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Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro

An Erratum to this article was published on 01 May 2000

Abstract

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

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Figure 1: Phase contrast micrographs of ES cells and their differentiated progeny
Figure 2: Marker expression in ES cells and their differentiated progeny.
Figure 3: RT-PCR analysis of gene expression in ES cells and their differentiated derivatives.
Figure 4: Histology of differentiated elements found in teratomas formed in the testis of SCID mice following inoculation of HES-1 or HES-2 colonies.
Figure 5: Phase contrast microscopy and immunochemical analysis of marker expression in neural progenitor cells isolated from differentiating ES cultures.
Figure 6: Phase contrast appearance and marker expression in cultures of neurons derived from progenitor cells shown in Fig. 5.

References

  1. 1

    Evans, M.J. & Kaufman, M. Establishment in culture of pluripotential stem cells from mouse embryos. Nature 292, 151–156 (1981).

    Google Scholar 

  2. 2

    Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci USA 78, 7634–7638 ( 1981).

    CAS  Article  Google Scholar 

  3. 3

    Andrews, P.W. et al Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Lab. Invest. 50, 147–162 (1984).

    CAS  PubMed  Google Scholar 

  4. 4

    Pera, M.F., Cooper, S., Mills, J. & Parrington, J.M. Isolation and characterization of a multipotent clone of human embryonal carcinoma-cells . Differentiation 42, 10– 23 (1989).

    CAS  Article  Google Scholar 

  5. 5

    Thompson, S. et al. Cloned human teratoma cells differentiate into neuron-like cells and other cell types in retinoic acid. J. Cell Sci. 72, 37–64 (1984).

    CAS  PubMed  Google Scholar 

  6. 6

    Pera, M.F. & Herszfeld, D. Differentiation of pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2. Reprod. Fertil. Devel. 10, 551–556 (1999).

    Article  Google Scholar 

  7. 7

    Thomson, J.A. et al. Isolation of a primate embryonic stem cell line. Proc. Natl. Acad. Sci. USA 92, 7844– 7844 (1995).

    CAS  Article  Google Scholar 

  8. 8

    Thomson, J.A. et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55, 254–259 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Bongso, A., Fong, C.Y., Ng, S.C. & Ratnam, S. Isolation and culture of inner cell mass cells from human blastocysts. Hum. Reprod. 9, 2110–2117 (1994).

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Andrews, P.W. et al. Comparative-analysis of cell-surface antigens expressed by cell-lines derived from human germ-cell tumors. Int. J. Cancer 66, 806–816 ( 1996).

    CAS  Article  Google Scholar 

  12. 12

    Cooper, S., Pera, M.F., Bennett, W. & Finch, J.T. A novel keratan sulfate proteoglycan from a human embryonal carcinoma cell-line. Biochem. J. 286, 959–966 (1992).

    CAS  Article  Google Scholar 

  13. 13

    Pera, M.F. et al. Analysis of cell-differentiation lineage in human teratomas using new monoclonal-antibodies to cytostructural antigens of embryonal carcinoma-cells . Differentiation 39, 139– 149 (1988).

    CAS  Article  Google Scholar 

  14. 14

    Badcock, G., Pigott, C., Goepel, J. & Andrews, P.W. The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulphate proteoglycan . Cancer Res. 59, 4715– 4719 (1999).

    CAS  PubMed  Google Scholar 

  15. 15

    Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    CAS  Article  Google Scholar 

  16. 16

    Roach, S., Cooper, S., Bennett, W. & Pera, M.F. Cultured cell lines from human germ cell tumours: windows into tumour growth and differentiation and early human development. Eur. Urol. 23, 82–88 (1993).

    CAS  Article  Google Scholar 

  17. 17

    Shamblott, M.J. et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95, 13726–13731 (1998).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    Andrews, P.W. et al. Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein 7). Lab. Invest. 71, 243–251 (1994).

    CAS  PubMed  Google Scholar 

  20. 20

    Caricasole, A.D. et al. In Inhibin, activin and follistatin: regulatory functions in system and cell biology (eds Aono, T., Sugino, H. & Vale, W.W.) 308–311 (Springer, New York, NY; 1997 ).

    Book  Google Scholar 

  21. 21

    Flax, J.D. et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat. Biotechnol. 16, 1033–1039 ( 1998).

    CAS  Article  Google Scholar 

  22. 22

    Kukekov, V.G. et al. Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp. Neurol. 156, 333–344 ( 1999).

    CAS  Article  Google Scholar 

  23. 23

    Dani, C. et al. Paracrine induction of stem cell renewal by LIF-deficient cells–a new ES cell regulatory pathway. Dev. Biol. 203, 149–162 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Rathjen, J. et al. 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 (1999).

    CAS  PubMed  Google Scholar 

  25. 25

    McWhir, J. et al. Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background. Nat. Genet. 14, 223– 226 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Li, M., Pevny, L., Lovell-Badge, R. & Smith, A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr. Biol. 8, 971– 974 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Fong, C.Y. & Bongso, A. Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture . Hum. Reprod. 14, 774– 781 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Fong, C.Y. et al. Ongoing pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human. Hum. Reprod. 12, 557–560 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Solter, D. & Knowles, B. Immunosurgery of mouse blastocyst . Proc. Natl. Acad. Sci. USA 72, 5099– 5102 (1975).

    CAS  Article  Google Scholar 

  30. 30

    Buehr, M. & Mclaren, A. Isolation and culture of primordial germ cells. Methods Enzymol. 225, 58– 76 (1993).

    CAS  Article  Google Scholar 

  31. 31

    van Eijk, M.J. et al. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol. Reprod. 60, 1093 –1103 (1999).

    CAS  Article  Google Scholar 

  32. 32

    Vescovi, A.L. et al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp. Neurol. 156, 71–83 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Neelands, T.R. et al. GABAa receptor pharmacology and subtype expression in human neuronal NT2-N cells. J. Neurosci. 18, 4993–5007 (1998).

    CAS  Article  Google Scholar 

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Acknowledgements

We gratefully acknowledge the assistance of Dr. Gary Dawson and the staff of the cytogenetics department, Monash Medical Centre, in the analysis of the karyotypes of the cell lines, and Mr. Peter Edwards of the biochemistry department, Monash Medical Centre, for immunoassay of human chorionic gonadotrophin and α-fetoprotein. Tianhao Xiang assisted with the RT-PCR assay for Oct-4. We thank Ms. Jacqui Johnson for assistance with cell culture.

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Correspondence to Martin F. Pera.

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Reubinoff, B., Pera, M., Fong, CY. et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399–404 (2000). https://doi.org/10.1038/74447

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