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Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Abstract

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+Sox17+ definitive endoderm and GscSox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

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Figure 1: Establishment of ES-GscgfpSox17huCD25 cell lines.
Figure 2: Two distinctive Sox17+ endoderm cells were differentially induced by the two distinct serum-free culture conditions.
Figure 3: Identification of cell surface markers for distinguishing definitive and visceral endoderm in ES cell differentiation.
Figure 4: Expression of Cxcr4 in vivo and coexpression of Cxcr4 with goosecoid (Gsc) during differentiation of mesendoderm in vitro.
Figure 5: Purification of definitive endoderm from genetically unmanipulated ES cells.

References

  1. Beddington, R.S. & Robertson, E.J. Axis development and early asymmetry in mammals. Cell 96, 195–209 (1999).

    CAS  Article  PubMed  Google Scholar 

  2. Rossant, J. Lineage development and polar asymmetries in the peri-implantation mouse blastocyst. Semin. Cell Dev. Biol. 15, 573–581 (2004).

    Article  PubMed  Google Scholar 

  3. Gardner, R.L. Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo. J. Embryol. Exp. Morphol. 68, 175–198 (1982).

    CAS  PubMed  Google Scholar 

  4. Tam, P.P., Kanai-Azuma, M. & Kanai, Y. Early endoderm development in vertebrates: lineage differentiation and morphogenetic function. Curr. Opin. Genet. Dev. 13, 393–400 (2003).

    CAS  Article  PubMed  Google Scholar 

  5. Wells, J.M. & Melton, D.A. Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15, 393–410 (1999).

    CAS  Article  PubMed  Google Scholar 

  6. Zernicka-Goetz, M. Patterning of the embryo: the first spatial decisions in the life of a mouse. Development 129, 815–829 (2002).

    CAS  PubMed  Google Scholar 

  7. Duncan, S.A. et al. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst. Proc. Natl. Acad. Sci. USA 91, 7598–7602 (1994).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Abe, K. et al. Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp. Cell Res. 229, 27–34 (1996).

    CAS  Article  PubMed  Google Scholar 

  9. Dufort, D., Schwartz, L., Harpal, K. & Rossant, J. The transcription factor HNF3beta is required in visceral endoderm for normal primitive streak morphogenesis. Development 125, 3015–3025 (1998).

    CAS  PubMed  Google Scholar 

  10. Patient, R.K. & McGhee, J.D. The GATA family (vertebrates and invertebrates). Curr. Opin. Genet. Dev. 12, 416–422 (2002).

    CAS  Article  PubMed  Google Scholar 

  11. Fujikura, J. et al. Differentiation of embryonic stem cells is induced by GATA factors. Genes Dev. 16, 784–789 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Sousa-Nunes, R. et al. Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection. Genome Res. 13, 2609–2620 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Asahina, K. et al. Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells. Genes Cells 9, 1297–1308 (2004).

    CAS  Article  PubMed  Google Scholar 

  14. Lumelsky, N. et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292, 1389–1394 (2001).

    CAS  Article  PubMed  Google Scholar 

  15. Blyszczuk, P. et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc. Natl. Acad. Sci. USA 100, 998–1003 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Hamazaki, T. et al. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett. 497, 15–19 (2001).

    CAS  Article  PubMed  Google Scholar 

  17. Jones, E.A., Tosh, D., Wilson, D.I., Lindsay, S. & Forrester, L.M. Hepatic differentiation of murine embryonic stem cells. Exp. Cell Res. 272, 15–22 (2002).

    CAS  Article  PubMed  Google Scholar 

  18. Lawson, K.A., Meneses, J.J. & Pedersen, R.A. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development 113, 891–911 (1991).

    CAS  PubMed  Google Scholar 

  19. Kinder, S.J. et al. The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm. Development 128, 3623–3634 (2001).

    CAS  PubMed  Google Scholar 

  20. Belo, J.A. et al. Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45–57 (1997).

    CAS  Article  PubMed  Google Scholar 

  21. Perea-Gomez, A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev. Cell 3, 745–756 (2002).

    CAS  Article  PubMed  Google Scholar 

  22. Kispert, A. & Herrmann, B.G. Immunohistochemical analysis of the Brachyury protein in wild-type and mutant mouse embryos. Dev. Biol. 161, 179–193 (1994).

    Article  PubMed  Google Scholar 

  23. Kimelman, D. & Griffin, K.J. Vertebrate mesendoderm induction and patterning. Curr. Opin. Genet. Dev. 10, 350–356 (2000).

    CAS  Article  PubMed  Google Scholar 

  24. Kubo, A. et al. Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1662 (2004).

    CAS  Article  PubMed  Google Scholar 

  25. Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000).

    CAS  Article  PubMed  Google Scholar 

  26. Tada, S. et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374 (2005).

    CAS  Article  PubMed  Google Scholar 

  27. Tam, P.P., Khpp, P.L., Wong, N., Tsang, T.E. & Behringer, R.R. Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function. Dev. Biol. 274, 171–187 (2004).

    CAS  Article  PubMed  Google Scholar 

  28. Kanai-Azuma, M. et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129, 2367–2379 (2002).

    CAS  PubMed  Google Scholar 

  29. Pearce, J.J. & Evans, M. Mml, a mouse Mix-like gene expressed in the primitive streak. Mech. Dev. 87, 189–192 (1999).

    CAS  Article  PubMed  Google Scholar 

  30. Hart, A.H. et al. Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo. Development 129, 3597–3608 (2002).

    CAS  PubMed  Google Scholar 

  31. Ng, E.S. et al. The primitive streak gene Mixl1 is required for efficient haematopoiesis and BMP4-induced ventral mesoderm patterning in differentiating ES cells. Development 132, 873–884 (2005).

    CAS  Article  PubMed  Google Scholar 

  32. Kalantry, S. et al. The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain. Nat. Genet. 27, 412–416 (2001).

    CAS  Article  PubMed  Google Scholar 

  33. Couchman, J.R. & Woods, A. Syndecan-4 and integrins: combinatorial signaling in cell adhesion. J. Cell Sci. 112, 3415–3420 (1999).

    CAS  PubMed  Google Scholar 

  34. Wadehra, M., Goodglick, L. & Braun, J. The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion. J. Biol. Chem. 277, 41094–41100 (2002).

    CAS  Article  PubMed  Google Scholar 

  35. Crambert, G. & Geering, K. FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. Sci. STKE 2003, RE1 (2003).

    PubMed  Google Scholar 

  36. Verheijen, M.H. et al. Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor. Mech. Dev. 81, 151–161 (1999).

    CAS  Article  PubMed  Google Scholar 

  37. Vaarala, M.H., Porvary, K., Kellokumpu, S., Kyllonen, A.P. & Vihko, P.T. Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues. J. Pathol. 193, 134–140 (2001).

    CAS  Article  PubMed  Google Scholar 

  38. Hemler, M.E. Specific tetraspanin functions. J. Cell Biol. 155, 1103–1107 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. McGrath, K.E., Koniski, A.D., Maltby, K.M., McGann, J.K. & Palis, J. Embryonic expression and function of the chemokine SDF-1 and its receptor, Cxcr4. Dev. Biol. 213, 442–456 (1999).

    CAS  Article  PubMed  Google Scholar 

  40. Filmus, J. Glypicans in growth control and cancer. Glycobiology 11, 19R–23R (2001).

    CAS  Article  PubMed  Google Scholar 

  41. Dor, Y., Brown, J., Martinez, O.I. & Melton, D.A. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41–46 (2004).

    CAS  Article  PubMed  Google Scholar 

  42. Trucco, M. Regeneration of the pancreatic beta cell. J. Clin. Invest. 115, 5–12 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Leitges, M., Neidhardt, L., Haenig, B., Herrmann, B.G. & Kispert, A. The paired homeobox gene Uncx4.1 specifies pedicles, transverse processes and proximal ribs of the vertebral column. Development 127, 2259–2267 (2000).

    CAS  PubMed  Google Scholar 

  44. Beck, F., Tara, F. & Chawengsaksophak, K. Homeobox genes and gut development. Bioessays 22, 431–441 (2000).

    CAS  Article  PubMed  Google Scholar 

  45. Tachibana, K. et al. The chemokine receptor Cxcr4 is essential for vascularization of the gastrointestinal tract. Nature 393, 591–594 (1998).

    CAS  Article  PubMed  Google Scholar 

  46. Imitola, J. et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc. Natl. Acad. Sci. USA 101, 18117–18122 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    CAS  Article  PubMed  Google Scholar 

  48. Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).

    CAS  Article  PubMed  Google Scholar 

  49. Shirayoshi, Y., Nose, A., Iwasaki, K. & Takeichi, M. N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin. Cell Struct. Funct. 11, 245–252 (1986).

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Hitoshi Niwa for EB5 cells, to Yoshiakira Kanai for the in situ probe of Sox17, to Muneaki Miyata for his help in the cell sorting and to Yasuhiro Tosaka for obtaining qPCR data. This work was supported by grant (to N.S.) from the project for realization of regenerative medicine, grant (No.17045039 to E.T.) from the Ministry of Education, grant (to E.T. and M.Y.) from RIKEN research collaborations with industry program and grant (Kobe city collaboration of regional entities for advancement of technological excellence to M.Y.) from Japan Science and Technology Agency.

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Correspondence to Shin-Ichi Nishikawa.

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Supplementary information

Supplementary Fig. 1

Fate analysis of mesendoderm cells and two derivatives; derivative endoderm progenitors and mesoderm progenitors. (PDF 36 kb)

Supplementary Table 1

List of tenfold upregulated genes in derivative endoderm or visceral endoderm using Affymetrix MGU74v2 Gene Chips. (PDF 40 kb)

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Yasunaga, M., Tada, S., Torikai-Nishikawa, S. et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotechnol 23, 1542–1550 (2005). https://doi.org/10.1038/nbt1167

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