Yuko Hoki

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. STEM CELLS 2011; 29:1362–1370

Conversion of Ancestral Fibroblasts to Induced Pluripotent Stem Cells

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. Interestingly, this conversion occurred within 48 hours after gene introduction in most iPSC generations. This is the first report to directly observe a cell lineage conversion of somatic cells to stem cells and provides a critical new insight into the “black box” of iPSCs, that is, the first three days of their generation. STEM CELLS 2010;28:213–220

Induced Pluripotent Stem Cell Generation-Associated Point Mutations Arise during the Initial Stages of the Conversion of These Cells

Summary A 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.

Effect of Atm Disruption on Spontaneously Arising and Radiation-Induced Deletion Mutations in Mouse Liver

Abstract Furuno-Fukushi, I., Masumura, K., Furuse, T., Noda, Y., Takahagi, M., Saito, T., Hoki, Y., Suzuki, H., Wynshaw-Boris, A., Nohmi, T. and Tatsumi, K. Effect of Atm Disruption on Spontaneously Arising and Radiation-Induced Deletion Mutations in Mouse Liver. Radiat. Res. 160, 549–558 (2003). 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 2.8 × 10−6. The livers from Atm−/− mice yielded a virtually identical dose–response curve for X rays with a background fraction of 2.4 × 10−6. 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.

X‐Irradiation Induces Up‐regulation of ATM Gene Expression in Wild‐type Lymphoblastoid Cell Lines, but Not in Their Heterozygous or Homozygous Ataxia‐telangiectasia Counterparts

Ataxia‐telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradi‐ated and X‐irradiated EBV (Epstein‐Barr virus)‐transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM+/+), AT heterozygotes (ATM+/−), or AT homozy‐gotes (ATM−/−), respectively. In ATM+/+ LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5‐fold within 1 h of exposure to 10 Gy of X‐rays, while the ATM protein levels had increased by 1.5‐ to 2.0‐fold within 2 to 3 h of irradiation. The wild‐type mRNA and protein levels both returned to their basal values fairly quickly after this tune. The results obtained with the ATM+/− LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X‐irradiation in any ATM+/− LCL. Twelve of the mutations in the ATM−/− LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM−/− LCLs produced mutant ATM proteins that were identical in molecular weight to the wild‐type ATM protein. When cells from three of these five clones were exposed to X‐rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up‐regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.