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L-Proline Supplementation Drives Self-Renewing Mouse Embryonic Stem Cells to a Partially Primed Pluripotent State: The Early Primitive Ectoderm-Like Cell

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Epiblast Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2490))

Abstract

Mouse embryonic stem cells (mESCs) can be grown under a variety of culture conditions as discrete cell states along the pluripotency continuum, ranging from the least mature “ground state” to being “primed” to differentiate. Cells along this continuum are demarcated by differences in gene expression, X chromosome inactivation, ability to form chimeras and epigenetic marks. We have developed a protocol to differentiate “naïve” mESCs to a “partially primed” state by adding the amino acid L-proline to self-renewal medium. These cells express the primitive ectoderm markers Dnmt3b and Fgf5, and are thus called early primitive ectoderm-like (EPL) cells. In addition to changes in gene expression, these cells undergo a morphological change to flattened, dispersed colonies, have an increased proliferation rate, and a predisposition to neural fate. EPL cells can be used to study the cell states along the pluripotency continuum, peri-implantation embryogenesis, and as a starting point for efficient neuronal differentiation.

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References

  1. Rodda SJ, Kavanagh SJ, Rathjen J, Rathjen PD (2002) Embryonic stem cell differentiation and the analysis of mammalian development. Int J Dev Biol 46:449–458

    CAS  PubMed  Google Scholar 

  2. Ying Q, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281–292. https://doi.org/10.1016/S0092-8674(03)00847-X

    Article  CAS  PubMed  Google Scholar 

  3. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

    Article  CAS  Google Scholar 

  4. Williams RL, Hilton DJ, Pease S et al (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687. https://doi.org/10.1038/336684a0

    Article  CAS  PubMed  Google Scholar 

  5. Tomioka M, Nishimoto M, Miyagi S et al (2002) Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res 30:3202–3213

    Article  CAS  Google Scholar 

  6. Kuroda T, Tada M, Kubota H et al (2005) Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol Cell Biol 25:2475–2485. https://doi.org/10.1128/MCB.25.6.2475-2485.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F (2005) Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem 280:5307–5317. https://doi.org/10.1074/jbc.M410015200

    Article  CAS  PubMed  Google Scholar 

  8. Loh Y-H, Wu Q, Chew J-L et al (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38:431–440. https://doi.org/10.1038/ng1760

    Article  CAS  PubMed  Google Scholar 

  9. Ying Q, Wray J, Nichols J et al (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523. https://doi.org/10.1038/nature06968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hackett JA, Dietmann S, Murakami K et al (2013) Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency. Stem Cell Reports 1:518–531. https://doi.org/10.1016/j.stemcr.2013.11.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Glover HJ (2019) L-proline-induced transition of mouse ES cells to a spatially distinct primitive ectoderm-like cell population primed for neural differentiation. The University of Sydney

    Google Scholar 

  12. Tesar PJ, Chenoweth JG, Brook FA et al (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448:196–199. https://doi.org/10.1038/nature05972

    Article  CAS  PubMed  Google Scholar 

  13. Brons IGM, Smithers LE, Trotter MWB et al (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448:191–195. https://doi.org/10.1038/nature05950

    Article  CAS  PubMed  Google Scholar 

  14. Iacovino M, Bosnakovski D, Fey H et al (2011) Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells. Stem Cells 29:1580–1588

    Article  CAS  Google Scholar 

  15. Osteil P, Studdert J, Wilkie E et al (2016) Generation of genome-edited mouse epiblast stem cells via a detour through ES cell-chimeras. Differentiation 91:119–125. https://doi.org/10.1016/j.diff.2015.10.004

    Article  CAS  PubMed  Google Scholar 

  16. Hayashi K, de Sousa Lopes SMC, Tang F et al (2008) Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3:391–401. https://doi.org/10.1016/j.stem.2008.07.027

    Article  CAS  PubMed  Google Scholar 

  17. Greber B, Wu G, Bernemann C et al (2010) Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6:215–226. https://doi.org/10.1016/j.stem.2010.01.003

    Article  CAS  PubMed  Google Scholar 

  18. Greber B, Lehrach H, Adjaye J (2008) Control of early fate decisions in human ES cells by distinct states of TGFbeta pathway activity. Stem Cells Dev 17:1065–1077. https://doi.org/10.1089/scd.2008.0035

    Article  CAS  PubMed  Google Scholar 

  19. James D, Levine AJ, Besser D, Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132:1273–1282. https://doi.org/10.1242/dev.01706

    Article  CAS  PubMed  Google Scholar 

  20. Vallier L, Alexander M, Pedersen RA (2005) Activin/nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118:4495–4509. https://doi.org/10.1242/jcs.02553

    Article  CAS  PubMed  Google Scholar 

  21. Vallier L, Touboul T, Chng Z et al (2009) Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS One 4:e6082. https://doi.org/10.1371/journal.pone.0006082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kojima Y, Kaufman-Francis K, Studdert JB et al (2014) The transcriptional and functional properties of mouse epiblast stem cells resemble the anterior primitive streak. Cell Stem Cell 14:107–120. https://doi.org/10.1016/j.stem.2013.09.014

    Article  CAS  PubMed  Google Scholar 

  23. Coucouvanis E, Martin GR (1995) Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83:279–287

    Article  CAS  Google Scholar 

  24. Coucouvanis E, Martin GR (1999) BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126:535–546

    Article  CAS  Google Scholar 

  25. Rathjen J, Lake JA, Bettess MD et al (1999) Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 112:601–612

    Article  CAS  Google Scholar 

  26. Rathjen J, Haines BP, Hudson KM et al (2002) Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development 129:2649–2661

    Article  CAS  Google Scholar 

  27. Stead E, White J, Faast R et al (2002) Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities. Oncogene 21:8320–8333. https://doi.org/10.1038/sj.onc.1206015

    Article  CAS  PubMed  Google Scholar 

  28. Snow MHL (1977) Gastrulation in the mouse: growth and regionalization of the epiblast. Development 42

    Google Scholar 

  29. Lake J, Rathjen J, Remiszewski J, Rathjen PD (2000) Reversible programming of pluripotent cell differentiation. J Cell Sci 113:555–566

    Article  CAS  Google Scholar 

  30. Washington JM, Rathjen J, Felquer F et al (2010) L-proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture. Am J Physiol 298:982–992

    Article  Google Scholar 

  31. Casalino L, Comes S, Lambazzi G et al (2011) Control of embryonic stem cell metastability by L-proline catabolism. J Mol Cell Biol 3:108–122. https://doi.org/10.1093/jmcb/mjr001

    Article  CAS  PubMed  Google Scholar 

  32. D’Aniello C, Habibi E, Cermola F et al (2017) Vitamin C and L-proline antagonistic effects capture alternative states in the pluripotency continuum. Stem Cell Reports 8:1–10. https://doi.org/10.1016/j.stemcr.2016.11.011

    Article  CAS  PubMed  Google Scholar 

  33. Comes S, Gagliardi M, Laprano N et al (2013) L-proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation. Stem Cell Reports 1:307–321. https://doi.org/10.1016/j.stemcr.2013.09.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tan BS, Lonic A, Morris MB et al (2011) The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells. Am J Physiol 300:C1270–C1279. https://doi.org/10.1152/ajpcell.00235.2010

    Article  CAS  Google Scholar 

  35. Tan BSN, Kwek J, Wong CKE et al (2016) Src family kinases and p38 mitogen-activated protein kinases regulate pluripotent cell differentiation in culture. PLoS One 11:e0163244. https://doi.org/10.1371/journal.pone.0163244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lonic A (2006) Molecular mechanism of L-proline induced EPL-cell formation. University of Adelaide

    Google Scholar 

  37. D’Aniello C, Fico A, Casalino L et al (2015) A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-proline metabolism controls stem cell identity. Cell Death Differ 22:1094–1105. https://doi.org/10.1038/cdd.2015.24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Carey BW, Finley LWS, Cross JR et al (2015) Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 518:413–416. https://doi.org/10.1038/nature13981

    Article  CAS  PubMed  Google Scholar 

  39. Ryu JM, Han HJ (2011) L-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways. J Biol Chem 286:23667–23678. https://doi.org/10.1074/jbc.M110.216283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Shparberg RA, Glover HJ, Morris MB (2019) Embryoid body differentiation of mouse embryonic stem cells into neurectoderm and neural progenitors. Methods Mol Biol 2029:273–285

    Article  CAS  Google Scholar 

  41. Shiraki N, Shiraki Y, Tsuyama T et al (2014) Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells. Cell Metab 19:780–794. https://doi.org/10.1016/j.cmet.2014.03.017

    Article  CAS  PubMed  Google Scholar 

  42. Chen G, Wang J (2014) Threonine metabolism and embryonic stem cell self-renewal. Curr Opin Clin Nutr Metab Care 17:80–85. https://doi.org/10.1097/MCO.0000000000000007

    Article  CAS  PubMed  Google Scholar 

  43. Morris MB, Ozsoy S, Zada M et al (2020) Selected amino acids promote mouse pre-implantation embryo development in a growth factor-like manner. Front Physiol 11:140. https://doi.org/10.3389/fphys.2020.00140

    Article  PubMed  PubMed Central  Google Scholar 

  44. Shparberg R (2018) L-proline-mediated neural differentiation of mouse embryonic stem cells. University of Sydney

    Google Scholar 

  45. Bernemann C, Greber B, Ko K et al (2011) Distinct developmental ground states of epiblast stem cell lines determine different pluripotency features. Stem Cells 29:1496–1503. https://doi.org/10.1002/stem.709

    Article  CAS  PubMed  Google Scholar 

  46. Doetschman TC, Eistetter H, Katz M et al (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87:27–45

    CAS  PubMed  Google Scholar 

  47. Aubert J, Stavridis MP, Tweedie S et al (2003) Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1 – gfp knock-in mice. Proc Natl Acad Sci U S A 100:11836–11841

    Article  CAS  Google Scholar 

  48. Ying Q, Stavridis M, Griffiths D et al (2003) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21:183–186. https://doi.org/10.1038/nbt780

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Bosch Institute and Discipline of Physiology, School of Medical Sciences, University of Sydney, Camperdown, NSW, Australia

    Hannah J. Glover, Rachel A. Shparberg & Michael B. Morris

Authors
  1. Hannah J. Glover
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  2. Rachel A. Shparberg
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  3. Michael B. Morris
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Corresponding authors

Correspondence to Hannah J. Glover or Michael B. Morris .

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Editors and Affiliations

  1. Swiss Cancer Research Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Pierre Osteil

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Glover, H.J., Shparberg, R.A., Morris, M.B. (2022). L-Proline Supplementation Drives Self-Renewing Mouse Embryonic Stem Cells to a Partially Primed Pluripotent State: The Early Primitive Ectoderm-Like Cell. In: Osteil, P. (eds) Epiblast Stem Cells. Methods in Molecular Biology, vol 2490. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2281-0_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2281-0_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2280-3

  • Online ISBN: 978-1-0716-2281-0

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