(317a) Lactate Inhibits the Growth of Human Pluripotent Stem Cells without Affecting Pluripotency

Introduction: Advances in human pluripotent stem cell (hPSC) biology have intensified efforts for the development of cellular therapies against a gamut of currently incurable diseases. Bioprocess for the generation of stem cell-derived therapeutics entails two stages: hPSC expansion with preservation of their competence for downstream differentiation and directed specification to the cell or tissue type of interest [1]. The metabolism of hPSCs under propagative mode is glycolytic resulting in the production of excess lactate. Lactate is known to inhibit the growth of mammalian cells commonly employed in biopharmaceutical processes [2]. This metabolic product also induces signaling activity in highly proliferative tumor cells [3]. Nonetheless, the effects of lactate on the growth and pluripotent state of hPSCs, particularly in the context of biomanufacturing of cell therapeutics, remain understudied. To this end, results are presented from recent work combining planar and stirred suspension hPSC cultivation with biochemical and molecular analyses.

Materials & Methods: Lactate feed experiments were performed to establish if hPSC culture performance is affected by elevated lactate over 6-day passages in line with the lactate buildup observed in bioreactor cultures. The H9 human embryonic stem cells (hESCs) and iPSC(IMR90)-clone 4 (IMR90) human induced pluripotent stem cells (hiPSCs) were used in these studies. Experimental endpoints were subjected to qPCR, immunostaining, flow cytometry, and western blotting for relative expression of pluripotency genes (e.g., NANOG, OCT4) as well as genes relevant to lactate transport and hPSC metabolic activity (e.g., monocarboxylate transporters (MCTs), uncoupling protein 2 (UCP2)). Pluripotency was also assessed by directed differentiation of hPSCs to the three germ layers.

Results/Discussion: H9 hESCs and IMR90 hiPSCs in 5 g/L of exogenous lactate exhibited an 11.5- and 11.7-fold increase, respectively, compared to 21.4- and 22.4-fold for the control groups with no additional lactate over 6 days while viability was maintained over 85% for all conditions. Moreover, hPSC aggregates cultured at elevated lactate displayed cyst-like protrusions and membrane blebbing, an attribute we have observed to be a negative prognosticator of successful directed differentiation. Lactate exchange between cells and their environment is mediated primarily by MCTs 1 – 4, which are associated with the transfer of ketone bodies and lactic acid in and out of the cells[4]. MCT-1 and -2 are responsible for shuttling lactate inside the cells while MCT3 and MCT4 facilitate its removal. The expression of MCT1 and MCT3 was lower in both cell lines cultured with added lactate. Immunostaining and western blotting did not reveal appreciable changes in MCT3 among the control and 5 g/L lactate groups. Results for the expression of other MCTs were hPSC-line specific. For example, MCT2 and MCT4 increased in H9 hESCs, but the opposite was true in IMR90 hiPSCs. Moreover, we observed the behavior of the uncoupling protein 2 (UCP2), which is known to remove four-carbon substrates from the mitochondria, thereby reducing the tricarboxylic acid (TCA) cycle activity and the production of ATP. Literature reports UCP2 regression to coincide with the metabolic shift from glycolysis to oxidative phosphorylation when hPSCs are differentiated Additionally, pluripotency genes such as NANOG and OCT4 remained unchanged and there was even upregulation of NANOG in the presence of lactate. The maintenance of the pluripotent state was further assessed by subjecting cultured hPSCs to directed differentiation toward endoderm, ectoderm and mesoderm. Acknowledging that these results pertain to the short-term effects that lactate may have on hPSCs, ongoing experiments are aiming to investigate global transcriptomic changes inflicted over a series of passages via RNA sequencing.

Conclusion: Our study shows that lactate, a common metabolic product in mammalian cell bioprocessing, negatively impacts the growth of hPSCs in culture. Lactate also induces changes in the transport and metabolic networks of hPSCs. Elucidating the cascade of molecular events that lactate accumulation imposes on cells, will inform strategies for lactate control in hPSC cultures, efficient expansion, and robust directed differentiation to therapeutically useful cell types.

References

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