TY - JOUR
T1 - Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions
AU - Garitaonandia, Ibon
AU - Amir, Hadar
AU - Boscolo, Francesca Sesillo
AU - Wambua, Gerald K.
AU - Schultheisz, Heather L.
AU - Sabatini, Karen
AU - Morey, Robert
AU - Waltz, Shannon
AU - Wang, Yu Chieh
AU - Tran, Ha
AU - Leonardo, Trevor R.
AU - Nazor, Kristopher
AU - Slavin, Ileana
AU - Lynch, Candace
AU - Li, Yingchun
AU - Coleman, Ronald
AU - Romero, Irene Gallego
AU - Altun, Gulsah
AU - Reynolds, David
AU - Dalton, Stephen
AU - Parast, Mana
AU - Loring, Jeanne F.
AU - Laurent, Louise C.
N1 - Funding Information:
The research was funded by grants to JFL from the California Institute for Regenerative Medicine (CL1–00502, RT1–01108, TR1–01250, RM1–01717), the NIH (R33MH087925), and the UCSD Department of Reproductive Medicine. LCL was supported by an NIH/NICHD K12 Career Development Award, the Hartwell Foundation, and the UCSD Department of Reproductive Medicine. KS was supported by a Millipore Foundation grant to JFL and GA by an Esther O’Keefe Foundation grant to JFL. FSB, GW, SW, and JRS were supported by the CIRM Bridges to Stem Cell Research Internship program (TB1–01193). IG, HLS and IS were supported by CIRM Training grant TG2–01165. YCW was supported by the Marie Mayer Fellowship. KN was supported by an Autism Speaks Dennis Weatherstone Fellowship. IS was supported by the Pew Charitable Trust. IGR was supported by a Wellcome Trust Fellowship. SD was supported by the NIH (P01 GM085354 and PO1 HL089471). YL, LCL and MP were supported by CIRM grant RN2–00931–1 to M.P.
Publisher Copyright:
© 2015 Garitaonandia et al.
PY - 2015/2/25
Y1 - 2015/2/25
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84923869698&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0118307
DO - 10.1371/journal.pone.0118307
M3 - Article
C2 - 25714340
AN - SCOPUS:84923869698
SN - 1932-6203
VL - 10
JO - PLoS ONE
JF - PLoS ONE
IS - 2
M1 - e0118307
ER -