Lactate is a byproduct of cellular metabolism often viewed as a waste product with underappreciation of its potential functional effects. Recent advances are repositioning lactate as a versatile metabolite and fuel source that links glycolysis and oxidative phosphorylation1 (OXPHOS), mediates signaling as a ‘lactormone’, and partaking in post-translational and epigenetic regulation via lactylation2. Despite the glycolytic nature of the metabolism of hPSCs during their proliferation and the inevitable production of lactate, the specific effects of lactate on hPSC growth, physiology, and the stability of the pluripotent phenotype—remain underreported, warranting more detailed studies. In addition to the hPSCs’s pluripotent state, their differentiation propensity upon exposure to accumulating lactate is of great interest for stem cell bioprocessing. The metabolic shift from glycolysis to OXPHOS that hPSCs experience during the transition from naïve to specialized phenotypes3, and the reverse shift upon reprogramming of terminally differentiated cells4, underline the importance of lactate motivating further investigation of its role on stem cell physiology.
To test the effects of hPSC exposure to elevated lactate concentration, exogenous lactate was added to planar and suspension cultures at 2.5 and 5 g/L. These are concentrations observed during stirred suspension bioreactor cultivation5. At high lactate concentration, both human embryonic (H9 cells) and induced pluripotent (IMR90 clone 4 cells) stem cells experienced a significant reduction in their growth rate over 6 days: from (9.2 +/- 1.2) to (4.8 +/- 1.9) fold-increase for H9 cells and (14.1 +/- 2.7) to (7.6 +/- 1.7) fold-increase for IMR90 cells while maintaining viability over 90%. Yet, the expression of the pluripotency markers NANOG and OCT4 remained unchanged (over 97% of cells were NANOG and OCT4 positive) for both cell lines and exhibited no significant difference when compared to our control (no lactate added). Moreover, the propensity of the cells to differentiate toward endoderm, ectoderm and mesoderm progeny was determined following propagation with or without exogenous lactate. A downward trend was noted in the expression of germ-layer specific markers though the overall specification was unhindered. To obtain a more complete picture of the effects and potential changes, cells were cultivated in the presence of lactate over multiple passages and modulations in the global transcriptome were examined via RNA sequencing.
Lastly, we discovered that intracellular lactate levels are analogously varied with increasing glucose levels in culture as well as with the addition of chemicals such as oxamate and rotenone and demonstrated a correspondingly elevated lactylation of lysine residues in histone proteins. Exposing hPSCs to rotenone (100 μΜ) and oxamate (50 mM) for 8h resulted in significantly increased (p*< 0.05) and decreased (p**< 0.01) intracellular lactate levels respectively when compared to our control. Addition of exogenous lactate is also reflected intracellularly resulting in even higher histone lactylation levels.
This study demonstrates that lactate –a ubiquitous product of mammalian cell metabolism of the highly proliferative and glycolytic hPSCs– negatively impacts growth in high concentrations without significantly impairing pluripotency and differentiation capacity while serving as a substrate for the newly discovered process of lactylation.
Acknowledgment: The research was partially supported by the National Science Foundation (NSF) grant CBET-2326510.
References
1 Brooks, G. A. The Science and Translation of Lactate Shuttle Theory. Cell Metab 27, 757-785, doi:10.1016/j.cmet.2018.03.008 (2018).
2 Zhang, D. et al. Metabolic regulation of gene expression by histone lactylation. Nature 574, 575-580, doi:10.1038/s41586-019-1678-1 (2019).
3 Cliff, T. S. & Dalton, S. Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development. Curr Opin Genet Dev 46, 44-49, doi:10.1016/j.gde.2017.06.008 (2017).
4 Folmes, C. D. et al. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metab 14, 264-271, doi:10.1016/j.cmet.2011.06.011 (2011).
5 Jacobson, E. F. et al. Non-xenogeneic expansion and definitive endoderm differentiation of human pluripotent stem cells in an automated bioreactor. Biotechnol Bioeng 118, 979-991, doi:10.1002/bit.27629 (2021).