title: Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro.
abstract: Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Ovchinnikov
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Drew M
authors.initials: DM
authors.lastname: Titmarsh
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Patrick R J
authors.initials: PR
authors.lastname: Fortuna
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Hidalgo
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Samah
authors.initials: S
authors.lastname: Alharbi
authors.affiliation: School of Veterinary Sciences, The University of Queensland, Gatton, QLD 4343, Australia.
authors.forename: Deanne J
authors.initials: DJ
authors.lastname: Whitworth
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Cooper-White
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia. Electronic address: e.wolvetang@uq.edu.au.
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Wolvetang
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D024881:Drug Resistance, Bacterial / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D046210:Microfluidic Analytical Techniques; D010641:Phenotype; D011691:Puromycin / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011691:Puromycin