title: Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.
abstract: The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.
authors.affiliation: Department of Cardiovascular Surgery, Division of Experimental Surgery, German Heart Center Munich, Munich Heart Alliance.
authors.forename: Harald
authors.initials: H
authors.lastname: Lahm
authors.forename: Stefanie
authors.initials: S
authors.lastname: Doppler
authors.forename: Martina
authors.initials: M
authors.lastname: Dreßen
authors.forename: Astrid
authors.initials: A
authors.lastname: Werner
authors.forename: Klaudia
authors.initials: K
authors.lastname: Adamczyk
authors.forename: Dominic
authors.initials: D
authors.lastname: Schrambke
authors.forename: Thomas
authors.initials: T
authors.lastname: Brade
authors.forename: Karl-Ludwig
authors.initials: KL
authors.lastname: Laugwitz
authors.forename: Marcus-André
authors.initials: MA
authors.lastname: Deutsch
authors.forename: Matthias
authors.initials: M
authors.lastname: Schiemann
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Lange
authors.forename: Alessandra
authors.initials: A
authors.lastname: Moretti
authors.forename: Markus
authors.initials: M
authors.lastname: Krane
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology*; D065150:Cellular Reprogramming; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009841:Oligonucleotides / Q000494:pharmacology*; D013552:Swine
chemical_list: D002232:Carbocyanines; D009841:Oligonucleotides; C089187:cyanine dye 3
keywords: Embryonic stem cells; Fluorescent nanoparticles; Induced pluripotent stem cells; Live staining; Pluripotency