title: FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.
abstract: Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage.
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium. Electronic address: kim.vanuytsel@med.kuleuven.be.
authors.forename: Kim
authors.initials: K
authors.lastname: Vanuytsel
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Qing
authors.initials: Q
authors.lastname: Cai
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Nisha
authors.initials: N
authors.lastname: Nair
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Satish
authors.initials: S
authors.lastname: Khurana
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Swati
authors.initials: S
authors.lastname: Shetty
authors.affiliation: KU Leuven Center for Human Genetics, 3000 Leuven, Belgium.
authors.forename: Joris R
authors.initials: JR
authors.lastname: Vermeesch
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Laura
authors.initials: L
authors.lastname: Ordovas
authors.affiliation: KU Leuven Stem Cell Institute, Department of Development and Regeneration, Cluster Stem Cell Biology and Embryology, Herestraat 49, Onderwijs en Navorsing 4, Box 804, 3000 Leuven, Belgium.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Verfaillie
mesh_terms: D059447:Cell Cycle Checkpoints; D002460:Cell Line; D049109:Cell Proliferation; D018920:Coculture Techniques; D015536:Down-Regulation; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000473:pathology; D005091:Exons; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics / Q000378:metabolism*; D061252:Feeder Cells; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D005838:Genotype; D006801:Humans; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein