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Amino acid intake strategies define pluripotent cell states

Nature Metabolism volume 6, pages 127–140 (2024)Cite this article

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Abstract

Mammalian preimplantation development is associated with marked metabolic robustness, and embryos can develop under a wide variety of nutrient conditions, including even the complete absence of soluble amino acids. Here we show that mouse embryonic stem cells (ESCs) capture the unique metabolic state of preimplantation embryos and proliferate in the absence of several essential amino acids. Amino acid independence is enabled by constitutive uptake of exogenous protein through macropinocytosis, alongside a robust lysosomal digestive system. Following transition to more committed states, ESCs reduce digestion of extracellular protein and instead become reliant on exogenous amino acids. Accordingly, amino acid withdrawal selects for ESCs that mimic the preimplantation epiblast. More broadly, we find that all lineages of preimplantation blastocysts exhibit constitutive macropinocytic protein uptake and digestion. Taken together, these results highlight exogenous protein uptake and digestion as an intrinsic feature of preimplantation development and provide insight into the catabolic strategies that enable embryos to sustain viability before implantation.

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Fig. 1: Naïve ESCs can proliferate without essential amino acids.
Fig. 2: ESCs engage in constitutive macropinocytosis.
Fig. 3: Leucine independence is a feature of naïve pluripotency.
Fig. 4: Lysosomal metabolism facilitates amino acid independence.
Fig. 5: Digestive amino acid metabolism is attenuated with developmental progression.
Fig. 6: Cells in preimplantation blastocyst engage in macropinocytosis.

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Data availability

RNA-seq data supporting the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE216356 and GSE234802. Alignment was performed against the mouse mm10 genome assembly. EpiLC conversion RNA-seq was obtained from previously published data48 (GSE92412). E3.5–5.5 scRNA-seq data were obtained from endoderm-explorer.com45. RNA-seq from female ESCs was obtained from previously published data75 (GSE84164). RNA-seq from human ESCs was obtained from previously published data (E-MTAB-2857, GSE59435, GSE174771)72,73,74. All unique reagents generated in this study will be made available, following request, from the corresponding author with a completed material transfer agreement. Source data are provided with this paper.

Code availability

No custom code was generated for this study. All previously published code used in this study is referred to in Methods.

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Acknowledgements

We thank members of the Finley Laboratory, W. Palm, B. King, A. Intlekofer and S. Vardhana for helpful discussions, the Media Preparation Facility at MSKCC for specialized media production and A. Smith and R. Jaenisch for ESC lines. B.T.J. is a National Institute of Child Health and Human Development Ruth L. Kirschstein Predoctoral fellow (no. F30HD107943) and is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award no. T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. K.I.P. was supported by a Bruce Charles Forbes Pre-Doctoral Fellowship (MSKCC). J.S.B. is supported by a Human Frontier Science Program Fellowship (no. LT000200/2021-L). L.W.S.F. is a New York Stem Cell Foundation – Robertson Investigator and was a Searle Scholar. This research was additionally supported by MSKCC Support Grant no. P30CA008748, by grants to A.-K.H. from the National Institutes of Health (nos. R01DK127821 and R01HD094868) and by grants to L.W.S.F. from the Starr Foundation (no. I12-0051), the Tri-Institutional Stem Cell Initiative (no. 2019-007), the Basic Research Innovation Award from the Sloan Kettering Institute and the New York Stem Cell Foundation.

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Authors and Affiliations

  1. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Pavlina K. Todorova, Benjamin T. Jackson, Katrina I. Paras, Julia S. Brunner, Anna E. Bridgeman, Yanyang Chen, Sanjeethan C. Baksh, Jielin Yan & Lydia W. S. Finley

  2. Louis V. Gerstner Jr Graduate School of Biomedical Sciences, New York, NY, USA

    Benjamin T. Jackson & Jielin Yan

  3. Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Vidur Garg & Anna-Katerina Hadjantonakis

  4. Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY, USA

    Katrina I. Paras

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Contributions

P.K.T. and L.W.S.F. conceived the study. P.K.T and B.T.J. performed all of the experiments, with assistance from J.S.B., A.E.B., Y.C. and J.Y. V.G. performed blastocyst experiments. K.I.P. performed and analysed RNA-seq experiments. S.C.B. performed image analysis. A.-K.H. provided additional study guidance. L.W.S.F. supervised the project. P.K.T. and L.W.S.F. wrote the paper with input from all authors.

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Correspondence to Lydia W. S. Finley.

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Nature Metabolism thanks T. Rodriguez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Naïve ESCs grow without exogenous leucine.

(a) Population doublings of S/L- and S/L + 2i-cultured ESCs subjected to glutamine (Gln) replete or depleted conditions for 48 h. (b) Relative viability (measured by PI exclusion) of S/L- and S/L + 2i-cultured ESCs subjected to leucine (Leu) deprivation for 48 h. Viability is expressed relative to leucine-replete controls. (c) Teratoma formation from S/L + 2i-cultured ESCs subjected to leucine (Leu) replete or depleted conditions for 24 h. Each injection resulted in teratoma formation within two weeks (6 per group); two tumors per group were subjected to histology analysis with similar findings. Representative images of hematoxylin and eosin stained tumor tissue showing differentiation into endoderm, mesoderm and ectoderm-derived tissues. (d) Population doublings of S/L- and S/L + 2i-cultured ESCs (ESC-2) subjected to leucine (Leu) depleted conditions for 48 h. (e) Population doublings of S/L- and S/L + 2i-cultured ESCs subjected to isoleucine (Ile), phenylalanine (Phe), or valine (Val) depleted conditions for 48 h. (f) Cell volume of S/L- and S/L + 2i-cultured ESCs subjected to leucine (Leu) replete or depleted conditions for 48 h. (g) Change in total cellular volume of S/L- and S/L + 2i-cultured ESCs (ESC-1 and ESC-2) subjected to leucine (Leu) depleted conditions for 48 h. (h) Change in protein content of S/L- and S/L + 2i-cultured ESCs subjected to leucine (Leu) depleted conditions for the indicated times. Data are mean ± SD, n = 3 independent samples. Significance was assessed by unpaired two-tailed t test (b,d-g) and two-way ANOVA with Sidak’s multiple comparisons post-test (h). Scale bar, 50 µm.

Source data

Extended Data Fig. 2 Protein uptake and hydrolysis in ESCs.

(a) DQ-BSA signal in S/L + 2i ESCs (ESC-2) subjected to leucine (Leu) replete or depleted conditions. (b,c) Dextran uptake (b) and DQ-BSA hydrolysis (c) in S/L- and S/L + 2i-cultured ESCs (ESC-2). (d) Flow cytometry quantification of DQ-BSA signal in S/L-, S/L + 2i- and 2i/L (serum-free)-cultured ESCs. (e) Flow cytometry quantification of DQ-BSA signal in S/L- and S/L + 2i-cultured ESCs and PANC-1 pancreatic ductal adenocarcinoma cells. (f) Flow cytometry quantification of DQ-Ovalbumin signal in S/L- and S/L + 2i-cultured ESCs with or without pre-treatment with EIPA. (g,h) Population doublings of S/L- and S/L + 2i-cultured ESCs subjected to leucine (Leu) deficient conditions for 48 h in the presence or absence of EIPA (g) or chloroquine (h). Data are single measurements (b,c) or mean ± SD, n = 3 independent samples (d-h). Significance was assessed in comparison to S/L (d) or S/L + 2i (e) cells by one-way ANOVA with Sidak’s multiple comparisons post-test and two-way ANOVA with Sidak’s multiple comparisons post-test (g,h).

Source data

Extended Data Fig. 3 Amino acid deprivation affects features of ESC identity.

(a,b) Nanog-GFP ESCs were sorted into GFP-low and GFP-high populations, recovered for 24 h after seeding, and subjected to leucine (Leu) replete or depleted conditions for 48 h. The emergence of GFP-high ESCs from the GFP-low sorted population (a) and of GFP-low ESCs from the GFP-high sorted population (b) was quantified. (c) Nanog-GFP expression in S/L cultured ESCs treated with vehicle or bafilomycin A (BafA) for 24 h under leucine (Leu) replete (left panel) or depleted (right panel) conditions. (d) RT-qPCR of pluripotency-associated (Esrrb, Klf4, Nanog, Zfp42/Rex1, Pou5f1/Oct4) and early differentiation associated genes (Fgf5, Oct6, T) in S/L-cultured ESCs subjected to 24 h of lysine (Lys), phenylalanine (Phe), or valine (Val) replete or depleted conditions. (e) Quantification of undifferentiated colonies formed by ESCs seeded at clonal density in S/L medium following 24 h culture in leucine (Leu) replete or depleted conditions in the presence of EHop-016, chloroquine or vehicle controls. (f) Proliferation of S/L- and S/L + 2i-cultured ESCs provided physiological levels of leucine (30 µM) for 48 h relative to growth in leucine-replete (800 µM) conditions. Data are mean ± SD, n = 3 independent samples (a,b,e,f). Significance was assessed by unpaired two-tailed t test (a,b,f) and two-way ANOVA with Sidak’s multiple comparisons post-test (e) (ns=not significant).

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Extended Data Fig. 4 Tfe3 activation induces amino acid independence.

(a) Heat map showing expression of KEGG lysosome genes in Tfe3-ERT2-expressing S/L-cultured ESCs treated with vehicle (ethanol, EtOH) or tamoxifen (Tam). Sample genes are labeled; full gene list is available in Supplementary Table 3. (b) Population doublings of Tfe3-ERT2-expressing S/L-cultured ESCs treated with vehicle (ethanol, EtOH) or tamoxifen (Tam) for 48 h. (c) Population doublings of Tfe3-ERT2-expressing S/L-cultured ESCs subjected to phenylalanine (Phe), lysine (Lys) or valine (Val) depleted conditions for 48 h. Data are mean ± SD, n = 3 independent samples (b,c). Significance was assessed by unpaired two-tailed t test (b,c), (ns=not significant).

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Extended Data Fig. 5 Lysosomal and plasma amino acid transporter gene signatures.

(a) RNA-seq analysis of S/L + 2i- and 2i/L-cultured ESCs subjected to exit from pluripotency conditions (-2i/L) for 12, 24 and 40 h. Genes related to naïve pluripotency and early post-implantation development are shown. (b,c) Heat maps showing expression of KEGG lysosome genes (b) or plasma membrane amino acid transporters (c) in S/L + 2i-cultured ESCs subjected to exit from pluripotency conditions for 12, 24 and 40 h. Sample genes are labeled; full gene list is available in Supplementary Tables 6 and 7. (d,e) Linear regression of KEGG lysosome genes or plasma membrane amino acid transporter genes in S/L + 2i- (d) or 2i/L- (e) cultured ESCs subjected to exit from pluripotency conditions for 12, 24 and 40 h. 95% confidence intervals are shown in grey. (f) Violin plots of RNAseq data75 showing the expression distribution of KEGG lysosome and plasma amino acid transporter genes in S/L + 2i- and S/L-cultured ESCs derived from female mouse embryos. (g,h) Violin plots of three RNAseq data sets72–74 showing the expression distribution of KEGG lysosome (g) and plasma amino acid transporter (h) genes in naïve and primed human ESCs. Data are mean ± SD, n = 3 independent samples (a,b); n = 2 independent samples (c-e). Significance was assessed by unpaired two-tailed t test (a-c) and by paired two-tailed t test (f,g) (ns=not significant).

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Extended Data Fig. 6 Conversion of naïve ESCs to EpiLCs.

(a) Schematic of the conversion of S/L + 2i-cultured ESCs to EpiLCs. (b) RT-qPCR of naïve pluripotency-associated (Klf4, Nanog) and EpiLC-associated (Oct6, Otx2) genes in S/L + 2i-cultured ESCs maintained under control or EpiLC-inducing conditions for 48 h. n = 3 independent samples.

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Extended Data Fig. 7 Quantification of macropinocytosis in epiblast and primitive endoderm cells.

Quantification of DQ-BSA staining in control and EIPA pre-treated ICM cells dissociated from E3.75 PdgfraH2B-GFP/+ blastocysts. GFP demarcates primitive endoderm.

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Supplementary information

Supplementary Information

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Reporting Summary

Supplementary Tables 1–7.

Supplementary Table 1: List of all RT–qPCR primer sequences. Supplementary Table 2: List of all gene names comprising the plasma membrane amino acid transporter gene set. Supplementary Table 3: Expression of KEGG lysosome genes (z-scores) in Tfe3–ERT2-expressing, S/L-cultured ESCs treated with vehicle (EtOH) or tamoxifen (Tam). Supplementary Table 4: Expression of KEGG lysosome genes (z-scores) in ESCs following 2i/L withdrawal for the indicated times. Supplementary Table 5: Expression of genes encoding plasma membrane amino acid transporters (z-scores) in ESCs following 2i/L withdrawal for the indicated times. Supplementary Table 6: Heat maps showing expression of KEGG lysosome genes (z-scores) in S/L + 2i-cultured ESCs subjected to exit from pluripotency conditions for 12, 24 and 40 h. Supplementary Table 7: Heat maps showing expression of plasma membrane amino acid transporters (z-scores) in S/L + 2i-cultured ESCs subjected to exit from pluripotency conditions for 12, 24 and 40 h.

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Todorova, P.K., Jackson, B.T., Garg, V. et al. Amino acid intake strategies define pluripotent cell states. Nat Metab 6, 127–140 (2024). https://doi.org/10.1038/s42255-023-00940-6

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