Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Isolation of amniotic stem cell lines with potential for therapy

Abstract

Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid–derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Clonal human AFS cells have a normal karyotype and retain long telomeres.
Figure 2: Clonal human AFS cells are broadly multipotent.
Figure 3: Neurogenic differentiation of human AFS cells in culture.
Figure 4: Engraftment of neurogenically differentiated human AFS cells in mouse brain.
Figure 5: Urea secretion by human AFS cells after hepatogenic in vitro differentiation.
Figure 6: Tissue engineered bone from human AFS cells.

Similar content being viewed by others

References

  1. Priest, R.E., Marimuthu, K.M. & Priest, J.H. Origin of cells in human amniotic fluid cultures: ultrastructural features. Lab. Invest. 39, 106–109 (1978).

    CAS  PubMed  Google Scholar 

  2. Polgar, K. et al. Characterization of rapidly adhering amniotic fluid cells by combined immunofluorescence and phagocytosis assays. Am. J. Hum. Genet. 45, 786–792 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. DeCoppi, P. et al. Human fetal stem cell isolation from amniotic fluid. In American Academy of Pediatrics National Conference, p. 210–211, (San Francisco, 2001).

  4. In 't Anker, P.S. et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 102, 1548–1549 (2003).

    Article  CAS  Google Scholar 

  5. Tsai, M.S., Lee, J.L., Chang, Y.J. & Hwang, S.M. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum. Reprod. 19, 1450–1456 (2004).

    Article  Google Scholar 

  6. Prusa, A.R. et al. Neurogenic cells in human amniotic fluid. Am. J. Obstet. Gynecol. 191, 309–314 (2004).

    Article  Google Scholar 

  7. Taylor, R.M. & Snyder, E.Y. Widespread engraftment of neural progenitor and stem-like cells throughout the mouse brain. Transplant. Proc. 29, 845–847 (1997).

    Article  CAS  Google Scholar 

  8. Zsebo, K.M. et al. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213–224 (1990).

    Article  CAS  Google Scholar 

  9. Barry, F.P., Boynton, R.E., Haynesworth, S., Murphy, J.M. & Zaia, J. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem. Biophys. Res. Commun. 265, 134–139 (1999).

    Article  CAS  Google Scholar 

  10. Barry, F., Boynton, R., Murphy, M., Haynesworth, S. & Zaia, J. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem. Biophys. Res. Commun. 289, 519–524 (2001).

    Article  CAS  Google Scholar 

  11. Kannagi, R. et al. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 2, 2355–2361 (1983).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Carpenter, M.K., Rosler, E. & Rao, M.S. Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 5, 79–88 (2003).

    Article  CAS  Google Scholar 

  14. 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).

    Article  CAS  Google Scholar 

  15. Pan, G.J., Chang, Z.Y., Scholer, H.R. & Pei, D. Stem cell pluripotency and transcription factor Oct4. Cell Res. 12, 321–329 (2002).

    Article  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  18. Cowan, C.A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

    Article  CAS  Google Scholar 

  19. Bryan, T.M., Englezou, A., Dunham, M.A. & Reddel, R.R. Telomere length dynamics in telomerase-positive immortal human cell populations. Exp. Cell Res. 239, 370–378 (1998).

    Article  CAS  Google Scholar 

  20. Siddiqui, M.M. & Atala, A. Amniotic fluid-derived pluripotential cells: adult and fetal. In Handbook of Stem Cells, Vol. 2. (eds. R. Lanza et al.) 175–180, (Elsevier Academic Press, Amsterdam, 2004).

    Chapter  Google Scholar 

  21. Wu, X. & Burgess, S.M. Integration target site selection for retroviruses and transposable elements. Cell. Mol. Life Sci. 61, 2588–2596 (2004).

    Article  CAS  Google Scholar 

  22. Lendahl, U., Zimmerman, L.B. & McKay, R.D. CNS stem cells express a new class of intermediate filament protein. Cell 60, 585–595 (1990).

    Article  CAS  Google Scholar 

  23. Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).

    Article  CAS  Google Scholar 

  24. Liao, Y.J., Jan, Y.N. & Jan, L.Y. Heteromultimerization of G-protein-gated inwardly rectifying K+ channel proteins GIRK1 and GIRK2 and their altered expression in weaver brain. J. Neurosci. 16, 7137–7150 (1996).

    Article  CAS  Google Scholar 

  25. Taylor, R.M. et al. Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: evidence from a mouse model of Krabbe leukodystrophy. J. Neurochem. 97, 1585–1599 (2006).

    Article  CAS  Google Scholar 

  26. Suzuki, K. & Suzuki, K. The twitcher mouse: a model for Krabbe disease and for experimental therapies. Brain Pathol. 5, 249–258 (1995).

    Article  CAS  Google Scholar 

  27. Rodan, G.A. & Noda, M. Gene expression in osteoblastic cells. Crit. Rev. Eukaryot. Gene Expr. 1, 85–98 (1991).

    CAS  PubMed  Google Scholar 

  28. Roth, E.A. et al. Inkjet printing for high-throughput cell patterning. Biomaterials 25, 3707–3715 (2004).

    Article  CAS  Google Scholar 

  29. Xu, T., Jin, J., Gregory, C., Hickman, J.J. & Boland, T. Inkjet printing of viable mammalian cells. Biomaterials 26, 93–99 (2005).

    Article  Google Scholar 

  30. Shay, J.W. & Wright, W.E. Hayflick, his limit, and cellular ageing. Nat. Rev. Mol. Cell Biol. 1, 72–76 (2000).

    Article  CAS  Google Scholar 

  31. Gosden, C.M. Amniotic fluid cell types and culture. Br. Med. Bull. 39, 348–354 (1983).

    Article  CAS  Google Scholar 

  32. Chabot, B., Stephenson, D.A., Chapman, V.M., Besmer, P. & Bernstein, A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335, 88–89 (1988).

    Article  CAS  Google Scholar 

  33. Fleischman, R.A. From white spots to stem cells: the role of the Kit receptor in mammalian development. Trends Genet. 9, 285–290 (1993).

    Article  CAS  Google Scholar 

  34. Hoffman, L.M. & Carpenter, M.K. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23, 699–708 (2005).

    Article  CAS  Google Scholar 

  35. Guo, C.S., Wehrle-Haller, B., Rossi, J. & Ciment, G. Autocrine regulation of neural crest cell development by steel factor. Dev. Biol. 184, 61–69 (1997).

    Article  CAS  Google Scholar 

  36. Crane, J.F. & Trainor, P.A. Neural crest stem and progenitor cells. Annu. Rev. Cell Dev. Biol. 22, 267–286 (2006).

    Article  CAS  Google Scholar 

  37. Hipp, J. & Atala, A. GeneChips in regenerative medicine. In Principles of Regenerative Medicine. (eds. A. Atala, R. Lanza, J.A. Thomson & R.M. Nerem) in press (Elsevier, Philadelphia, 2006).

    Google Scholar 

  38. Atala, A. Recent developments in tissue engineering and regenerative medicine. Curr. Opin. Pediatr. 18, 167–171 (2006).

    Article  Google Scholar 

  39. Morris, S.M., Jr. Regulation of enzymes of the urea cycle and arginine metabolism. Annu. Rev. Nutr. 22, 87–105 (2002).

    Article  CAS  Google Scholar 

  40. Klein, C., Bueler, H. & Mulligan, R.C. Comparative analysis of genetically modified dendritic cells and tumor cells as therapeutic cancer vaccines. J. Exp. Med. 191, 1699–1708 (2000).

    Article  CAS  Google Scholar 

  41. Jaiswal, N., Haynesworth, S.E., Caplan, A.I. & Bruder, S.P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J. Cell. Biochem. 64, 295–312 (1997).

    Article  CAS  Google Scholar 

  42. Ferrari, G. et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528–1530 (1998).

    Article  CAS  Google Scholar 

  43. Rosenblatt, J.D., Lunt, A.I., Parry, D.J. & Partridge, T.A. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell. Dev. Biol. Anim. 31, 773–779 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Schwartz, R.E. et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J. Clin. Invest. 109, 1291–1302 (2002).

    Article  CAS  Google Scholar 

  46. Woodbury, D., Schwarz, E.J., Prockop, D.J. & Black, I.B. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370 (2000).

    Article  CAS  Google Scholar 

  47. Hampson, R.E., Zhuang, S.Y., Weiner, J.L. & Deadwyler, S.A. Functional significance of cannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) in the hippocampus. J. Neurophysiol. 90, 55–64 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge support from the Joshua Frase Foundation, Fondazione Citta' della Speranza, the Crown Foundation and the March of Dimes. We are grateful to Paola Dal Cin and Mark Pettenati for access to amniocentesis specimens, Sam Deadwyler and Robert Hampson for electrophysiology, and Daragh Conrad and Heather Mertz for the cover image.

Author information

Authors and Affiliations

Authors

Contributions

P.D.C., G.B. and M.M.S., cell isolation and in vitro differentiation; T.X. and J.J.Y., in vivo bone engineering; C.C.S. and S.S., neuronal differentiation; L.P., telomere length; G.M. and M.M.S., retroviral marking; A.C.S. and E.Y.S., brain engraftment; S.S. and M.E.F., molecular analysis; A.A., principal investigator.

Corresponding author

Correspondence to Anthony Atala.

Ethics declarations

Competing interests

A.A. assigned to Children's Hospital Boston a patent involved with this technology and Children's Hospital licensed the patent to Plureon, Inc. A.A. serves as a member of the board of directors of Plureon, Inc.

Supplementary information

Supplementary Fig. 1

Marker expression by human AFS cells.

Supplementary Fig. 2

Marker expression by mouse AFS cells.

Supplementary Fig. 3

Osteogenic differentiation of human AFS cells.

Supplementary Table 1

Marker expression of cells from human amniotic fluid.

Supplementary Methods

Supplementary Video

Rights and permissions

Reprints and permissions

About this article

Cite this article

De Coppi, P., Bartsch, G., Siddiqui, M. et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25, 100–106 (2007). https://doi.org/10.1038/nbt1274

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1274

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing