with unequal variance and false discovery rate (FDR) correction. The DIA uncovered amino acid and lipid metabolism as being the most affected pathways during adipogenesis. Among lipid-related pathways, the DIA indicated a strong induction during differentiation of unsaturation of fatty acids (FA) and metabolism of lipids, particularly triacylglycerol formation (TAG). The metabolism of tryptophan, phenylalanine, and histidine was strongly induced among amino acid-related metabolism. Among the lipid molecules identified in the mass-to-charge ratio (m/z) range of 250–1000 by the DESI-MS, >200 had significantly (FDR <0.01) lower abundance and ~30 lipids had higher abundance in differentiated compared with undifferentiated ASC. The lipid profile of undifferentiated cells was featured by a large amount of lipid molecules in the lower (m/z 780) mass range but with almost no signal in the intermediate range. The differentiated cells had a consistent large increase in amount of molecules in the range from m/z 500 to 650 and a decreased amount in the lower and upper ranges. Among the lipid molecules that decreased during differentiation, we identified several phospholipids, particularly phosphoserine and phosphoinositol species, but also linoleic, linolenic, arachidic, and eicosapentaenoic acids. The unsaturated FA oleate and docosahexaenate increased >10-fold and 2.2-fold, respectively, due to adipogenic differentiation. High-resolution experiments are being performed in order to annotate more, if not all, lipid species identified in the samples by DESI-MS. The DIA indicated greater unsaturation of FA during adipogenesis, confirmed by DESI-MS, which corresponds to the characteristic increase in lipid fractions with intermediate molecular mass. This can be considered a signature of adipogenesis in porcine ASC and essential for TAG formation. The DESI-MS appears to be a valuable tool for rapid lipid profile of cells and with the potential to be used to identify cell differentiation.