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.

SummaryA large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations.

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 cMyc (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 highquality iPSCs.

The emergence of induced pluripotent stem cells (iPSCs) from an ancestral somatic cell is one of the most important processes underlying their generation, but the mechanism has yet to be identified. This is principally because these cells emerge at a low frequency, about 0.1% in the case of fibroblasts, and in a stochastic manner. In our current study, we succeeded in identifying ancestral fibroblasts and the subsequent processes leading to their conversion to iPSCs. The ancestral fibroblasts were found to divide several times in a morphologically symmetric manner, maintaining a fibroblastic shape, and then gradually transform into embryonic stem-like cells.

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.