title: Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.
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 CD13(NEG)SSEA4(POS)Tra-1-60(POS) 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.
authors.affiliation: The New York Stem Cell Foundation, New York, New York, United States of America. dkahler@nyscf.org
authors.forename: David J
authors.initials: DJ
authors.lastname: Kahler
authors.forename: Faizzan S
authors.initials: FS
authors.lastname: Ahmad
authors.forename: Anita
authors.initials: A
authors.lastname: Ritz
authors.forename: Haiqing
authors.initials: H
authors.lastname: Hua
authors.forename: Dorota N
authors.initials: DN
authors.lastname: Moroziewicz
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Sproul
authors.forename: Carmen R
authors.initials: CR
authors.lastname: Dusenberry
authors.forename: Linshan
authors.initials: L
authors.lastname: Shang
authors.forename: Daniel
authors.initials: D
authors.lastname: Paull
authors.forename: Matthew
authors.initials: M
authors.lastname: Zimmer
authors.forename: Keren A
authors.initials: KA
authors.lastname: Weiss
authors.forename: Dieter
authors.initials: D
authors.lastname: Egli
authors.forename: Scott A
authors.initials: SA
authors.lastname: Noggle
mesh_terms: D000328:Adult; D000818:Animals; D001706:Biopsy; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D007621:Karyotyping; D008297:Male; D051379:Mice; D008875:Middle Aged; D012867:Skin / Q000166:cytology* / Q000473:pathology; D013724:Teratoma / Q000473:pathology; D013997:Time Factors
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