Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.
Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine.
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK; Sheffield Diagnostic Genetic Services, Sheffield Children's Hospital, Sheffield S10 2TH, UK.
Duncan
D
Baker
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK.
Adam J
AJ
Hirst
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK.
Paul J
PJ
Gokhale
School of Mathematics and Statistics, The University of Sheffield, Sheffield S3 7RH, UK.
Miguel A
MA
Juarez
Sheffield Diagnostic Genetic Services, Sheffield Children's Hospital, Sheffield S10 2TH, UK.
Steve
S
Williams
Sheffield Diagnostic Genetic Services, Sheffield Children's Hospital, Sheffield S10 2TH, UK.
Mark
M
Wheeler
Sheffield Diagnostic Genetic Services, Sheffield Children's Hospital, Sheffield S10 2TH, UK.
Kerry
K
Bean
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK.
Thomas F
TF
Allison
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK.
Harry D
HD
Moore
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK.
Peter W
PW
Andrews
Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK. Electronic address: i.barbaric@sheffield.ac.uk.
Ivana
I
Barbaric
D018929:Cell Culture Techniques; D002460:Cell Line; D002877:Chromosomes, Human; D002886:Chromosomes, Human, Pair 17; D002890:Chromosomes, Human, Pair 20; D056915:DNA Copy Number Variations; D014644:Genetic Variation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D059785:Karyotype; D009030:Mosaicism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014314:Trisomy
Human pluripotent stem cells; detection methods; genetic changes; sensitivity