title: Overcoming Pluripotent Stem Cell Dependence on the Repair of Endogenous DNA Damage.
abstract: Pluripotent stem cells (PSCs) maintain a low mutation frequency compared with somatic cell types at least in part by preferentially utilizing error-free homologous recombination (HR) for DNA repair. Many endogenous metabolites cause DNA interstrand crosslinks, which are repaired by the Fanconi anemia (FA) pathway using HR. To determine the effect of failed repair of endogenous DNA lesions on PSC biology, we generated iPSCs harboring a conditional FA pathway. Upon FA pathway loss, iPSCs maintained pluripotency but underwent profound G2 arrest and apoptosis, whereas parental fibroblasts grew normally. Mechanistic studies revealed that G2-phase FA-deficient iPSCs possess large γH2AX-RAD51 foci indicative of accrued DNA damage, which correlated with activated DNA-damage signaling through CHK1. CHK1 inhibition specifically rescued the growth of FA-deficient iPSCs for prolonged culture periods, surprisingly without stimulating excessive karyotypic abnormalities. These studies reveal that PSCs possess hyperactive CHK1 signaling that restricts their self-renewal in the absence of error-free DNA repair.
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Chlon
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Sonya
authors.initials: S
authors.lastname: Ruiz-Torres
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Logan
authors.initials: L
authors.lastname: Maag
authors.affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Mayhew
authors.affiliation: Divisions of Pathology and Laboratory Medicine and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA; Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45220, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Wikenheiser-Brokamp
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Stella M
authors.initials: SM
authors.lastname: Davies
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Parinda
authors.initials: P
authors.lastname: Mehta
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA.
authors.forename: Kasiani C
authors.initials: KC
authors.lastname: Myers
authors.affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Wells
authors.affiliation: Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC-7013, Cincinnati, OH 45229, USA. Electronic address: susanne.wells@cchmc.org.
authors.forename: Susanne I
authors.initials: SI
authors.lastname: Wells
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D006657:Histones / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D012867:Skin / Q000378:metabolism / Q000473:pathology
chemical_list: D051856:Fanconi Anemia Complementation Group Proteins; C468783:H2AX protein, human; D006657:Histones; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051135:Rad51 Recombinase