Mutation Frequency Dynamics inHPRTLocus in Culture-Adapted Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Correspond to Their Differentiated Counterparts

Abstract

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing

“…The hiPSC line cl1 was obtained from Dr. Majlinda Lako, Institute of Genetic Medicine, Newcastle University and the iPSC lines MDMD2Se were derived in our laboratory from human skin biopsies (Krutá et al 2014). The hESCs and iPSC lines were propagated on mitotically inactivated mouse embryonic fibroblasts (Dvorak et al, 2005).…”

Effect of ionizing radiation on the proliferation of human embryonic stem cells

“…the colony is the true unit of the hESC population 21 . Therefore, dissociation of a colony to a single cell suspension, which is commonly used in the literature to assess hESC growth rate 30,31 may result in a different estimation of DT, at least due to the lag period needed for re-establishing the colony. Certainly more studies are required to test this hypothesis.…”

Ligase 3–mediated end‐joining maintains genome stability of human embryonic stem cells

“…hESCs were shown to accumulate various types of mutations (2)(3)(4). At the same time and based mostly on the reporter gene assays, hESCs were reported to have lower mutant frequency (MF) than their differentiated counterparts (5)(6)(7). In order to maintain their genomic integrity, hESCs were shown to have increased mRNAs and protein levels of several DNA repair genes (8)(9)(10).…”

“…In mammalian cells, BER attempts to repair clustered damaged bases that are on the opposing strands within a few helical turns by generating double-strand breaks (DSBs) (19,20), which are characterized by short homologous overhangs. We previously demonstrated that hESCs (with elevated BER) release large amounts of ABBREVIATIONS: gH2AX, H2A histone family member X phosphorylated on Ser139; 53BP1, p53 binding protein 1; BER, base excision repair; CCTL, Centre for Cell Therapy line; Cdc25C, cell division cycle 25C; Chk, checkpoint kinase; cNHEJ, canonical nonhomologous EJ; DNA PKcs, DNA-dependent protein kinase catalytic subunit; DSB, DNA doublestrand break; EJ, end-joining; GFP, green fluorescent protein; hESC, human embryonic stem cell; HPRT, hypoxanthine phosphoribosyltransferase; HR, homologous recombination; ICC, immunocytochemistry; IR, irradiation; Lig, ligaseMF, mutant frequency; MMEJ, microhomologymediated EJ; Nu7026, 2-(morpholin-4-yl)-benzo[h]chromen-4-one; PARP1, poly (ADP-ribose) polymerase 1; pChk1, phosphorylated Chk1; pChk2, phosphorylated Chk2; PFGE, pulse-field gel electrophoresis; PolQ, DNA polymerase u; Rad51, Rad51 recombinase (recombination protein A homolog); SCE, sister chromatid exchange; siRNA, small interfering RNA; WB, Western blot DSBs upon induction with IR (5,14). Such DSBs trigger phosphorylation of histone H2A family member X at Ser139 and form gH2AX foci, which serve as a biomarker of DSBs (21) and can lead to retardation of cell cycle progression.…”

“…Therefore, BER-mediated DSBs are important for DNA damage response signaling in hESCs. Both downregulation associated with in vitro cultivation and intentional down-regulation of apurinic/apyrimidinic endonuclease 1 can contribute to genome instability in hESCs (5).…”