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Induced pluripotent stem cells are sensitive to DNA damage.

ABSTRACT: Induced pluripotent stem cells (iPSCs) resemble embryonic stem cells (ESCs) in morphology, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts have been made to improve reprogramming efficiency, significant problems such as genomic instability of iPSCs need to be addressed before clinical therapy. In this study, we try to figure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation (IR). We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fibroblast (MEF) cells, from which 3FB4-1line was derived. After the introduction of DNA damage by IR, the number of ?-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF, albeit both significantly (P<0.01). In addition, whole-genome sequencing analysis showed that after IR, 3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes. These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity. The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.

SUBMITTER: Zhang M

PROVIDER: S-EPMC4357839 | biostudies-other | 2013 Oct

REPOSITORIES: biostudies-other

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Induced pluripotent stem cells are sensitive to DNA damage.

Zhang Minjie M Yang Caiyun C Liu Huixian H Sun Yingli Y

Genomics, proteomics & bioinformatics 20131004 5

Induced pluripotent stem cells (iPSCs) resemble embryonic stem cells (ESCs) in morphology, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts have been made to improve reprogramming efficiency, significant problems such as genomic instability of iPSCs need to be addressed before clinical therapy. In this study, we try to figure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation (IR). We found t ...[more]

PMID: 24100274

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Project description:BackgroundInduced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Methodology/principal findingsHere, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after ?-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle, displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of ?-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.Conclusions/significanceHigh degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

Project description:UnlabelledComprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest.SignificanceThe results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent stem cells (iPSCs). The data suggest developmental age-dependent differences in DNA damage repair capacity. In iPSCs (closest to embryonic stem cells), the highest expression level of DNA damage response and repair genes was found, followed by neonatal stromal cells and adult stromal cells with the lowest overall expression. In addition, a differentiation-dependent downregulation of repair capacity was observed during osteogenic differentiation in neonatal stromal cells. Notably, the impact of genotoxic stress on osteogenic differentiation depended on the time the genotoxic insult took place and, moreover, was agent-specific. These results strongly support the necessity of offering and establishing adequate cell sources for informative toxicological testing matching to the developmental age and differentiation status of the respective cell of interest.

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Induced pluripotent stem cells are sensitive to DNA damage.

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Induced pluripotent stem cells are sensitive to DNA damage.

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