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.
Details
Title
Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions
Author
Garitaonandia, Ibon : The Scripps Research Institute Amir, Hadar : University of California San Diego Sesillo Boscolo, Francesca : The Scripps Research Institute Wambua, Gerald K. : The Scripps Research Institute Schultheisz, Heather L. : The Scripps Research Institute Sabatini, Karen : The Scripps Research Institute Morey, Robert : The Scripps Research Institute Waltz, Shannon : The Scripps Research Institute Wang, Yu-Chieh : University of North Texas Health Science Center Tran, Ha : The Scripps Research Institute Leonardo, Trevor R. : The Scripps Research Institute Nazor, Kristopher : The Scripps Research Institute Slavin, Ileana : The Scripps Research Institute Lynch, Candace : The Scripps Research Institute` Li, Yingchun : University of California San Diego Coleman, Ronald : The Scripps Research Institute Gallego Romero, Irene : University of Chicago Altun, Gulsah : The Scripps Research Institute Reynolds, David : University of Georgia Parast, Mana : University of California San Diego
Data availability statement
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.
Funding Information
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
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.