Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

David J. Kahler, Faizzan S. Ahmad, Anita Ritz, Haiqing Hua, Dorota N. Moroziewicz, Andrew A. Sproul, Carmen R. Dusenberry, Linshan Shang, Daniel Paull, Matthew Zimmer, Keren A. Weiss, Dieter Egli, Scott A. Noggle

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    26 Citations (Scopus)

    Abstract

    Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13NEGSSEA4POSTra-1-60POS on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

    Original languageEnglish
    Article numbere59867
    JournalPloS one
    Volume8
    Issue number3
    DOIs
    Publication statusPublished - 29 Mar 2013

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    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)
    • General

    Cite this

    Kahler, D. J., Ahmad, F. S., Ritz, A., Hua, H., Moroziewicz, D. N., Sproul, A. A., Dusenberry, C. R., Shang, L., Paull, D., Zimmer, M., Weiss, K. A., Egli, D., & Noggle, S. A. (2013). Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting. PloS one, 8(3), [e59867]. https://doi.org/10.1371/journal.pone.0059867