Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Crucial role of c-Myc in the generation of induced pluripotent stem cells.

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.

Generation of genome integration-free induced pluripotent stem cells from fibroblasts of C57BL/6 mice without c-Myc transduction.

Although the induction of genome integration-free induced pluripotent stem cells (iPSCs) has been reported, c-Myc was still required for the efficient generation of these cells. Herein, we report mouse strain-dependent differences in the c-Myc dependence for iPSC generation and the successful generation of genome integration-free iPSCs without c-Myc transduction using C57BL/6 mouse embryonic fibroblasts. We performed 49 independent experiments and obtained a total of 24 iPSC clones, including 18 genome integration-free iPSC clones. These iPSCs were indistinguishable from embryonic stem cells and from iPSCs generated using other methods. Furthermore, the generation of three-factor iPSCs free of virus vectors revealed the contribution of c-Myc to the genomic integration of external genes. C57BL/6 is an inbred mouse strain with substantial advantages for use in genetic and molecular biological studies due to its use in the whole mouse genome sequencing project. Thus, the present series of C57BL/6 iPSCs generated by various procedures will serve as a valuable resource for future genetic studies of iPSC generation.

Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver.

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Growth retardation and skin abnormalities of the Recql4-deficient mouse.

Mutations in the Recql4 gene are very likely responsible for a subset of Rothmund-Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. Knockout mice in which the Recql4 gene is disrupted at exons 5-8 exhibit embryonic lethality at embryonic day 3.5-6.5. We generated a helicase activity-inhibited mouse by deleting exon 13 of Recql4, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The exon 13-deleted Recql4-deficient mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. Abnormalities in the Recql4-deficient mice are similar to those in RTS patients, suggesting that defects in the Recql4 gene may indeed be responsible for RTS. We speculate that the loss of Recql4 helicase activity results in the prematurely aged appearance observed in some RecQ helicase diseases.