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The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2–GG1/A2–C1 (bases − 149 to − 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2–GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2–GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR–Cas9 technology. The mice with homozygous mutations in the GG2–GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2–GG1/A2 element in mice is important for Ins transcription in vivo., 論文

Kyoto probe 1 (KP-1) rapidly distinguishes between human ES/iPS (hES/iPS) cells and their differentiated cells. Recently, we generated induced tissue-specific stem cells from pancreas (iTS-P cells) using reprogramming factors and tissue-specific selection. The iTS-P cells have self-renewal potential, and subcutaneously transplanting them into immunodeficient mice did not generate teratomas. In this study, we applied KP-1 to analyze mouse ES (mES) cells and mouse iTS-P (miTS-P) cells. KP-1 completely stained mES cells in colonies, but only miTS-P cells at the edge of a colony. This difference was caused by cell type-specific expression of different ABC transporters. These finding suggest that KP-1 will be useful for distinguishing between iPS and iTS-P cells.

We previously reported that treatment with a JNK inhibitory peptide (11R-JNKI) prevents islet apoptosis and enhances the islet function in vivo. In the present study, we explored more efficient JNK inhibitors. The inhibition of the JNK activity by five types of deletion peptides in 11R-JNKI was investigated. One of the peptides, 8R-sJNKI(-9), significantly prevented JNK activation. At a concentration of 1 µM, 8R-sJNKI(-9) inhibited JNK activity similarly to 10 µM 11R-JNKI and the inhibition of the JNK activity by 10 µM 8R-sJNKI(-9) was significantly greater than that by 10 µM 11R-JNK. To evaluate the effects of 8R-sJNKI(-9), porcine islets were cultured with 1 µM of 8R-sJNKI(-9) or 8R-mutant sJNKI(-9) (8R-mJNKI(-9)). After 1 day of culture, the numbers of islets in the 8R-sJNKI(-9)-treated group was significantly higher than that in the 8R-mJNKI(-9)-treated group. After islet transplantation, the blood glucose levels reached the normoglycemic range in 58.3% of streptozotocin-induced diabetic mice in the 8R-sJNKI(-9) group and 0% of the mice in the 8R-mJNKI(-9)-treated group. These data suggest that 8R-sJNKI(-9) inhibits islet apoptosis and improves islet function.

Human tissue-specific stem cells (hTSCs), found throughout the body, can differentiate into several lineages under appropriate conditions in vitro and in vivo. By transfecting terminally differentiated cells with reprogramming factors, we previously produced induced TSCs from the pancreas and hepatocytes that exhibit additional properties than iPSCs, as exemplified by very low tumour formation after xenogenic transplantation. We hypothesised that hTSCs, being partially reprogrammed in a state just prior to iPSC transition, could be isolated from any terminally differentiated cell type through transient reprogramming factor overexpression. Cytochemical staining of human deciduous tooth-derived dental pulp cells (HDDPCs) and human skin-derived fibroblasts following transfection with Yamanaka’s factors demonstrated increased ALP activity, a stem cell marker, three weeks after transfection albeit in a small percentage of clones. Repeated transfections (≤3) led to more efficient iPSC generation, with HDDPCs exhibiting greater multipotentiality at two weeks post-transfection than the parental intact HDDPCs. These results indicated the utility of iPSC technology to isolate TSCs from HDDPCs and fibroblasts. Generally, a step-wise loss of pluripotential phenotypes in ESCs/iPSCs occurs during their differentiation process. Our present findings suggest that the reverse phenomenon can also occur upon repeated introduction of reprogramming factors into differentiated cells such as HDDPCs and fibroblasts.

Induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells. Furthermore, our recent study demonstrated the generation of induced tissue-specific stem (iTS) cells by transient overexpression of the reprogramming factors using a plasmid combined with tissue-specific selection. In this study, we were able to generate RNA-based iTS cells that utilize a single, synthetic, self-replicating VEE-RF RNA replicon expressing four reprogramming factors (OCT4, KLF4, SOX2, and GLIS1). A single VEE-RF RNA transfection into mouse pancreatic tissue resulted in efficient generation of iTS cells from pancreas (iTS-P cells) with genetic markers of endoderm and pancreatic progenitors and differentiation into insulin-producing cells more efficiently than ES cells. Subcutaneous transplantation of iTS-P cells into immunodeficient mice resulted in no teratoma formation. Bisulfite genomic sequencing demonstrated that the promoters of Oct4 and Nanog remained partially methylated in iTS-P cells. We compared the global gene-expression profiles of ES cells, iTS-P cells, and pancreatic islets. Microarray analyses confirmed that the iTS-P cells were similar but not identical to ES cells compared with islets. These data suggest that iTS-P cells are cells that inherit numerous components of epigenetic memory from pancreas cells and acquire self-renewal potential. The generation of iTS cells may have important implications for the clinical application of stem cells.