Biological Sciences Division
Published February 25, 2015 | Version v1
Journal article Open

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

Creators

  • 1. The Scripps Research Institute
  • 2. University of California San Diego
  • 3. University of North Texas Health Science Center
  • 4. The Scripps Research Institute`
  • 5. University of Chicago
  • 6. University of Georgia

Description

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.

Notes

Due to the large number of authors, only the first 20 and the University of Chicago authors are included on the above author list. Please download the article for the complete list of authors.

Data availability

The hESC DNA methylation, gene expression, and SNP genotyping array data are available at the NCBI GEO database under the accession designation GSE34982. The hiPSC SNP genotyping data are available under accession number GSE56834. The hESC and hiPSC DNA methylation data are also available under SuperSeries GSE56851.

Files

journal.pone.0118307.pdf

Files (22.3 MB)

Name Size Download all
Article
md5:6b53893fd8d526bbc67e7b29f4eb82ee
12.0 MB Preview Download
md5:d4f7669fd9a426fe8ef9e60b67c3bf12
10.3 MB Preview Download

Additional details

Identifiers

DOI
10.1371/journal.pone.0118307
Other
oai:uchicago.tind.io:10862

Funding

California Institute for Regenerative Medicine
National Institutes of Health
Marie Mayer Foundation
Autism Speaks Foundation
Pew Charitable Trust
Hartwell Foundation
Millipore Foundation
Esther O'Keeffe Foundation
Wellcome Trust
University of California San Diego

UChicago Information

Division(s)
Biological Sciences Division
Department(s)
Human Genetics