Determining the role of lactate in induced pluripotent stem cell metabolism

Induced pluripotent stem (iPS) cells hold the potential to dramatically improve cell-based therapies and in vitro drug screening applications in the near future. Yet, for iPS cells to have a clinical impact, these cells must be generated in sufficient quantity and quality that currently exceeds today’s capabilities. To meet these cell needs, a comprehensive understanding of how environmental conditions affect iPS cell metabolism and pluripotency is essential. Rapidly proliferating cells, including cancer and iPS cells, catabolize glucose and secrete lactate at elevated rates, even in the presence of sufficient oxygen, a process referred to as the Warburg effect (Vander Heiden, Cantley, Thompson 2009; Varum et al. 2011; WARBURG 1956). In cancer cell metabolism, lactate accumulation is associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients (Martinez-Outschoorn et al. 2011). In addition, lactate has previously been shown to stabilize hypoxia inducible factors and induce a hypoxic response for cells cultured in normoxic environments (Pérez-Escuredo et al. 2016). However, there remains an incomplete understanding of the metabolic role of lactate for iPS cells and its effects on pluripotency. This study examined the impact of extracellular lactate on cellular metabolism and pluripotency of iPS K3 cells grown with sufficient glucose. Extracellular glucose, lactate, and amino acid concentrations were monitored throughout the experiment to determine the extracellular consumption or production fluxes. In addition, [1,2-13C] glucose, [U-13C] glutamine, and [U-13C] lactate isotope tracers were used in parallel labeling experiments to determine the intracellular metabolic contribution of each carbon source to iPS cell metabolism. High extracellular lactate resulted in altered cell metabolism, including a decrease in lactate production and glucose consumption. This was coupled with a decrease in glucose contribution to the TCA cycle. Also, lactate was catabolized to pyruvate, alanine, and TCA intermediate metabolites in the high-lactate condition. Furthermore, high extracellular lactate did not affect iPS cell pluripotency. These results suggest that lactate partially serves as a metabolic substrate for iPS even as it continues to accumulate in the extracellular media. The implications of these findings towards understanding iPS cell metabolism and improving future cell culture conditions will be discussed. Martinez-Outschoorn UE, Prisco M, Ertel A, Tsirigos A, Lin Z, Pavlides S, Wang C, Flomenberg N, Knudsen ES, Howell A. 2011. Ketones and lactate increase cancer cell “stemness,” driving recurrence, metastasis and poor clinical outcome in breast cancer: Achieving personalized medicine via metabolo-genomics. Cell Cycle 10(8):1271-86. Pérez-Escuredo J, Dadhich RK, Dhup S, Cacace A, Van Hée VF, De Saedeleer CJ, Sboarina M, Rodriguez F, Fontenille M, Brisson L. 2016. Lactate promotes glutamine uptake and metabolism in oxidative cancer cells. Cell Cycle 15(1):72-83. Vander Heiden MG, Cantley LC, Thompson CB. 2009. Understanding the warburg effect: The metabolic requirements of cell proliferation. Science 324(5930):1029-33. Varum S, Rodrigues AS, Moura MB, Momcilovic O, Easley IV CA, Ramalho-Santos J, Van Houten B, Schatten G. 2011. Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PloS One 6(6):e20914. WARBURG O. 1956. On the origin of cancer cells. Science 123(3191):309-14

Two small-scale perfusion models for the ambr250 to enable the study of production stability

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Effect of lactate media concentration on induced pluripotent stem cell proliferation and metabolism

Induced pluripotent stem (iPS) cells hold the potential to drastically improve cell-based therapies in the near future. Yet, in order for stem cell therapies to become clinically feasible, these cells must be generated in sufficient quantity and quality. Rapidly proliferating cells, including cancer and iPS cells, consume glucose and secrete lactate at high rates, even in the presence of sufficient oxygen; this process is called the Warburg effect (Vander Heiden, Cantley, Thompson 2009; Varum et al. 2011; WARBURG 1956) . In cancer cell metabolism, lactate accumulation is associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis (Martinez-Outschoorn et al. 2011) . Yet, there remains an incomplete understanding of the role of lactate in stem cell metabolism and pluripotency. The objective of this study was to determine the impact of lactate on stem cell metabolism and pluripotency. Metabolic responses to high and low extracellular lactate concentrations were examined in iPS K3 cells, where these responses included metabolic activity and pluripotency. Specifically, the respective extracellular consumption and production fluxes for glucose, lactate, and amino acids were determined. Growth rates were controlled to not be different between the high and low lactate cultures, which facilitated normalization of the extracellular fluxes. The high extracellular lactate concentrations resulted in a shift in cell metabolism, including a slight decrease in lactate production and glucose consumption fluxes. The high extracellular lactate concentrations also caused significant decreases in pyruvate and glutamine consumption fluxes. These altered fluxes due to the high extracellular lactate concentrations suggest decreased metabolic activity through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Consequently, it is likely that under high extracellular lactate the mitochondrial activity is also lower, a metabolic characteristic of pluripotent stem cells (Folmes et al. 2011) . The impact of high extracellular lactate on iPS cell metabolism and pluripotency will be discussed. The implications of these findings towards understanding iPS cell metabolism and designing large-scale cell culture conditions to limit lactate accumulation will be discussed

Investigating the reverse Warburg effect: How high extracellular lactate alters breast cancer metabolism

Metabolism plays a critical role in the progression of cancer malignancy, with nutrient source flexibility allowing cancer cells to outcompete normal cells and survive harsh environmental conditions. Lactate accumulation in poorly vascularized tumors has predominately been considered a waste product resulting from the Warburg effect; however, recent studies have shown that lactate can additionally serve as a metabolic substrate for some cancer cells, a process referred to as the Reverse Warburg effect. Lactate accumulation is also associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients. Unfortunately, little is known about the role of lactate in cancer cell metabolism and progression. This study examined the impact of extracellular lactate on the metabolic activity of normal breast cells (MCF-10A), early stage breast cancer cells (MCF-7), and advanced, malignant breast cancer cells (MDA-MB-231) by utilizing steady-state 13C-metabolic flux analysis. To quantify individual metabolic pathway activity, metabolic flux maps were constructed for each cell line for both control and high lactate conditions. Cellular composition studies were performed to determine the effect of high lactate on cellular amino acid composition and lipid content. The high lactate condition resulted in altered cell metabolism for each of the cell lines, including a decrease in glycolytic flux, decreased net lactate production and an increase in reductive carboxylation of glutamine. Reductive carboxylation has previously been observed to increase under hypoxia; but lactate’s effect on reductive carboxylation has not been previously reported. The implications of these findings towards understanding cancer metabolism flexibility and its impact on the tumor microenvironment will be discussed

Utilizing RNA-Seq technique to improve molecular understanding of Chinese Hamster ovary (CHO) cell bioprocessing

Chinese Hamster Ovary (CHO) cells are an important biopharmaceutical cell line, accounting for the production of over 70% of the approved protein therapeutics. However several limitations exist with the use of CHO cell lines including low product titers. Understanding of CHO cells in bioprocessing has up until now relied heavily on empirical results with a limited knowledge of the intracellular dynamics. With the recent establishment of both Chinese hamster and CHO-K1 cell line genome assemblies, it is now possible to leverage the genomic resources to better understand and further improve CHO cell bioprocessing. In this study, RNA-Seq, next-generation transcriptome sequencing, was used to characterize the gene expression profile of three different CHO cell lines under several industrially relevant conditions including low culture temperature and pH. Each culture condition sample was sequenced by HiSeq 2000 and contained over 15 million short reads, which were assembled using the Chinese hamster reference genome (v1.01). Differential gene expression between conditions was statistically quantified based on generalized linear models using edgeR software for the replicate samples. One of the applications of the RNA-Seq analysis method, in this study, was to observe and understand the impact of low culture temperature on CHO gene expression behavior. In a CHO-K1 cell line adapted to protein-free medium, the cultures grown at 33°C had higher expression of 251 genes and lower expression of 15 genes (filter criteria were a minimum of two-fold expression change and FDR ≤ 0.05) compared to cultures grown at 37°C. These genes could be utilized as potential targets for cellular and metabolic engineering to further improve CHO cell lines. The current investigation presents the potential of next-generation sequencing techniques for advanced characterization of CHO cell bioprocessing

High Gravity Fermentation of Sugarcane Bagasse Hydrolysate by \u3ci\u3eSaccharomyces pastorianus\u3c/i\u3e to Produce Economically Distillable Ethanol Concentrations: Necessity of Medium Components Examined

A major economic obstacle in lignocellulosic ethanol production is the low sugar concentrations in the hydrolysate and subsequent fermentation to economically distillable ethanol concentrations. We have previously demonstrated a two-stage fermentation process that recycles xylose with xylose isomerase to increase ethanol productivity, where the low sugar concentrations in the hydrolysate limit the final ethanol concentrations. In this study, three approaches are combined to increase ethanol concentrations. First, the medium-additive requirements were investigated to reduce ethanol dilution. Second, methods to increase the sugar concentrations in the sugarcane bagasse hydrolysate were undertaken. Third, the two-stage fermentation process was recharacterized with high gravity hydrolysate. It was determined that phosphate and magnesium sulfate are essential to the ethanol fermentation. Additionally, the Escherichia coli extract and xylose isomerase additions were shown to significantly increase ethanol productivity. Finally, the fermentation on hydrolysate had only slightly lower productivity than the reagent-grade sugar fermentation; however, both fermentations had similar final ethanol concentrations. The present work demonstrates the capability to produce ethanol from high gravity sugarcane bagasse hydrolysate using Saccharomyces pastorianus with low yeast inoculum in minimal medium. Moreover, ethanol productivities were on par with pilot-scale commercial starch-based facilities, even when the yeast biomass production stage was included

Impact of high extracellular lactate on induced pluripotent stem cell metabolism and pluripotency

Induced pluripotent stem (iPS) cells hold the potential to drastically improve cell-based therapies in the near future. However, in order for stem cell therapies to become clinically feasible, these cells must be generated in sufficient quantity and quality. This aim will require a comprehensive understanding of how environmental conditions affect iPS cell metabolism and pluripotency. Rapidly proliferating cells, including cancer and iPS cells, consume glucose and secrete lactate at high rates, even in the presence of sufficient oxygen, a process referred to as the Warburg effect. In cancer cell metabolism, lactate accumulation is associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients. Elevated lactate conditions have also been shown to preferentially cause iPS cells to differentiate into cardiomyocytes in glucose-deficient media. Yet, there remains an incomplete understanding of the role of lactate in stem cell metabolism and pluripotency in glucose containing media. This study examined the impact of extracellular lactate on the metabolic activity and pluripotency of iPS K3 cells grown with sufficient glucose. Extracellular glucose, lactate, and amino acid concentrations were monitored throughout the experiment to determine the extracellular consumption or production fluxes. High extracellular lactate resulted in altered cell metabolism, including decreased lactate production while glucose consumption remaining unchanged. These results support hypotheses that there is a possible redistribution of carbons within metabolism under high extracellular lactate, with a larger portion of carbon entering the tricarboxylic acid cycle. The implications of these findings towards understanding iPS cell metabolism and designing cell culture conditions to limit lactate accumulation will be discussed

Metabolic Regulatory Network Kinetic Modeling with Multiple Isotopic Tracers for iPSCs

The rapidly expanding market for regenerative medicines and cell therapies highlights the need to advance the understanding of cellular metabolisms and improve the prediction of cultivation production process for human induced pluripotent stem cells (iPSCs). In this paper, a metabolic kinetic model was developed to characterize underlying mechanisms of iPSC culture process, which can predict cell response to environmental perturbation and support process control. This model focuses on the central carbon metabolic network, including glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and amino acid metabolism, which plays a crucial role to support iPSC proliferation. Heterogeneous measures of extracellular metabolites and multiple isotopic tracers collected under multiple conditions were used to learn metabolic regulatory mechanisms. Systematic cross-validation confirmed the model's performance in terms of providing reliable predictions on cellular metabolism and culture process dynamics under various culture conditions. Thus, the developed mechanistic kinetic model can support process control strategies to strategically select optimal cell culture conditions at different times, ensure cell product functionality, and facilitate large-scale manufacturing of regenerative medicines and cell therapies.Comment: 26 pages, 16 figure

Using next-generation sequencing technology, RNA-Seq, to understand the Chinese Hamster Ovary (CHO) cell transcriptome under industrially relevant conditions

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Stochastic Biological System-of-Systems Modelling for iPSC Culture

Large-scale manufacturing of induced pluripotent stem cells (iPSCs) is essential for cell therapies and regenerative medicines. Yet, iPSCs form large cell aggregates in suspension bioreactors, resulting in insufficient nutrient supply and extra metabolic waste build-up for the cells located at core. Since subtle changes in micro-environment can lead to cell stress and heterogeneous cell population, a novel Biological System-of-Systems (Bio-SoS) framework is proposed to characterize cell-to-cell interactions, spatial heterogeneity, and cell response to micro-environmental variation. Building on stochastic metabolic reaction network, aggregation kinetics, and reaction-diffusion mechanisms, the Bio-SoS model can quantify the impact of factors (i.e., aggregate size) on cell product health and quality heterogeneity, accounting for causal interdependencies at individual cell, aggregate, and cell population levels. This framework can accurately predict iPSC culture conditions for both monolayer and aggregate cultures, where these predictions can be leveraged to ensure the control of culture processes for successful cell growth and expansion.Comment: 36 pages, 10 figure