title: Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells.
abstract: The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs).
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Ellen
authors.initials: E
authors.lastname: Scheerlinck
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Steendam
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Mado
authors.initials: M
authors.lastname: Vandewoestyne
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Trees
authors.initials: T
authors.lastname: Lepez
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Gobin
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Paulien
authors.initials: P
authors.lastname: Meert
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Vossaert
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
authors.affiliation: Department of Nutrition, Genetics, and Ethology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Peelman
authors.affiliation: Department for Reproductive Medicine, Ghent University Hospital, B-9000 Ghent, Belgium.
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
authors.affiliation: Department for Reproductive Medicine, Ghent University Hospital, B-9000 Ghent, Belgium.
authors.forename: Petra
authors.initials: P
authors.lastname: De Sutter
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dhaenens
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium. Electronic address: dieter.deforce@ugent.be.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D055879:Gene Knock-In Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology; D019096:Vitronectin / Q000494:pharmacology
chemical_list: D050814:Octamer Transcription Factor-3; D019096:Vitronectin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin
keywords: Flow cytometry; Fluorescence microscopy; MEF CM; OCT4; OCT4–eGFP knock-in hESC