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Clonal tracking of hESCs reveals differential contribution to functional assays

Nature Methods volume 7, pages 917–922 (2010)Cite this article

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Abstract

Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogenous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.

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Figure 1: Detection of clones in retrovirally transduced hESC cultures.
Figure 2: Differential contribution of hESC clones to in vivo and in vitro pluripotency assays.
Figure 3: C-hESCs and nc-hESCs are functionally distinct.
Figure 4: hESC self-renewal capacity affects contribution to in vitro versus in vivo differentiation assays.
Figure 5: Phenotypic distribution of hESC subclasses.
Figure 6: Proposed model of developmental potentials of self-renewing hESCs.

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References

  1. Reynolds, B.A. & Weiss, S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175, 1–13 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. Purton, L.E. & Scadden, D.T. Limiting factors in murine hematopoietic stem cell assays. Cell Stem Cell 1, 263–270 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  PubMed  Google Scholar 

  4. Itskovitz-Eldor, J. et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88–95 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Trounson, A. The production and directed differentiation of human embryonic stem cells. Endocr. Rev. 27, 208–219 (2006).

    Article  PubMed  Google Scholar 

  6. Lensch, M.W., Schlaeger, T.M., Zon, L.I. & Daley, G.Q. Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera. Cell Stem Cell 1, 253–258 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Stewart, M.H. et al. Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nat. Methods 3, 807–815 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Pyle, A.D., Lock, L.F. & Donovan, P.J. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344–350 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Bendall, S.C. et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448, 1015–1021 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Keller, G., Paige, C., Gilboa, E. & Wagner, E.F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature 318, 149–154 (1985).

    Article  CAS  PubMed  Google Scholar 

  15. Dick, J.E., Magli, M.C., Huszar, D., Phillips, R.A. & Bernstein, A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell 42, 71–79 (1985).

    Article  CAS  PubMed  Google Scholar 

  16. Guenechea, G., Gan, O.I., Dorrell, C. & Dick, J.E. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat. Immunol. 2, 75–82 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Palmer, T.D., Takahashi, J. & Gage, F.H. The adult rat hippocampus contains primordial neural stem cells. Mol. Cell. Neurosci. 8, 389–404 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. Morshead, C.M., Craig, C.G. & van der Kooy, D. In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development 125, 2251–2261 (1998).

    CAS  PubMed  Google Scholar 

  19. Wang, Z. & Jaenisch, R. At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Dev. Biol. 275, 192–201 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Schmidt, M. et al. A model for the detection of clonality in marked hematopoietic stem cells. Ann. NY Acad. Sci. 938, 146–155 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Mazurier, F., Gan, O.I., McKenzie, J.L., Doedens, M. & Dick, J.E. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103, 545–552 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Schmidt, M. et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 100, 2737–2743 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Blum, B. & Benvenisty, N. Clonal analysis of human embryonic stem cell differentiation into teratomas. Stem Cells 25, 1924–1930 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Blum, B., Bar-Nur, O., Golan-Lev, T. & Benvenisty, N. The anti-apoptotic gene survivin contributes to teratoma formation by human embryonic stem cells. Nat. Biotechnol. 27, 281–287 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Baum, C., Schambach, A., Bohne, J. & Galla, M. Retrovirus vectors: toward the plentivirus? Mol. Ther. 13, 1050–1063 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Ma, Y., Ramezani, A., Lewis, R., Hawley, R.G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21, 111–117 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Schmidt, M. et al. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum. Gene Ther. 12, 743–749 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. de Hoon, M.J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Saldanha, A.J. Java Treeview–extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

M.H.S. was supported by a Canadian Institutes of Health Research Canada Graduate Scholarship doctoral award. This work was supported by Canadian Institutes of Health Research, Ontario Ministry of Research Innovation and a Canada Research Chair award to M.B. We thank T. Schuesler and C. von Kalle for providing and troubleshooting the LAM-PCR methods and applications to hESC lines.

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Author notes

  1. Morag H Stewart

    Present address: Present address: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.,

Authors and Affiliations

  1. Stem Cell and Cancer Research Institute, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada

    Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia

  2. Center for Clinical Sciences Research, Stanford School of Medicine, Stanford, California, USA

    Sean C Bendall

  3. Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada

    Mickie Bhatia

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Contributions

M.H.S. designed, performed, and analyzed all experiments, and wrote and revised the manuscript. S.C.B. contributed intellectual input to design and analysis of experiments and results. M.L.-M. performed all FACS. M.B. provided intellectual support toward design and interpretation of the results, and wrote and revised the manuscript.

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Correspondence to Mickie Bhatia.

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The authors declare no competing financial interests.

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Supplementary Figures 1–11 and Supplementary Tables 1–4 (PDF 3951 kb)

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Stewart, M., Bendall, S., Levadoux-Martin, M. et al. Clonal tracking of hESCs reveals differential contribution to functional assays. Nat Methods 7, 917–922 (2010). https://doi.org/10.1038/nmeth.1519

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