(262b) Label-Free Optical Methods for Assessing Metabolic Changes during Human Pluripotent Stem Cell Differentiation.

Background: Human pluripotent stem cells (hPSCs) can be coaxed toward therapeutically relevant progeny utilizing signaling cues and relevant pathways but the accompanied changes in metabolism remain underappreciated. Even in the presence of ample oxygen¹, hPSCs rely on glycolysis similarly to cancer cells employing the Warburg eCect. However, stem cell specification is linked to a shift toward oxidative phosphorylation (OxPhos). Interestingly, the reverse shift from OxPhos to glycolysis is essential for the reprogramming of terminally differentiated cells² to a pluripotent phenotype.

Sugar catabolism and the TCA cycle are also linked to lipid metabolism whose role has been underappreciated in the context of hPSC stemness and commitment^3,4. De novo cellular fatty acid synthesis (FAS) starts with acetyl-coenzyme A (acetyl-CoA) as the substrate and requires NADH⁵. Conversely, fatty acid oxidation (FAO)⁶ leads to the breakdown of lipids producing acetyl-CoA in conjunction with the consumption of NAD and FAD, and the production of NADH and FADH2. Fatty acid utilization and intracellular acetyl- CoA change during diCerentiation of ESCs and FAO decreases while FAS is augmented when naïve hESCs transition to a primed state^7,8.

Results: The metabolic activities of hPSCs and hPSC-derived cells are typically gauged via biochemical assays which are lengthy, laborious, and require sample destruction, making challenging the timely assessment of the progression of hPSC specification. Label-free multiphoton microscopy (MPM) imaging provides unique sensitivity to detect metabolic changes that occur with cellular transformation⁹. In this study, we report the application of intracellular fluorescence-based methods to investigate the metabolic changes in hPSCs diCerentiating toward definitive endoderm (DE) cells, which are precursors to several cell types of the digestive tract including pancreatic and hepatic cells. Consistently, DE cells exhibited a mean optical redox ratio (representing the fluorescence intensity of NADH vs. that of FAD¹⁰) of 0.202±0.009 vs. 0.264±0.01 for their undiCerentiated counterparts (p<0.0005). This finding is contradictory to previous reports suggesting that the DE cells have a more glycolytic profile. However, hPSCs and DE cells were also analyzed via the radially- sampled power spectral density revealing a more fragmented network of mitochondria in hPSCs indicative of glycolysis. Quantification of the mitochondrial clustering revealed a beta value of 1.300±0.023 for hPSCs and 1.257±0.025 for DE cells (p=0.1). Employing fluorescence lifetime imaging (FLIM) uncovered diCerences in FAS/FAO which were further examined via biochemical assays.

Conclusions: The findings demonstrate the potential of label-free optical methods for appraising the metabolic state of human stem cells and pave the way for further development of relevant process analytical tools for the biomanufacturing of hPSC therapeutics.