Background: Human Amniotic Mesenchymal Stromal Cells (hAMSCs) secrete a complex mixture of bioactive factors—collectively known as the secretome—that promote tissue repair and regeneration, primarily through immunomodulation and resolution of inflammation. While the therapeutic potential of the hAMSC secretome is increasingly recognized, the biochemical identity of its active components remains incompletely defined. Integrated multi-omics approaches, including metabolomics and lipidomics, offer a powerful strategy for characterizing the soluble factors released by hAMSCs. 

Method: Conditioned media (CM) were collected from hAMSCs cultured in DMEM/F12 (n=12, all with anti-proliferative effects on activated PBMC) and UltraCULTURE™ (n=5, with variable anti-proliferative profiles), then lyophilized. Their anti-proliferative activity was assessed using anti-CD3 activated PBMCs. Lipidomic and metabolomic profiling was conducted using the Folch extraction method, followed by UHPLC-QTOF/MS Ion Mobility analysis of the organic phase and NMR analysis of the hydrophilic phase. Lipid species were classified with Lipid Annotator software. The biological activity of selected lipid species identified through lipidomic analysis was subsequently validated by testing their effects on activated PBMCs. 

Results: Principal Component Analysis (PCA) of UltraCULTURE™-derived CM revealed a clear separation between non-immunomodulatory and active samples. Cross-comparison of both media types identified shared lipid classes—phosphatidylcholines (PCs), sphingomyelins (SMs), and ceramides—implicated in inflammation regulation. Among these, three specific lipids—SM(d34:1), PC(34:1), and PC(32:0)—were selected for functional validation. When tested individually on anti-CD3-activated PBMCs, only SM(d34:1) exhibited a significant antiproliferative effect, suggesting it may be a key active component of the hAMSC secretome. 

Conclusion: This study highlights SM(d34:1) as a potential contributor to the immunomodulatory properties of the hAMSC secretome. These findings lay the groundwork for further validation and mechanistic studies aimed at elucidating the role of specific metabolites in hAMSC-mediated immune regulation.