title: Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.
abstract: The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Ibon
authors.initials: I
authors.lastname: Garitaonandia
authors.affiliation: Department of Reproductive Medicine, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, San Diego, CA 92093, United States of America.
authors.forename: Hadar
authors.initials: H
authors.lastname: Amir
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America; Department of Reproductive Medicine, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, San Diego, CA 92093, United States of America.
authors.forename: Francesca Sesillo
authors.initials: FS
authors.lastname: Boscolo
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Gerald K
authors.initials: GK
authors.lastname: Wambua
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Schultheisz
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America; Department of Reproductive Medicine, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, San Diego, CA 92093, United States of America.
authors.forename: Karen
authors.initials: K
authors.lastname: Sabatini
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America; Department of Reproductive Medicine, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, San Diego, CA 92093, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Morey
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Shannon
authors.initials: S
authors.lastname: Waltz
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Yu-Chieh
authors.initials: YC
authors.lastname: Wang
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Ha
authors.initials: H
authors.lastname: Tran
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Leonardo
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Kristopher
authors.initials: K
authors.lastname: Nazor
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Ileana
authors.initials: I
authors.lastname: Slavin
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Candace
authors.initials: C
authors.lastname: Lynch
authors.affiliation: Department of Pathology, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, La Jolla, CA 92093-0612, United States of America.
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Li
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Ronald
authors.initials: R
authors.lastname: Coleman
authors.affiliation: Department of Human Genetics, University of Chicago, 920 E 58th St, CLSC 317, Chicago, IL, 60637, United States of America.
authors.forename: Irene
authors.initials: I
authors.lastname: Gallego Romero
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Gulsah
authors.initials: G
authors.lastname: Altun
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA, 30602, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Reynolds
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA, 30602, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Dalton
authors.affiliation: Department of Pathology, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, La Jolla, CA 92093-0612, United States of America.
authors.forename: Mana
authors.initials: M
authors.lastname: Parast
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.
authors.forename: Jeanne F
authors.initials: JF
authors.lastname: Loring
authors.affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America; Department of Reproductive Medicine, UCSD Healthcare, 9500 Gilman Drive, Mail Code 0695, San Diego, CA 92093, United States of America.
authors.forename: Louise C
authors.initials: LC
authors.lastname: Laurent
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D000066673:Cell Self Renewal; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured; D002869:Chromosome Aberrations; D002872:Chromosome Deletion; D058674:Chromosome Duplication; D002881:Chromosomes, Human, Pair 12; D002886:Chromosomes, Human, Pair 17; D002890:Chromosomes, Human, Pair 20; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D020869:Gene Expression Profiling; D015894:Genome, Human; D042822:Genomic Instability; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D016159:Tumor Suppressor Protein p53