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 the changes in iPSC lipid profiles during the initial loss of pluripotency over the course of spontaneous differentiation using the co-registration of confocal microscopy and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. We identified phosphatidylethanolamine (PE) and phosphatidylinositol (PI) species that are highly informative of the temporal stage of differentiation and can reveal iPS cell lineage bifurcation occurring metabolically. Several PI species emerged from the machine learning analysis of MS data as the early metabolic markers of pluripotency loss, preceding changes in the pluripotency transcription factor Oct4. The manipulation of phospholipids via PI 3-kinase inhibition during differentiation manifested in the spatial reorganization of the iPS cell colony and elevated expression of NCAM-1. In addition, the continuous inhibition of phosphatidylethanolamine N-methyltransferase during differentiation resulted in the enhanced maintenance of pluripotency. Our machine learning analysis highlights the predictive power of lipidomic metrics for evaluating the early lineage specification in the initial stages of spontaneous iPSC differentiation.

Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch-to-batch variability, presenting challenges in current cell manufacturing contexts. Real-time, label-free control quality attributes (CQAs) are required to ensure efficient iPSC-derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC-derived CM one step closer to clinical use.

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.