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

Research output: Contribution to journalArticle

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
Externally publishedYes

Fingerprint

Induced Pluripotent Stem Cells
Fibroblasts
Stem cells
Sorting
fibroblasts
flow cytometry
Flow Cytometry
Fluorescence
Cells
cell lines
Cell Line
Skin
methodology
infection
cells
Sendai virus
Biopsy
Virus Diseases
Infection
induced pluripotent stem cells

ASJC Scopus subject areas

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

Cite this

Kahler, D. J., Ahmad, F. S., Ritz, A., Hua, H., Moroziewicz, D. N., Sproul, A. A., ... 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

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting. / Kahler, David J.; Ahmad, Faizzan S.; Ritz, Anita; Hua, Haiqing; Moroziewicz, Dorota N.; Sproul, Andrew A.; Dusenberry, Carmen R.; Shang, Linshan; Paull, Daniel; Zimmer, Matthew; Weiss, Keren A.; Egli, Dieter; Noggle, Scott A.

In: PLoS One, Vol. 8, No. 3, e59867, 29.03.2013.

Research output: Contribution to journalArticle

Kahler, DJ, Ahmad, FS, Ritz, A, Hua, H, Moroziewicz, DN, Sproul, AA, Dusenberry, CR, Shang, L, Paull, D, Zimmer, M, Weiss, KA, Egli, D & Noggle, SA 2013, 'Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting', PLoS One, vol. 8, no. 3, e59867. https://doi.org/10.1371/journal.pone.0059867
Kahler, David J. ; Ahmad, Faizzan S. ; Ritz, Anita ; Hua, Haiqing ; Moroziewicz, Dorota N. ; Sproul, Andrew A. ; Dusenberry, Carmen R. ; Shang, Linshan ; Paull, Daniel ; Zimmer, Matthew ; Weiss, Keren A. ; Egli, Dieter ; Noggle, Scott A. / Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting. In: PLoS One. 2013 ; Vol. 8, No. 3.
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