Volume 23, Issue 4, Pages 663-674 (April 2016) Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells  Shugo Tohyama, Jun Fujita, Takako Hishiki, Tomomi Matsuura, Fumiyuki Hattori, Rei Ohno, Hideaki Kanazawa, Tomohisa Seki, Kazuaki Nakajima, Yoshikazu Kishino, Marina Okada, Akinori Hirano, Takuya Kuroda, Satoshi Yasuda, Yoji Sato, Shinsuke Yuasa, Motoaki Sano, Makoto Suematsu, Keiichi Fukuda  Cell Metabolism  Volume 23, Issue 4, Pages 663-674 (April 2016) DOI: 10.1016/j.cmet.2016.03.001 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Consumption Profiles and Effects of Amino Acids in hESCs (A and B) To evaluate metabolite consumption profiles in hESCs, concentrations of all amino acids in the medium were analyzed before and after 3 days of culture (n = 4) (A). Serine (Ser), glutamine (Gln), arginine (Arg), and cystine (Cys2) were highly consumed (B). (C and D) We investigated whether four amino acids (Ser, Gly, Gln, and Arg) were essential for pluripotency and cell survival by alkaline phosphatase (ALP) staining. In glucose (Glc)-supplemented conditions, no obvious difference in staining was observed between cultures depleted in the different amino acids (C). In Glc-depleted medium, the lack of Gln resulted in rapid cell death (24 hr) (D). (E) LIVE/DEAD cell staining was performed to check cell viability. Under Glc- and Gln-depleted conditions, hESCs died within 24 hr and were completely eliminated within 48 hr. (F) The bar graphs show cell viability under various conditions after 24 hr (n = 4). Only Gln was essential to hESC viability under Glc-depleted conditions. Scale bars represent 100 μm (E). Data are shown as mean ± SD. See also Figure S1. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Metabolites from the Latter Steps of the TCA Cycle in hESCs Depend on Glutamine Metabolism (A) Heatmap image of metabolome data illustrating relative metabolite levels in hESCs under various conditions. Each condition was compared with Glc-depleted and Gln-supplemented conditions (Glc- and Gln-supplemented, n = 3; Glc-depleted and Gln-supplemented, n = 4; Gln-depleted, n = 4 each). Intermediate metabolites of the glycolytic pathway depended on Glc independent of Gln. Products from the latter steps of the TCA cycle, and reduced glutathione (GSH), depended on Gln. Red indicates increased expression, whereas green indicates decreased expression, and gray means undetectable. (B) Metabolome analysis of hESC metabolic pathways. Former steps in the TCA cycle: acetyl-CoA, isocitrate. Latter steps: αKG, oxaloacetate. (C) Intermediate metabolites from the former steps of the TCA cycle did not differ between conditions. Total and separate amounts of metabolites from the latter steps of the TCA cycle were significantly reduced in Gln-depleted cultures, independent of Glc. (D) Reduced glutathione (GSH), oxidized/reduced glutathione ratio (GSH/GSSG), and total glutathione (GSH+2GSSG) were significantly reduced under Gln-depleted conditions. (E) The production of ATP was reduced under Glc-depleted and Gln-supplemented conditions due to lower glycolytic activity. According to a reduced metabolites in TCA cycle, the production of ATP dropped markedly without Gln. Glc (−) Gln (+), blue bar; Glc (+) Gln (+), red bar; Glc (+) Gln (−), green bar; and Glc (−) Gln (−), purple bar. ∗p < 0.05; ∗∗p < 0.01. Data are shown as mean ± SEM. See also Figure S2. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 hESCs Depend on Glutamine Oxidation, and Glucose Depletion Affects the Increase in Glutamine Oxidation (A) hESCs were incubated in Gln-supplemented and Glc-depleted media with 13C6-Glc or in Glc-supplemented and Gln-depleted media with 13C5-Gln. After 1–8 hr, CE-MS-based metabolic flux analysis was performed. (B) Fraction of each metabolite labeled by 13C derived from 13C6-Glc or 13C5-Gln. (C) Oxygen consumption rate (OCR) changes under mitochondrial stress in each condition (Glc supplemented, n = 4; Glc depleted and Gln supplemented, n = 4; Glc depleted and Gln depleted, n = 5). We sequentially applied oligomycin (0.5 μM), FCCP (0.25 μM), and antimycin (1 μM) and rotenone (1 μM), according to the mitochondrial stress kit protocol. (D) ECAR was significantly increased under Glc-supplemented conditions. (E) Basal and maximal OCR were significantly increased under Gln-supplemented conditions, and basal OCR tended to be higher under the Glc-depleted and Gln-supplemented conditions. (F) Schematic drawing of 13C5-Gln-derived metabolites in the TCA cycle. (G) Metabolites derived from Gln in the TCA cycle (succinate, fumarate, and malate) were markedly increased under Glc-depleted conditions (n = 4). Glc (−) Gln (+), blue bar; Glc (+) Gln (+), red bar; Glc (+) Gln (−), green bar; Glc (−) Gln (−), purple bar. ∗p < 0.05; ∗∗p < 0.01. Data are shown as mean ± SEM. See also Figure S2. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Dimethyl-α-Ketoglutarate, but Not Pyruvate, Can Rescue hESCs under Glucose- and Glutamine-Depleted Conditions (A) Cell-permeable dimethyl-α-ketoglutarate (DM-αKG; 4 mM) or pyruvate (Pyr; 2 mM) was added to Glc- and Gln-depleted media. Cell survival was assessed by ALP staining. Only DM-αKG rescued hESCs. hESCs did not survive under Glc-depleted and Gln-supplemented conditions in the presence of oligomycin (0.5 μM). (B) Neither nucleosides nor GSH (1 mM) rescued hESCs, as judged by ALP staining. (C and D) Metabolome analysis revealed that Pyr supplementation significantly increased citrate (n = 3; C), but not metabolites from the latter steps of the TCA cycle (succinate, fumarate, and malate; D). DM-αKG supplementation significantly increased glutamate and its metabolites in the latter steps of the TCA cycle (n = 3). These results indicate that the TCA cycle in hESCs highly depends on Gln oxidation, but not Pyr oxidation. (E and F) Microarray analysis was performed in hESCs and hESC-derived purified cardiomyocytes (CMs) to identify differential gene expression in hESCs (E). Expression of metabolic-enzyme-related genes used in the former steps of the TCA cycle (ACO2, IDH2, and IDH3A) was particularly decreased in hESCs, while expression of metabolic enzyme-related genes involved in the lipid synthesis pathway (ACLY and FASN) and glutaminolysis (SLC1A5, GLS2, and GPT2) was increased (F). (G) Western blot analyses for IDH2, ACO2, and ACLY expression in hiPSCs (253G4) and hiPSC (253G4)-derived purified CMs. (H) The bar graphs show relative protein expression levels for IDH2, ACO2, and ACLY in hiPSCs and hiPSC-derived purified CMs. Protein levels were normalized using GAPDH expression levels. ∗p < 0.05; ∗∗p < 0.01. Data are shown as mean ± SEM. See also Figure S3. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Lactate Supplementation Can Rescue Cardiomyocytes Cultured under Glucose- and Glutamine-Depleted Conditions (A and B) Rat neonatal CMs were cultured under Glc-depleted conditions. Additional depletion of Gln and Arg accelerated the death of CMs. Supplementation of lactate (Lac) prevented the loss of cell viability in the absence of Glc (A). The bar graphs show cell viability under various conditions after 48 hr (n = 4; B). (C) Cardiac differentiation and purification protocol of hESCs. CMs were purified with Lac-supplemented and Glc- and Gln-depleted media. (D) Metabolic purification successfully eliminated cell types other than hESC-derived CMs. Before purification, non-cardiac cells existed in the interspace of cardiac fibers. After metabolic purification (day 3), only CMs remained. (E) Representative immunofluorescence staining for α-actinin (red) in hiPSCs (253G4)-derived dispersed cells after metabolic purification. Cell nuclei are stained with DAPI (blue). (F) Representative immunofluorescence staining for α-actinin (red) and troponin I (green) in hiPSC (253G4)-derived dispersed cells after metabolic purification. Cell nuclei were stained with DAPI (blue). (G) FACS analyses showed cardiac troponin T expression increased in hiPSC-derived cells during metabolic selection. (H) The proportion of troponin T-positive CMs in hiPSC (253G4)-derived dispersed cells before (n = 5) and after metabolic selection (n = 4). Human iPSC-derived dispersed cells after metabolic selection were cultivated for 14 days under Glc-supplemented conditions plus 10% FBS and analyzed by FACS (n = 3). All data were obtained from independent experiments. (I) The undifferentiated cell marker NANOG was not detected in purified CMs by qRT-PCR (n = 3). mRNA expression levels are relative to those in hiPSCs. (J) Immunofluorescence staining for TRA1-60 expression in dispersed cells (4 × 105 cells) from hiPSC-derived cells before and after metabolic purification (Glc-free with or without Gln). (K) Immunofluorescence staining for MLC2a (red) and MLC2v (green) in hiPSC (253G4)-derived purified CMs on day 35 and day 60 of differentiation. Cell nuclei were stained with DAPI (blue). Scale bars represent 100 μm (A, D, F, J, and K) or 500 μm (E). Data are shown as mean ± SD. See also Figure S4. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Lactate Supplementation Can Replenish Glutamate under Glutamine-Depleted Conditions in Cardiomyocytes (A) Schematic metabolic map of [U-13C]-labeled lactate (Lac) metabolism. (B) Lac-derived total metabolites in the TCA cycle of neonatal CMs (n = 5) were increased more than twice compared to those from human embryonic stem cells (hESCs) (n = 4). (C and D) Glutamate (C) and Gln (D) were efficiently generated from Lac in CMs, but not in hESCs. (E) Transfection of ACO2 and IDH2 siRNA decreased protein expression compared to controls in hiPSC (253G4)-derived purified CMs. Protein levels were normalized using expression of GAPDH. (F) The oxygen consumption rate (OCR) of hiPSC (253G4)-derived purified CMs varied with each culture condition (control, n = 4; ACO2 and IDH2 knockdown, n = 4). We sequentially applied oligomycin (1.5 μM), FCCP (0.5 μM), and antimycin (1 μM) and rotenone (1 μM), according to the mitochondrial stress kit protocol. Basal OCR tended to decrease, and maximal OCR was significantly decreased, in ACO2 and IDH2 knocked-down CMs. (G and H) Summary of Glc and Gln metabolism in human pluripotent stem cells (hPSCs) and CMs. hPSCs depend highly on both Glc and Gln metabolism. Glc metabolism contributes not only to ATP generation but also to the synthesis of nucleotides, amino acids, and fatty acids. Gln metabolism contributes not only to synthesis of nucleotides and glutathione but also to ATP production via OXPHOS (G, left). Under Glc-depleted conditions, Gln oxidation is activated because of a lack of ATP (G, right). Under Glc- and Gln-depleted conditions with Pyr or Lac supplementation, hPSCs cannot utilize Pyr efficiently because of low ACO2 and IDH2/3 expression (H, left). In contrast, CMs can utilize Pyr or Lac efficiently for OXPHOS (H, right). ∗p < 0.05; ∗∗p < 0.01. Data are shown as mean ± SEM. See also Figure S5. Cell Metabolism 2016 23, 663-674DOI: (10.1016/j.cmet.2016.03.001) Copyright © 2016 Elsevier Inc. Terms and Conditions