多能性幹細胞を用いたダウン症候群の早期造血系譜における短型GATA1タンパクの量的効果の解析

京都大学新制・課程博士博士(医科学)甲第23472号医科博第131号新制||医科||9(附属図書館)京都大学大学院医学研究科医科学専攻(主査)教授 滝田 順子, 教授 髙折 晃史, 教授 江藤 浩之学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

A New Device Facilitating Intracorporeal Purse-string Suture during Endoscopic Surgery

Standard laparoscopic colorectal surgery requires additional incision or enlargement of the trocar incision for the retrieval of the surgical specimen. A natural orifice specimen extraction （NOSE） procedure, in which the specimen is retrieved through the anus or vagina without any additional skin incision, requires purse-string suture （PSS） of the rostral intestinal segment in order to fix the anvil head of the stapler and perform extracorporeal mechanical anastomosis. Colorectal surgery has a limited NOSE in cases where the end of the rostral segment could be pulled through the anus. Broader application of NOSE depends on intracorporeal PSS. We developed a new forceps for intracorporeal PSS during NOSE and evaluated its efficacy. The PSS instrument was refined to pass through a 12-mm trocar in an intracorporeal PSS and achieve anastomosis using double stapling. In trials utilizing an endoscopic practice box, regular spacing of stitches during PSS were consistent （n＝10）, and tight intracorporeal anastomosis of the porcine colon was successfully performed （n＝2）. We then confirmed efficacy through an operation on a pig. Our novel PSS device will help us perform NOSE not only in laparoscopic colorectal surgery but also in any operation requiring intracorporeal PSS, which should contribute to further advances in endoscopic digestive surgery

Body mass index and colorectal cancer risk : A Mendelian randomization study

Traditional observational studies have reported a positive association between higher body mass index (BMI) and the risk of colorectal cancer (CRC). However, evidence from other approaches to pursue the causal relationship between BMI and CRC is sparse. A two-sample Mendelian randomization (MR) study was undertaken using 68 single nucleotide polymorphisms (SNPs) from the Japanese genome-wide association study (GWAS) and 654 SNPs from the GWAS catalogue for BMI as sets of instrumental variables. For the analysis of SNP-BMI associations, we undertook a meta-analysis with 36 303 participants in the Japanese Consortium of Genetic Epidemiology studies (J-CGE), comprising normal populations. For the analysis of SNP-CRC associations, we utilized 7636 CRC cases and 37 141 controls from five studies in Japan, and undertook a meta-analysis. Mendelian randomization analysis of inverse-variance weighted method indicated that a one-unit (kg/m2) increase in genetically predicted BMI was associated with an odds ratio of 1.13 (95% confidence interval, 1.06-1.20; P value <.001) for CRC using the set of 68 SNPs, and an odds ratio of 1.07 (1.03-1.11, 0.001) for CRC using the set of 654 SNPs. Sensitivity analyses robustly showed increased odds ratios for CRC for every one-unit increase in genetically predicted BMI. Our MR analyses strongly support the evidence that higher BMI influences the risk of CRC. Although Asians are generally leaner than Europeans and North Americans, avoiding higher BMI seems to be important for the prevention of CRC in Asian populations

Efficient long-term survival of cell grafts after myocardial infarction with thick viable cardiac tissue entirely from pluripotent stem cells

心臓組織シートを、細胞が生きた状態で簡便に積層化する方法の開発. 京都大学プレスリリース. 2015-11-27.Poor engraftment of cells after transplantation to the heart is a common and unresolved problem in the cardiac cell therapies. We previously generated cardiovascular cell sheets entirely from pluripotent stem cells with cardiomyocytes, endothelial cells and vascular mural cells. Though sheet transplantation showed a better engraftment and improved cardiac function after myocardial infarction, stacking limitation (up to 3 sheets) by hypoxia hampered larger structure formation and long-term survival of the grafts. Here we report an efficient method to overcome the stacking limitation. Insertion of gelatin hydrogel microspheres (GHMs) between each cardiovascular cell sheet broke the viable limitation via appropriate spacing and fluid impregnation with GHMs. Fifteen sheets with GHMs (15-GHM construct; >1mm thickness) were stacked within several hours and viable after 1 week in vitro. Transplantation of 5-GHM constructs (≈2×106 of total cells) to a rat myocardial infarction model showed rapid and sustained functional improvements. The grafts were efficiently engrafted as multiple layered cardiovascular cells accompanied by functional capillary networks. Large engrafted cardiac tissues (0.8mm thickness with 40 cell layers) successfully survived 3 months after TX. We developed an efficient method to generate thicker viable tissue structures and achieve long-term survival of the cell graft to the heart

Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

Transforming DNA repair errors into assets. Kyoto University press release. 08 Nov. 2019.The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening

Pluripotent stem cell model of early hematopoiesis in Down syndrome reveals quantitative effects of short-form GATA1 protein on lineage specification.

Children with Down syndrome (DS) are susceptible to two blood disorders, transient abnormal myelopoiesis (TAM) and Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL). Mutations in GATA binding protein 1 (GATA1) have been identified as the cause of these diseases, and the expression levels of the resulting protein, short-form GATA1 (GATA1s), are known to correlate with the severity of TAM. On the other hand, despite the presence of GATA1 mutations in almost all cases of DS-AMKL, the incidence of DS-AMKL in TAM patients is inversely correlated with the expression of GATA1s. This discovery has required the need to clarify the role of GATA1s in generating the cells of origin linked to the risk of both diseases. Focusing on this point, we examined the characteristics of GATA1 mutant trisomy-21 pluripotent stem cells transfected with a doxycycline (Dox)-inducible GATA1s expression cassette in a stepwise hematopoietic differentiation protocol. We found that higher GATA1s expression significantly reduced commitment into the megakaryocytic lineage at the early hematopoietic progenitor cell (HPC) stage, but once committed, the effect was reversed in progenitor cells and acted to maintain the progenitors. These differentiation stage-dependent reversal effects were in contrast to the results of myeloid lineage, where GATA1s simply sustained and increased the number of immature myeloid cells. These results suggest that although GATA1 mutant cells cause the increase in myeloid and megakaryocytic progenitors regardless of the intensity of GATA1s expression, the pathways vary with the expression level. This study provides experimental support for the paradoxical clinical features of GATA1 mutations in the two diseases