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Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.

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Figure 1: FACS and immunofluorescence time-course analysis of human fibroblast reprogramming.
Figure 2: Characterization of types I, II and III colonies.

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Acknowledgements

G.Q.D. was supported by grants from the National Institutes of Health (NIH), the NIH Director's Pioneer Award of the NIH Roadmap for Medical Research, and private funds contributed to the Harvard Stem Cell Institute and the Children's Hospital Stem Cell Program. G.Q.D. is a recipient of Clinical Scientist Awards in Translational Research from the Burroughs Wellcome Fund and the Leukemia and Lymphoma Society, and is an investigator of the Howard Hughes Medical Institute. T.M.S. was supported by the Harvard Stem Cell Institute and the Children's Hospital Stem Cell Program. Y.-H.L. is funded by the Agency of Science, Technology and Research International Fellowship and the Institute of Medical Biology, Singapore. E.M.C. was funded by The Canadian Institutes of Health Research. T.A.I. was supported by Breast Cancer Research Foundation, NY, and Department of Defense, Congressionally Directed Medical Research Programs (CDMRPs) and Breast Cancer Research Program (BCRP). We thank T. Woo (BWH) for her assistance with immnuhistopathological stains of the teratoma sections.

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Contributions

Co-direction of the project: G.Q.D. and T.M.S. Study concept and design: T.M.S., G.Q.D., E.M.C., S.R. and I.-H.P. Live imaging reprogramming studies: T.M.S., E.M.C., S.R. and P.D.M. Isolation of cell lines: E.M.C. and S.R. Characterization of cell lines: E.M.C., S.R., I.H.P., Y.H.-L., H.H. and J.R. Human iPS cell reprogramming comparison on chemically defined conditions on Matrigel versus KOSR media on feeders: S.R., J.D.M. and O.H. Teratoma interpretation and characterization: T.A.I.

Corresponding authors

Correspondence to George Q Daley or Thorsten M Schlaeger.

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Competing interests

G.Q.D. is founder of iPierian, a biotechnology company that is using iPS cells in drug discovery in neurodegenerative diseases.

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Chan, E., Ratanasirintrawoot, S., Park, IH. et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol 27, 1033–1037 (2009). https://doi.org/10.1038/nbt.1580

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