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Enzymatic Passaging of Human Embryonic Stem Cells Alters Central Carbon Metabolism and Glycan Abundance

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Enzymatic Passaging of Human Embryonic Stem Cells Alters Central Carbon Metabolism and Glycan Abundance UC San Diego UC San Diego Previously Published Works Title Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance Permalink https://escholarship.org/uc/item/1x27t1sx Journal Biotechnology Journal, 10(10) ISSN 1860-6768 Authors Badur, MG Zhang, H Metallo, CM Publication Date 2015-10-01 DOI 10.1002/biot.201400749 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Biotechnology Biotechnol. J. 2015, 10 DOI 10.1002/biot.201400749 Journal www.biotechnology-journal.com Research Article Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance Mehmet G. Badur1, Hui Zhang1 and Christian M. Metallo1,2 1 Department of Bioengineering, University of California, San Diego, La Jolla, USA 2 Moores Cancer Center, University of California, San Diego, La Jolla, USA To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug Received 17 MAR 2015 discovery applications, large numbers of cells that accurately recapitulate cell and tissue function Revised 10 JUL 2015 must be robustly produced. Previous studies have suggested that genetic instability and epigenetic Accepted 18 AUG 2015 changes occur as a consequence of enzymatic passaging. However, the potential impacts of such Accepted passaging methods on the metabolism of hESCs have not been described. Using stable isotope article online 18 AUG 2015 tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regen- Supporting information available online erative medicine applications. Keywords: Glycans · Lipids · Pluripotent stem cells · Stable isotope tracing · Stem cell metabolism 1 Introduction and expansion of hESC lines on feeder layer conditions [1], more recently developed, chemically-defined culture Human embryonic stem cells (hESCs) are characterized conditions have become commonly used for the isolation by their ability to differentiate into the three terminal and culture of hESCs and induced pluripotent stem cells germ layers and self-renew indefinitely. This makes them (iPSCs) [2]. Along with the engineering of defined culture ideal cell types for regenerative medicine and drug conditions, a concomitant development of enzymatic discovery applications. However, the impact of in vitro passaging methods to replace laborious mechanical pas- culture conditions on cell performance and stability must saging methods also occurred. Analysis of cells cultivated be monitored and validated to ensure these cells reca- using these enzymatic passaging techniques has sug- pitulate actual tissue functions. Since the initial isolation gested these approaches increase karyotypic instability of hESCs [3–5]. Another recent study also indicated that enzymatic passaging may cause increased genetic insta- Correspondence: Prof. Christian M. Metallo, Department of Bioengineer- bility and differential DNA methylation [6]. While these ing, University of California, San Diego, 9500 Gilman Dr. #0412, PFBH studies have addressed the effects of enzymatic passag- 204, 92093, La Jolla, CA USA ing on cellular genetics and epigenetic alterations, the E-mail: [email protected] impacts of passaging method on cellular metabolism have not been examined. Abbreviations: AcCoA, acetyl coenzyme A; GC/MS, gas chromatography/ mass spectrometry; MID, mass isotopomer distribution; hESC, human HESC metabolism is characterized by a greater reli- embryonic stem cell; ISA, isotopomer spectral analysis; MFA, metabolic flux ance on glycolysis as compared to cells in the differentiat- 13 analysis; UGlc, [U- C6]glucose; TCA, tricarboxylic acid. ed state [7–9]. Glycolytic flux is essential for maintenance © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Biotechnology Biotechnol. J. 2015, 10 Journal www.biotechnology-journal.com www.biotecvisions.com of the stem cell phenotype, as evidenced by the impact Glucose (99%; Cambridge Isotopes, Cambridge, MA) at of inhibitors of glycolysis on pluripotency marker expres- DMEM/F-12 levels. sion and cellular reprogramming [10, 11]. While glu- cose metabolism provides cellular energy by producing 2.2 Enzymatic passaging experiments adenosine triphosphate (ATP) and reducing equivalents, glucose is also the primary carbon source for a myriad H9s (p29-35) were grown on Synthemax II-SC coated of biosynthetic precursors, including ribose for nucleo- plates to 50–70% confluency. Cells were rinsed with 1 mL tides, non-essential amino acids, and acetyl-coenzyme PBS and then exposed at 37°C to either 1 mL Versene for A (AcCoA) for lipids. Though commonly overlooked in 10 min, Accutase (Innovative Cell Technology, San Diego, studies of intermediary metabolism, glucose (in addition CA) for 5 min, or Trypsin-EDTA (0.25%; Gibco, Grand to glutamine) provides the necessary building blocks Island, NY) for 5 min. Versene-treated cells were then split for the synthesis of glycan moieties, which are essential to three wells by aspirating Versene and resuspending in for protein function and trafficking. Glycans are also the 6 mL mTESR. Accutase-treated cells were split to three key components that comprise the glycocalyx, which wells by adding 9 mL PBS to Accutase solution, centri- surrounds the cell membrane of some cells [12]. Through fuging at 300 ×g for 5 min, and resuspending in 6 mL the cleavage of extracellular proteins and their associated mTESR after aspiration. Trypsin-treated cells were split glycans, enzymatic digestion could affect the pheno- to three wells by adding 9 mL PBS to Trypsin solution, type of hESCs by impacting their metabolic demands centrifuging at 300 ×g for 5 min, and resuspendingpellet or ability to respond to extracellular signaling cues in 6 mL mTESR after aspiration. Cells traced immediately [13–17]. after passaging were resuspended in tracer mTESR. Cells To understand the influence of enzymatic passaging traced 24 h after passaging were resuspended in mTESR1 on hESC metabolism we utilized stable isotope tracing immediately after passaging, rinsed with PBS 24 h later, and mass spectrometry-based metabolomics to charac- and changed into tracer mTESR before extracting 4 h lat- terize central carbon metabolism after passaging. We er. For experiments with ROCK inhibitor, 5 μM of Y-27632 developed and applied a method for rapid quantitation of (Tocris, Avon, UK) was added to media. glycan, nucleotide, and amino acid pools to explore the For quantitation of biomass abundances after pas- impact of passaging reagents on hESC biomass. Finally, saging, cellsin triplicate were rinsed with 1 mL PBS and we determined how enzymatic passaging affects bio- exposed at 37°C to 1 mL Versene for 10 min, TrypLE synthetic flux to lipids, nucleotides and carbohydrates. Express (Gibco, Grand Island, NY) for 5 min, Accutase Our results demonstrate that enzymatic passaging alters for 5 min, or Trypsin-EDTA for 5 min. 1 mL of PBS was hESC metabolism and the cell’s ability to synthesize bio- immediately added after incubation to quench enzymatic synthetic intermediates while highlighting the quantita- digestion and then transferred to 15 mL conical tube con- tive importance of metabolic flux to glycan pools. taining 7 mL PBS. Each well was then washed with 1 mL PBS and added to the respective conical tube. Cells were then centrifuged at 300 ×g for 5 min and supernatant was 2 Materials and methods aspirated. Cells were then washed twice by resuspension of the pellet in 1 mL 0.9% w/v saline, centrifugation at 2.1 Cell culture 300 ×g for 5 min, and aspiration of supernatant. Pellets were then stored at -20°C for metabolite extraction. WA09 hESCs (H9s) were maintained on Synthemax II-SC coated (Corning, Corning, NY) plates in mTESR1 (Stem 2.3 Metabolite extraction and GC-MS analysis Cell Technologies, Vancouver, BC). hESCs were passaged every five days by exposure to Versene (Gibco, Grand Polar metabolites and fatty acids were extracted using Island, NY) for 10 min at 37°C. Synthemax II-SC coating methanol/water/chloroform as previously described [18]. was performed by dispensing 2 mL of working dilution Briefly, cells were rinsed with 0.9% w/v saline and (25 μg/mL) to each well of a six-well of a tissue culture 250 μL of -80°C MeOH was added to quench metabolic polystyrene plate and incubating for 2 h. For isotopic reactions. 100 μL of ice-cold water supplemented with labeling experiments, cells were maintained in mTESR1 10 μg/mL norvaline was then added to each well and media with uniformly-labeled glucose(tracer mTESR) by cells were collected by scraping. The lysate was moved adding 5x mTESR1 supplement to custom DMEM/F-12. to a fresh 1.5 mL Eppendorf tube and 250 μL of
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