Mass spectrometry imaging reveals early metabolic priming of cell lineage in differentiating human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine; however, few algorithms of quality control at the earliest stages of differentiation have been established. Despite lipids having known functions in cell signaling, their role in pluripotency maintenance and lineage specification is underexplored. We investigated changes in iPSC lipid profiles during initial loss of pluripotency over the course of spontaneous differentiation using co-registration of confocal microscopy and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. We identified lipids that are highly informative of the temporal stage of the differentiation and can reveal lineage bifurcation occurring metabolically. Several phosphatidylinositol species emerged from machine learning analysis as early metabolic markers of pluripotency loss, preceding changes in Oct4. Manipulation of phospholipids via PI 3-kinase inhibition during differentiation manifested in spatial reorganization of the colony and elevated expression of NCAM-1. In addition, continuous inhibition of phosphatidylethanolamine N-methyltransferase during differentiation resulted in increased pluripotency maintenance. Our machine learning analysis highlights the predictive power of metabolic metrics for evaluating lineage specification in the initial stages of spontaneous iPSC differentiation.