Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines

Defining Hypo-Methylated Regions of Stem Cell-Specific Promoters in Human iPS Cells Derived from Extra-Embryonic Amnions and Lung Fibroblasts

BACKGROUND: Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived. Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of "stemness" remain unknown in iPS cells derived from extra-embryonic and embryonic cells. METHODOLOGY/PRINCIPAL FINDINGS: We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells. CONCLUSIONS/SIGNIFICANCE: We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for "stemness" and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications

大腸癌肺転移切除例の検討

The objective of this study was to evaluate prognostic factors after pulmonary resection for metastasis of colorectal cancer. We retrospectively analyzed the clinicopathological factors and the prognosis of 36patients who received pulmonary resection for metastasis of colorectal cancer. The 5-year overall survival after pulmonary resection was 75.4％, and the 3-year disease free survival after pulmonary resection was 53.5％. There was no significant prognostic factor regarding overall survival after pulmonary resection by multivariate analysis. However, regarding disease-free survival after pulmonary resection, T4 stage colorectal cancer showed significant poorer prognosis by multivariate analysis（p＝0.014）. Patients who received reoperation for pulmonary recurrence showed better prognosis than patients who did not receive reoperation (p＝ 0.04). Prognosis after pulmonary resection for metastasis of colorectal cancer is favorable owing to progresses of chemotherapies. Metastasectomy may not be the primary therapy for patients with pulmonary metastasis from T4 stage colorectal cancer because of their short disease-free survival after metastasectomy. Reoperation for resectable recurrence of pulmonary metastasis may improve overall survival

ハイガン ロボット シュジュツ ノ シヒ リョウキン セッテイ

In Japan, the only robot surgery covered by health insurance is prostatectomy. Robot surgery for pulmonary cancer has not been approved as advanced health care, and hospitals are required to set out-of-pocket fees for surgery on their own. We calculated the fee not covered by insurance to be 1.28million yen, taking into account expenses for : （1） the use of medical equipment, （2） maintenance, （3） labor costs, and （4） medical materials and drugs. Since the fees for surgery plus hospitalization are actually incurred by patients, whether or not the hospitalization fee should be included in out-of-pocket medical fees is decided by each health care institution. In any case, it is necessary to provide patients with a summary of medical fees prior to surgery. Hospitals are required to collect data on pulmonary robot surgical cases while operating the system with medical fees incurred by individual patients

Kansei Evaluation in Agent Rearing Game

In this paper， We have studied the Kansei evaluation of the agent rearing game. The agent rearing game is a game by which the characters who are the agents are brouht up. The Kansei evaluation is an evaluation by Kansei engineering like the sensibility and feelings， etc. to treat technological1y. In this research， We produced the agent rearing game. We propose the method of the interesting the game using the technique of Kansei engineering for the evaluation

Dynamic movement of the Golgi unit and its glycosylation enzyme zones

Harada A., Kunii M., Kurokawa K., et al. Dynamic movement of the Golgi unit and its glycosylation enzyme zones. Nature Communications 15, 4514 (2024); https://doi.org/10.1038/S41467-024-48901-1.Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small ‘Golgi units’ that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call ‘zones’. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis

DNA Methylation Dynamics in Human Induced Pluripotent Stem Cells over Time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the “convergence” of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs

Nap1 regulates proper CENP-B binding to nucleosomes

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1

Erlotinib デ induction therapy オ オコナッタ IIIAキ ヒショウサイボウ ハイガン ノ 1シュジュツレイ

［Background ］Erlotinib, epidermal growth factor receptor-tyrosine kinase inhibitor（EGFR-TKI）, is effective for advanced and metastatic non-small cell lung cancer（NSCLC）with EGFR mutation. However, the report of Erlotinib as induction therapy is rare. We report a surgical case of NSCLC with Erlotinib as induction therapy. ［Case ］A４１-years-old man, diagnosed left lung adenocarcinoma with EGFR mutation（exon１９deletion）, was referred to our hospital. CT showed that the tumor was ３５mm in S８ of the left lung and ＃７ lymphnode was swelling markedly（cT２aN２M０ stage ⅢA）. He took Erlotinib（１５０mg／day）for１２weeks at first because of EGFR mutation positive. The evaluation of Erlotinib was partial response in RESIST. He could take radical operation as lower lobe and lingual segment resection, because CT showed bulky ＃７got smaller significantly. There was no postoperative complication. The pathological finding was adenocarcinoma（papillary& acinartype）, PL０, v（＋）, ly（＋）, br（－）, pa（－）, pv（－）, Ef :１b,（ypT１aN２M０stage ⅢA）. He has taken adjuvant therapy（Erlotinib１５０mg／day）for２８weeks. There is no recurrence six months after operation. ［Conclusion ］It is possible that Erlotinib as induction-therapy is very effective in patients with EGFR mutation like this case ; however there is no evidence of EGFR-TKI as induction therapy. It is necessary to validate the effectiveness of Erlotinib as induction therapy