Identification of Inappropriately Reprogrammed Genes by Large-Scale Transcriptome Analysis of Individual Cloned Mouse Blastocysts

Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos

A mouse strain that is sensitive for iPS cellgeneration: C57BL/6

There are many similar aspects between cancer cells and iPS cells (iPSCs), and it is well known that some biological phenomena, including tumorigenesis, depend on the mouse strain. Therefore, we examined six mouse strains including three inbred strains, C3H, 129, and C57BL/6, and found a clear difference in iPS generation between these strains. C57BL/6 was revealed to be a strain of which embryonic fibroblasts (MEF) cause iPSC with a high incidence rate, and an efficient iPS cells generation without c-Myc transduction was also shown in the strain. \nHere we succeeded in generating iPSCs from C57BL/6 mice MEF using the virus-free and c-Myc-free gene transduction system with 2A-3F vector that contains Oct3/4, Klf4 and Sox2 genes. As expected, several integration-free colonies were included among these iPS clones, and such clones were indistinguishable from other iPS cells which have been established with various methods thus far, using retrovirus-mediated gene transduction system and with the defined four factors including c-Myc. In addition to the gene expression profiling analysis, their developmental potential was examined by the teratomas formation and chimera mice formation tests. \nFurthermore, we also succeeded in generating iPSCs with three factors excluding Sox2 from the MEF of C57BL/6 with retro virus-mediated gene transduction system. Characterization of the iPSCs (-Sox2) was performed with the same analyses for the iPSCs generated by 2A-3F plasmid, and then their quality was verified. \nVarious cells, including embryonic fibroblasts, prepared from C57BL/6 strain would allow us to conduct more in-depth and interesting investigations on the iPSC generation mechanism. In addition, C57BL/6 is suitable for longitudinal studies such as cancer study, and also suitable and powerful for genetic studies because C57BL/6 is the mouse strain that was selected for the whole genome sequencing project. Finally, our present results may suggest that c-Myc- and genome integration-free iPS cells can be established in a portion of human population, since mouse strain difference conceptually corresponds to the difference among human individuals.ISSCR 8th Annual Meetin

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

Sex Specification and Heterogeneity of Primordial Germ Cells in Mice.

In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells

Crucial Role of c-Myc in the Generation of Induced Pluripotent Stem Cells

c-Myc transduction has been considered previously to be non-essential for induced pluripotent stem cell (iPSC) generation. In our current study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred 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 withot c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F- 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 HDAC 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

DNA methylation and histone modifications in female- and male-specific PGC-expressed genes.

<p><b>(A)</b> Average methylation levels of TSS and the shore values (±5 kb of the TSS) in female- and male-specific PGC-expressed genes. Our previous methylation data of PGCs were obtained from the DDBJ database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144836#pone.0144836.ref012" target="_blank">12</a>]. <b>(B)</b> Occupancy of H3K4me3- and H3K27me3-bound TSS and TTS shore regions (±5 kb of the TSS and TTS) in female- and male-specific PGC-expressed genes. <b>(C)</b> Proportion of genes showing histone-modification patterns. The number of genes classified into each histone-modification pattern is shown. <b>(D)</b> Three typical histone-modification patterns. The red regions indicate ChIP/input enrichment-peak regions that were calculated using the DROMPA peak-calling program.</p

Identification of female and male PGC-specific-expressed genes.

<p><b>(A)</b> Top: scatter plot of female and male PGC samples. The green lines indicate a 2-FC in the average expression levels between the 2 samples. Bottom: volcano plot of differentially expressed genes in female and male PGC samples. Red spheres indicate statistically significant genes. FSGs (651) and MSGs (428). <b>(B)</b> Top 20 differentially expressed genes in female- and male-specific PGCs. Expression levels are shown in log2 values. The genes are ranked in the order of decreasing log FC values. <b>(C)</b> Pie chart showing the composition and quantity of each reference gene type in the UCSC Genome Browser and Ingenuity Pathway analysis tool for FSGs and MSGs.</p