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.

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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.

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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

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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.

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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

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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

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