Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism (original) (raw)

Nature volume 481, pages 516–519 (2012)Cite this article

Subjects

Abstract

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the ‘immortal strand hypothesis’, which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with 15N-thymidine from gestation until post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute the 15N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Schoenheimer, R. & Rittenberg, D. The application of isotopes to the study of intermediary metabolism. Science 87, 221–226 (1938)
    Article ADS CAS Google Scholar
  2. Schoenheimer, R. et al. The application of the nitrogen isotope N15 for the study of protein metabolism. Science 88, 599–600 (1938)
    Article ADS CAS Google Scholar
  3. Lechene, C. et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J. Biol. 5, 20 (2006)
    Article Google Scholar
  4. Lechene, C. P., Luyten, Y., McMahon, G. & Distel, D. L. Quantitative imaging of nitrogen fixation by individual bacteria within animal cells. Science 317, 1563–1566 (2007)
    Article ADS CAS Google Scholar
  5. Lark, K. G., Consigli, R. A. & Minocha, H. C. Segregation of sister chromatids in mammalian cells. Science 154, 1202–1205 (1966)
    Article ADS CAS Google Scholar
  6. Cairns, J. Mutation selection and the natural history of cancer. Nature 255, 197–200 (1975)
    Article ADS CAS Google Scholar
  7. Potten, C. S., Hume, W. J., Reid, P. & Cairns, J. The segregation of DNA in epithelial stem cells. Cell 15, 899–906 (1978)
    Article CAS Google Scholar
  8. Potten, C. S., Owen, G. & Booth, D. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J. Cell Sci. 115, 2381–2388 (2002)
    CAS PubMed Google Scholar
  9. Shinin, V., Gayraud-Morel, B., Gomes, D. & Tajbakhsh, S. Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells. Nature Cell Biol. 8, 677–682 (2006)
    Article CAS Google Scholar
  10. Conboy, M. J., Karasov, A. O. & Rando, T. A. High incidence of non-random template strand segregation and asymmetric fate determination in dividing stem cells and their progeny. PLoS Biol. 5, e102 (2007)
    Article Google Scholar
  11. Quyn, A. J. et al. Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6, 175–181 (2010)
    Article CAS Google Scholar
  12. Rando, T. A. The immortal strand hypothesis: segregation and reconstruction. Cell 129, 1239–1243 (2007)
    Article CAS Google Scholar
  13. Kiel, M. J. et al. Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU. Nature 449, 238–242 (2007)
    Article ADS CAS Google Scholar
  14. Sotiropoulou, P. A., Candi, A. & Blanpain, C. The majority of multipotent epidermal stem cells do not protect their genome by asymmetrical chromosome segregation. Stem Cells 26, 2964–2973 (2008)
    Article CAS Google Scholar
  15. Schepers, A. G. et al. Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes. EMBO J. 30, 1104–1109 (2011)
    Article CAS Google Scholar
  16. Lansdorp, P. M. Immortal strands? Give me a break. Cell 129, 1244–1247 (2007)
    Article CAS Google Scholar
  17. Pech, M. F. & Artandi, S. E. TRAPping telomerase within the intestinal stem cell niche. EMBO J. 30, 986–987 (2011)
    Article CAS Google Scholar
  18. Cheng, H. & Leblond, C. P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141, 537–561 (1974)
    Article CAS Google Scholar
  19. Totafurno, J., Bjerknes, M. & Cheng, H. The crypt cycle. Crypt and villus production in the adult intestinal epithelium. Biophys. J. 52, 279–294 (1987)
    Article CAS Google Scholar
  20. Bjerknes, M. & Cheng, H. Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology 116, 7–14 (1999)
    Article CAS Google Scholar
  21. Lopez-Garcia, C., Klein, A. M., Simons, B. D. & Winton, D. J. Intestinal stem cell replacement follows a pattern of neutral drift. Science 330, 822–825 (2010)
    Article ADS CAS Google Scholar
  22. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010)
    Article CAS Google Scholar
  23. Gutierrez, E., Wiggins, D., Fielding, B. & Gould, A. P. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445, 275–280 (2007)
    Article ADS CAS Google Scholar
  24. Schick, P. et al. Labelling of human resting lymphocytes by continuous infusion of [3H]thymidine. I. Characterization of cytoplasmic label. J. Cell Sci. 33, 351–362 (1978)
    CAS PubMed Google Scholar
  25. Stürup, S., Hansen, H. R. & Gammelgaard, B. Application of enriched stable isotopes as tracers in biological systems: a critical review. Anal. Bioanal. Chem. 390, 541–554 (2008)
    Article Google Scholar
  26. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5 . Nature 449, 1003–1007 (2007)
    Article ADS CAS Google Scholar
  27. Sangiorgi, E. & Capecchi, M. R. Bmi1 is expressed in vivo in intestinal stem cells. Nature Genet. 40, 915–920 (2008)
    Article CAS Google Scholar
  28. Montgomery, R. K. et al. Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc. Natl Acad. Sci. USA 108, 179–184 (2011)
    Article ADS CAS Google Scholar

Download references

Acknowledgements

We thank A. Mudge, M. Raff and A. Aperia for critical reading of the manuscript; M. Raff for numerous enlightening discussions; T. Bloom for her strong support at the origin of MIMS development; M. Wang for MIMS analysis; J. Poczatek and Z. Kaufman for MIMS analysis software development; L. Trakimas, C. MacGillivray, S. Clark and E. Hurst for histology; W. Wang for statistics advice. We thank Cambridge Isotope Laboratories for their generous gift of thymidine (15N2, 96–98%). M.L.S. is funded by the American Heart Association and Future Leaders in Cardiovascular Medicine. A.P.G. is funded by the Medical Research Council (U117584237). R.T.L. is funded by the National Institutes of Health (AG032977) and a grant from the Harvard Stem Cell Institute. C.P.L. is funded by the National Institutes of Health (EB001974, AG034641), the Ellison Medical Foundation and the Human Frontier Science Program.

Author information

Authors and Affiliations

  1. Department of Medicine, Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, 02115, Massachusetts, USA
    Matthew L. Steinhauser, Samuel E. Senyo, Todd S. Perlstein & Richard T. Lee
  2. Harvard Medical School, Boston, 02115, Massachusetts, USA
    Matthew L. Steinhauser, Samuel E. Senyo, Christelle Guillermier, Todd S. Perlstein, Richard T. Lee & Claude P. Lechene
  3. Division of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, UK,
    Andrew P. Bailey & Alex P. Gould
  4. National Resource for Imaging Mass Spectroscopy, 65 Landsdowne St., Cambridge, 02139, Massachusetts, USA
    Christelle Guillermier & Claude P. Lechene
  5. Department of Medicine, Division of Genetics, Brigham and Women’s Hospital, Boston, 02115, Massachusetts, USA
    Christelle Guillermier & Claude P. Lechene
  6. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA
    Richard T. Lee

Authors

  1. Matthew L. Steinhauser
    You can also search for this author inPubMed Google Scholar
  2. Andrew P. Bailey
    You can also search for this author inPubMed Google Scholar
  3. Samuel E. Senyo
    You can also search for this author inPubMed Google Scholar
  4. Christelle Guillermier
    You can also search for this author inPubMed Google Scholar
  5. Todd S. Perlstein
    You can also search for this author inPubMed Google Scholar
  6. Alex P. Gould
    You can also search for this author inPubMed Google Scholar
  7. Richard T. Lee
    You can also search for this author inPubMed Google Scholar
  8. Claude P. Lechene
    You can also search for this author inPubMed Google Scholar

Contributions

M.L.S. designed the experiments to study the ‘immortal strand hypothesis’ in the small intestine with input from C.P.L.; M.L.S. performed in vivo mouse experiments with help from S.E.S.; A.P.B. and A.P.G. designed and performed the Drosophila experiments. M.L.S. designed the human experiment with input from R.T.L. and T.S.P.; M.L.S. and T.S.P. conducted the human protocol. S.E.S. was involved in study design. M.L.S. analysed the data with C.P.L. input. C.G. operated the instrument and assisted with analysis of Drosophila lipid droplets. M.L.S. and C.P.L. wrote the manuscript; A.P.B. and A.P.G. contributed the section on Drosophila. R.T.L. was involved in study design and provided critical feedback at all junctures. C.P.L. conceived of the general application of MIMS to metabolism, cell turnover and human experimentation. C.P.L. designed and performed in vitro experiments.

Corresponding author

Correspondence toClaude P. Lechene.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-13 with legends and an additional reference. (PDF 5698 kb)

PowerPoint slides

Rights and permissions

About this article

Cite this article

Steinhauser, M., Bailey, A., Senyo, S. et al. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism.Nature 481, 516–519 (2012). https://doi.org/10.1038/nature10734

Download citation

This article is cited by

Editorial Summary

Quantifying metabolism

Multi-isotope imaging mass spectrometry (MIMS) is a new and generally applicable method for the study of DNA replication, lipid and protein turnover and cell fate in animals and humans. In a proof-of-principle study, MIMS was used to test the 'immortal strand hypothesis', which proposes that stem cells maintain a master genetic template that is protected from cancer-causing mutations. The hypothesis remains hotly debated, in part because of the difficulties involved in testing it experimentally. Stable isotope incorporation was viewed and measured by MIMS in mammalian intestinal cell division, Drosophila melanogaster lipid metabolism and human lymphopoiesis.

Associated content