Metabolomic and Lipidomic Profiling of the Amniotic Stromal Cell Secretome
Giulia Tolle1, Samuele Cheri2, Pietro Romele4, Roberta Pisu2, Ornella Parolini3,5, Paola Scano1, Pierluigi Caboni1, Fabio Marongiu2.
1Life and environmental sciences, Università degli Studi di Cagliari, Cagliari, Italy; 2Biomedical sciences, Università degli Studi di Cagliari, Cagliari, Italy; 3Life Science and Public Health, Università Cattolica del Sacro Cuore, Roma, Italy; 4Centro di Ricerca E.Menni; Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy; 5Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy
Introduction: It is increasingly more evident that the bioactive factors produced by human Amniotic Mesenchymal Stromal Cells (hAMSCs) (i.e. secretome) can favor tissue repair and regeneration through the modulation of the immune system and the resolution of inflammation. To date, the biochemical nature of these factors has yet to be defined. The integration of several -omics technologies (e.g. proteomics, genomics, transcriptomics, lipidomics and metabolomics) can enable a multiapproach profiling of the hAMSC secretome.
This study focuses on the metabolomic and lipidomic characterization of the soluble factors produced and released by hAMSCs.
Method: Conditioned media (CM) were obtained from hAMSC cultures and lyophilized upon harvest. Their immunomodulatory effect was validated on activated PBMCs. For these studies, the following CM samples were analyzed: 12 from hAMSCs grown in DMEM/F12 (all with anti-inflammatory effect) as well as 5 from hAMSCs grown in UltraCULTURE™ (with varying immunomodulatory effects). Samples were processed by standard Folch method, and the organic phase was then reconstituted for LCQTOF-MS Ion Mobility analysis, while the hydrophilic phase was analyzed by NMR. In order to obtain a classification of the lipid components, Ion Mobility data were processed with Lipid Annotator software.
Results: NMR analysis revealed the presence, in both media, of lactic acid, alanine, glutamic acid and proline. Importantly, PCA analysis of UltraCULTURE™-based CM revealed clear separation between samples that had no effect vs. ones that had mild or strong immunomodulatory activity.
Direct comparison between DMEM/F12- and UltraCULTURE™-based CM, allowed to identify shared metabolites belonging to the lipidic classes of phosphatidylcholines, sphingomyelins and ceramides which are known for their role in regulating the inflammatory process. Relative abundance of these metabolites in the UltraCULTURE™-based CM strongly correlated with their biological activity.
Conclusion: These preliminary results, allowed to identify a number of molecules that might have a possible direct role in the immunomodulatory activity of hAMSCs. Future studies will allow to validate this hypothesis and to directly test the specific effect of individual metabolites.
