Yasushi Kondo

DNA segments mapped by reciprocal use of microsatellite primers between mouse and rat.

Rat microsatellite primers were used for detection of homologous DNA segments in the mouse species (Mus laboratorius, Mus musculus musculus, and Mus spretus). Twenty five (16.3%) of 153 rat primer pairs amplified specific DNA segments, when genomic DNA of mice was used as a template in the polymerase chain reaction (PCR). Size variation among inbred strains of mice was found for 13 DNA segments (8.5%). Eight out of the 13 polymorphic DNA segments were mapped to a particular chromosome with two sets of recombinant inbred strains, AKXL or BXD. Similarly, mouse microsatellite primers were used for detection of homologous DNA segments in rats (Rattus norvegicus). Twenty (12.0%) of 166 primer pairs amplified specific DNA segments from rat genome. Size variation among inbred strains of rats was found for seven DNA segments (4.2%). Eleven of these 20 DNA segments were mapped with a rat x mouse somatic cell hybrid clone panel and/or linkage analysis by use of backcross progeny. Our results suggest that the mapped DNA segments are really homologs between mouse and rat. These polymorphic DNA segments are useful genetic markers.

Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells.

Embryonic pancreatic bud cells, the earliest pancreas-committed cells, generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into mature pancreatic β-cells in vivo, indicating the feasibility of hESC/iPSC-based cell therapy for diabetes. However, the key factors required for the differentiation of these cells into pancreatic bud cells are incompletely understood. The purpose of this study was to establish culture conditions that efficiently induce PDX1(+)NKX6.1(+) pancreatic bud cells from hESCs/iPSCs. We differentiated a hESC line, KhES-3, into pancreatic lineages with a stepwise protocol recapitulating developmental process. The induction rate of PDX1(+)NKX6.1(+) cells was correlated with cell density in adherent cultures, and markedly improved with cell aggregation cultures. The positive effects of cell aggregation cultures on the differentiation of pancreatic bud cells were reproduced in multiple hESC/iPSC lines. The human PDX1(+)NKX6.1(+) cells developed into pancreatic epithelia after implantation into immunocompromised mice. Moreover, human C-peptide secretion into mouse bloodstream was stimulated by glucose challenges after in vivo maturation. Taken together, these results suggest that cultures with high cell density are crucial for the differentiation of pancreas-committed progenitor cells from hESCs/iPSCs. Our findings may be applicable for the development of hESC/iPSC-based cell therapy for diabetes.

Exome sequencing identifies a new candidate mutation for susceptibility to diabetes in a family with highly aggregated type 2 diabetes

The aim of this study was to investigate the genetic background of familial clustering of diabetes using genome-wide linkage analysis combined with exome sequencing. We recruited a Japanese family with a 3-generation history of diabetes. The family comprised 16 members, 13 having been diagnosed with diabetes. Nine members had been diagnosed before the age of 40. Linkage analysis was performed assuming an autosomal dominant model. Linkage regions were observed on chromosomes 4q34, 5q11-q13, and 12p11-q22 and the logarithm of odds (LOD) scores were 1.80. To identify the susceptibility variants, we performed exome sequencing of an affected family member. We predicted that the familial clustering of diabetes is caused by a rare non-synonymous variant, and focused our analysis on non-synonymous variants absent in dbSNP131. Exome sequencing identified 10 such variants in the linkage regions, 7 of which were concordant with the affection status in the family. One hundred five normal subjects and 67 lean diabetes subjects were genotyped for the 7 variants; the only variant found to be significantly more frequent in the diabetes subjects than in the normal subjects was the N1072K variant of the early endosome antigen 1 (EEA1) gene (0 in normal subjects and 4 in diabetes subjects, p=0.022). We therefore propose that the N1072K variant of the EEA1 gene is a candidate mutation for susceptibility to diabetes in the Japanese population.

iPSC technology-based regenerative therapy for diabetes

The directed differentiation of human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), into pancreatic endocrine lineages has been vigorously examined by reproducing the in vivo developmental processes of the pancreas. Recent advances in this research field have enabled the generation from hESCs/iPSCs of functionally mature β‐like cells in vitro that show glucose‐responsive insulin secretion ability. The therapeutic potentials of hESC/iPSC‐derived pancreatic cells have been evaluated using diabetic animal models, and transplantation methods including immunoprotective devices that prevent immune responses from hosts to the implanted pancreatic cells have been investigated towards the development of regenerative therapies against diabetes. These efforts led to the start of a clinical trial that involves the implantation of hESC‐derived pancreatic progenitors into type 1 diabetes patients. In addition, patient‐derived iPSCs have been generated from diabetes‐related disorders towards the creation of novel in vitro disease models and drug discovery, although few reports so far have analyzed the disease mechanisms. Considering recent advances in differentiation methods that generate pancreatic endocrine lineages, we will see the development of novel cell therapies and therapeutic drugs against diabetes based on iPSC technology‐based research in the next decade.

Measurements of surface relaxation time of solid, liquid and adsorbed 3He in porous glasses

Abstract The surface relaxation time T ls has been investigated in solid, liquid and adsorbed 3 He in porous glasses for a wide frequency range by using pulsed SQUID-NMR. ALL T ls agreed with each other and were proportional to the frequency at least at low frequencies. A model is proposed to explain the above results of T ls .

Study of surface relaxation mechanism of liquid3He in porous glass by using SQUID NMR

Nuclear spin relaxation of liquid3He in porous glass has been studied. In addition to measurements of the longitudinal spin relaxation timeT1 by a usual pulsed SQUID NMR, measurements of the transverse spin relaxation timeT2 have been performed by using a newly developed SQUID NMR method to observe a spin echo signal. Temperature and frequency dependences ofT1 andT2 have been measured. A simple model is proposed which explains the main features of the experimental results.

Genetic polymorphism of tear proteins in the rat

Polymorphism of tear proteins was found by agarose gel electrophoresis among inbred strains of rats. The proteins (RTP-1) are inherited as a single autosomoal trait. The locus was designatedRtp-1 (rat tear protein-1) and it had two codominant alleles (Rtp-1a, Rtp-1b). Although we did not find any recombinant between theRtp-1 and theMup-1 loci among 67 backcross progeny, we found 3 strains with the recombinant type between them in 33 inbred strains tested. The results suggest that theRtp-1 locus is very closely linked with theMup-1 locus, which belongs to rat linkage group II. RTP-1 proteins strongly reacted with anti-MUP-1A serum on agarose gel electrophoretograms.

Solid-Liquid Phase Diagrams of 3He in Porous Glasses

Solidification and melting phenomena of 3He in restricted geometries were investigated by measuring the magnetization, Mz, and the spin lattice relaxation time, T1 of 3He with SQUID-NMR technique. Two porous glasses were used whose average pore diameters were 4 nm and 18 nm, respectively. Measurements were made at various pressures between S.V.P. and 9 MPa in the temperature range between 20 mK and 4 K. The melting curve minimum in the P-T phase diagram was observed for 18 nm porous glass, but not for 4 nm. We attributed this to the depression of Fermi temperature, in addition to the large hysteresis between solidification and melting, for 3He in the small pores.

Insulin‐producing cells derived from ‘induced pluripotent stem cells’ of patients with fulminant type 1 diabetes: Vulnerability to cytokine insults and increased expression of apoptosis‐related genes

The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine‐induced apoptotic reactions of β‐like insulin‐producing cells differentiated from the iPSCs.

Validation of Noninvasive Morphological and Diffusion Imaging in Mouse Emphysema by Micro–Computed Tomography and Hyperpolarized 129Xe Magnetic Resonance Imaging

Animal disease models are pivotal in investigating the pathogenesis of emphysema and developing novel drugs, but the modalities to evaluate murine emphysema models have been of limited validity and sensitivity. In this study, we evaluated hyperpolarized (129)Xe magnetic resonance imaging (MRI) and micro-computed tomography (micro-CT) compared with traditional methods, such as plethysmography and histology. Elastase-treated mice and adiponectin knockout mice were used as murine emphysema models to evaluate these modalities. Three weeks after elastase administration, significant and heterogeneous emphysema was evaluated according to the mean linear intercept and plethysmography parameters. Notably, the distribution of low-density areas, as examined by micro-CT, correlated with the mean linear intercept and plethysmography parameters in whole lungs. These correlations were also observed in regional areas. Furthermore, we introduced hyperpolarized (129)Xe MRI, which can evaluate gas exchange between the alveoli and blood during spontaneous breathing. Parameters of gas exchange (fD) and alveolar size (Vs/Va) were significantly decreased in elastase-treated mice, and moderately correlated with the plethysmography parameters. Of importance, we could detect a decrease of the fD value in low-density areas with micro-CT, suggesting that gas exchange decreased in emphysematous lesions. Likewise, these parameters (fD and Vs/Va) were also decreased in adiponectin knockout mice, which exhibit emphysema with a homogeneous distribution. We demonstrated the feasibility of (129)Xe MRI and micro-CT in combination with traditional modalities. These noninvasive modalities provide complementary data that can be used for repeated estimations of regional gas exchange and lung morphology.