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ABSTRACT Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells

in the presence of inhibitors against mitogen-activated protein kinase kinase and glycogen synthase kinase 3 beta to maintain pluripotency reduces cellular reliance on glutamine for

tricarboxylic acid cycle anaplerosis, enabling embryonic stem cells to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a

generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction,

decreases the use of glutamine-derived carbons in the tricarboxylic acid cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in

glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells.

These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for

mouse and human pluripotent stem cells within a heterogeneous population during both embryonic stem cell passage and induced pluripotent cell reprogramming. Access through your institution

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DIFFERENTIATION POTENTIAL OF PLURIPOTENT STEM CELLS BY OPTIMIZING CULTURE CONDITIONS Article Open access 19 August 2022 EPIGENETIC AND TRANSCRIPTIONAL REGULATIONS PRIME CELL FATE BEFORE

DIVISION DURING HUMAN PLURIPOTENT STEM CELL DIFFERENTIATION Article Open access 25 January 2023 DISSECTION OF TWO ROUTES TO NAÏVE PLURIPOTENCY USING DIFFERENT KINASE INHIBITORS Article Open

access 25 March 2021 DATA AVAILABILITY The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for all

gas-chromatography–mass spectrometry data are provided in Supplementary Table 1. CODE AVAILABILITY The MATLAB code that supports the findings of this study is also available from the

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We thank the Finley laboratory for discussion and A. Intlekofer for critical feedback. We thank A. Smith (University of Cambridge) for the gift of the chimeric LIF receptor and R. Jaenisch

(Whitehead Institute for Biomedical Research) for the gift of the Nanog-GFP ESCs. S.A.V. is a Parker Fellow with the Parker Institute of Cancer Immunotherapy. B.P.R. was supported by an

National Institutes of Health (NIH) T32 Training Grant in Molecular and Cellular Biology (no. T32GM008539). L.W.S.F. is a Searle Scholar and was a Dale F. Frey-William Raveis Charitable Fund

Scientist supported by the Damon Runyon Cancer Research Foundation (no. DFS-23-17). This work was additionally supported by the Concern Foundation and the Anna Fuller Fund (to L.W.S.F.),

The Starr Foundation (no. I11-0039 to L.W.S.F.), a Pathway to Independence Award from the NIH (no. R00 CA191021 to C.C.-F.), NIH/National Institute of Diabetes and Digestive and Kidney

Diseases (no. R01DK096239 to D.H.) and the Memorial Sloan Kettering Cancer Center Support Grant no. P30 CA008748. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cancer Biology and Genetics

Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Santosha A. Vardhana & Craig B. Thompson * Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New

York, NY, USA Santosha A. Vardhana, Paige K. Arnold, Yanyang Chen, Craig B. Thompson & Lydia W. S. Finley * Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY,

USA Paige K. Arnold, Yanyang Chen & Lydia W. S. Finley * Louis V. Gerstner, Jr., Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA Paige

K. Arnold * Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Bess P. Rosen & Danwei Huangfu * Laboratory of Chromatin Biology and Epigenetics, The

Rockefeller University, New York, NY, USA Bryce W. Carey * Center for Genomics & Systems Biology, Department of Biology, New York University, New York, NY, USA Carlos Carmona-Fontaine

Authors * Santosha A. Vardhana View author publications You can also search for this author inPubMed Google Scholar * Paige K. Arnold View author publications You can also search for this

author inPubMed Google Scholar * Bess P. Rosen View author publications You can also search for this author inPubMed Google Scholar * Yanyang Chen View author publications You can also

search for this author inPubMed Google Scholar * Bryce W. Carey View author publications You can also search for this author inPubMed Google Scholar * Danwei Huangfu View author publications

You can also search for this author inPubMed Google Scholar * Carlos Carmona-Fontaine View author publications You can also search for this author inPubMed Google Scholar * Craig B.

Thompson View author publications You can also search for this author inPubMed Google Scholar * Lydia W. S. Finley View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS S.A.V. and L.W.S.F. conceived the study. S.A.V., P.K.A and L.W.S.F. performed all the experiments with assistance from Y.C. B.W.C. assisted with the

reprogramming experiments. B.P.R. and D.H. performed the human ESC experiments. C.C.-F. performed the immunofluorescence and image analysis. C.B.T. provided additional work in study

conception and guidance. S.A.V. and L.W.S.F. wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Lydia W. S. Finley. ETHICS DECLARATIONS COMPETING INTERESTS C.B.T. is a founder of

Agios Pharmaceuticals and a member of its scientific advisory board. He also previously served on the board of directors of Merck and Charles River Laboratories. ADDITIONAL INFORMATION PEER

REVIEW INFORMATION: Primary Handling Editor: Ana Mateus PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–6 REPORTING SUMMARY SUPPLEMENTARY TABLE 1 Source data for all gas chromatography–mass spectrometry

experiments SUPPLEMENTARY TABLE 2 Calculation of reprogramming efficiency, related to Fig. 4c RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Vardhana,

S.A., Arnold, P.K., Rosen, B.P. _et al._ Glutamine independence is a selectable feature of pluripotent stem cells. _Nat Metab_ 1, 676–687 (2019). https://doi.org/10.1038/s42255-019-0082-3

Download citation * Received: 10 August 2018 * Accepted: 06 June 2019 * Published: 08 July 2019 * Issue Date: July 2019 * DOI: https://doi.org/10.1038/s42255-019-0082-3 SHARE THIS ARTICLE

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