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Dr. Shinya Yamanaka is recognized for his discovery of the induction of pluripotent stem cells from fibroblasts by a combination of defined factors. In this interview with Cell, he discusses the progress of the field, what's next for clinical applications of iPS cells, and the state of science in Japan and the rest of the world., Competing Interests: Declaration of interests S.Y. holds multiple advisory roles related to this topic: Altos Labs, Inc. (Scientific Advisory Board), iPS Academia Japan, Inc. (Scientific Advisor), Med, Elsevier Inc. (Scientific Advisory Board), Cell Press (Advisory Board), Orizuru Therapeutics, Inc. (Scientific Advisory Board), and The Uehiro Foundation on Ethics and Education (Scientific Advisor)., (Copyright © 2024.)

Background: I-cell disease is characterized by the presence of vacuole-like inclusions in lymphocytes. However, the nature and clinical significance of these inclusions have seldom been characterized. In this study, the authors tried to elucidate the distribution in different lymphocyte subpopulations, and the histological nature of the inclusions., Methods: Blood samples from three unrelated patients were analyzed. Lymphocyte subpopulations were separated using monoclonal antibodies conjugated to immunomagnetic beads. Cytochemical studies were performed using FITC-conjugated lectins. The expressions of surface and cytoplasmic class II molecules were analyzed by flow cytometry., Results: Virtually all B cells from the patients contained the inclusions. In contrast, CD4 + T cells, CD8 + T cells, natural killer cells, monocytes, or neutrophils did not contain the inclusions. Both fibroblasts and B cells from I-cell patients were stained intensely by multiple FITC-conjugated lectins with distinct binding profiles. The inclusions of B cells were stained intensely by fluorescence-conjugated antibodies against class II antigens., Conclusions: Inclusions in I-cell disease reflect the accumulation of HLA class II molecules within B cells. These results suggest a potential role for N-acetylglucosamine-1-phosphotransferase in immune functions. Furthermore, the fact that only B cells contain the inclusions provides a novel diagnostic aid for the diagnosis of I-cell disease.

Non-human primates are our closest relatives and are of special interest for ecological, evolutionary and biomedical research. The Japanese macaque (Macaca fuscata) has contributed to the progress of primatology and neurosciences over 60 years. Despite this importance, the molecular and cellular basis of the Japanese macaque remains unexplored since useful cellular tools are lacking. Here we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of the Japanese macaque with Sendai virus or plasmid vectors. The Japanese macaque iPSCs (jm-iPSCs) were established under feeder-free culture conditions, but feeder cells turned out to be essential for their maintenance. The jm-iPSCs formed human iPSC-like flat colonies which were positive for pluripotent antigens including alkaline phosphatase, SSEA4, and TRA-1-81. They also expressed endogenous OCT3/4, SOX2, L-MYC, and KLF4 and other pluripotent marker genes. The potential to differentiate into all three germ layers and neural stem cells was confirmed by embryoid body and neurosphere formation, respectively. The jm-iPSCs will provide a robust in vitro tool for investigating the underlying mechanisms of development and physiology studies with the Japanese macaque.

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.

The radiation induced bystander response is defined as a response in cells which have not been directly targeted by radiation, but which are in the neighborhood of cells which have been directly exposed. In many cases, the bystander response is saturated with increasing dose and is observed when only one cell in a population is targeted by high-LET particle radiations or ultrasoft X-rays (278 eV). However, in our studies using synchrotron X-ray microbeams (5.35 keV), the bystander cell killing effect in normal human fibroblast WI-38 cells had a parabolic relationship to the irradiating dose and was detected if 5 or more cell nuclei were irradiated. To evaluate the feature of the X-ray-induced bystander cell killing effect at a wider dose range and the existence of photon energy dependence, the effects were assessed by irradiating cell nuclei in confluent WI-38 cells with AlK X-ray microbeams (1.49 keV). The surviving fraction decreased when only a single cell nucleus was irradiated, suggesting the minimal number of targeted cells to induce the effect may depend on the energy of photons used. In this study, we found that the bystander cell killing effect showed a biphasic relationship to the irradiating dose. The decrease in bystander cell survival at the doses higher than 0.23 Gy was partially suppressed between 2.3 and 7.0 Gy, followed by level-off around 90% above 14 Gy, suggesting that the X-ray-induced bystander response is dose dependent. In addition, NO is one of chief initiators/mediators of the effect at least 0.47 Gy.

To identify plants with bioactive potential for skin care, methanol extracts of 56 plant parts from 47 medical and edible plants cultivated in Okinawa were tested for their proliferative effects on NB1RGB skin fibroblast cells. Extracts from six plants, Bischofia javanica, Colocasia esculenta, Melaleuca alternifolia, Piper angustifolia, Jasminum sambac, and Curcuma longa, showed higher NB1RGB cell proliferation activity (>10%) than the control, at various concentrations. Among the six extracts, only the C. longa extract caused an increase in collagen synthesis in NB1RGB cells, as compared to treatment with the positive control, ascorbic acid (AsA). Expression of the collagen synthesis marker, transforming growth factor-β1, was higher after treatment with the C. longa extract than with AsA.

Little is known about the ability of miso, to modulate mutability in human cells. We have observed increased levels of glucose-regulated protein 78 (GRP78) expression in association with suppression of mutation in human RSa cells irradiated with ultraviolet C (UVC). Here we examined to determine whether miso treatment results in increased GRP78 expression and suppression of UVC mutagenicity in RSa cells. Supernatants of water extracts of miso products and their components were tested. In the sample-treated cells, the amount of GRP78, as estimated by RT-PCR and immunoblotting analysis, increased, and the UVC-induced ouabain resistant mutation (Oua(R)) and the K-ras codon 12-base substitution mutation frequency decreased. This decrease was not observed in cells with downregulation of GRP78 by GRP78 siRNA transfection. The results suggest that miso suppresses UVC mutagenicity by increasing GRP78 expression in human cells.

Red sea bream iridovirus (RSIV) is a causative agent of red sea bream iridoviral disease (RSIVD) in marine fish species in Japan. Fibroblast cells were developed from a tail fin of red sea bream, Pagrus major, and then underwent single cell cloning. The successful cloned cells were named CRF-1 cells. Most CRF-1 cells had a normal diploid karyotype with 2n=48 by chromosomal analysis. RSIV-infected CRF-1 cells showed typical morphological changes that were associated with apoptosis by EGFP-annexin V staining. The serial viral passages were successful in CRF-1 cells but failed in BF-2 cells as judged by MTT assay. The expression of three genes obviously decreased in BF-2 cells compared with CRF-1 cells and finally was below detectable level. Because the expression of 591R gene showed the fastest decrease among three transcripts, the suppression of IE transcript may be responsible for the restricted replication in BF-2 cells. MCP and ATPase phylogenetic trees showed that RSIV strain U-1 belongs to a distinct group from RSIV strain ehime-1. Therefore, possibly recent epizootics of RSIVD in Japan do not originate directly from RSIV strain ehime-1. Taken together, this study confirmed that RSIV strain U-1 is more closely related to Korean RSIV isolates.

Background: The vascular type of Ehlers-Danlos syndrome (vEDS, EDS type IV; MIM#130050) is an autosomal dominantly inherited disorder that results from mutations in the genes for type III procollagen (COL3A1). Affected individuals with vEDS are at risk of arterial rupture, aneurysm, and/or dissection; gastrointestinal perforation or rupture; and uterine rupture during pregnancy, which may lead to sudden death., Methods and Results: Three unrelated Japanese individuals who exhibited symptoms of vEDS were analyzed. In order to identify mutations in the patients' RNA, one 3.8-kb reverse transcriptase polymerase chain reaction product containing the triple-helical domain of COL3A1 was prepared from cultured skin fibroblasts and then was sequenced directly. Three heterozygous mutations were identified; specifically, 2 novel missense base substitutions (Gly220Trp, Gly448Glu) in the (Gly-X-Y)n repeat of the triple-helical domain and a known splicing donor mutation of intron 20 (G+1, IVS20) of COL3A1. The genotype-phenotype correlations in Japanese vEDS individuals with COL3A1 mutations were also investigated., Conclusion: There was no association between the type of complications in vEDS and the related COL3A1 mutation found. After the genetic diagnosis of COL3A1, the establishment of both a network among medical specialists, including clinical geneticists to perform genetic counseling, and long-term follow-up systems of vEDS may help to improve the management of vascular and visceral complications.

The Ryukyu spiny rats (genus Tokudaia) inhabit only three islands in the Nansei Shoto archipelago in Japan, and have the variations of karyotype among the islands. The chromosome number of T. osimensis in Amami-Oshima Island is 2n = 25, and T. tokunoshimensis in Tokunoshima Island is 2n = 45, and the two species have X0 sex chromosome constitution with no cytogenetically visible Y chromosome in both sexes. We constructed the standard ideograms for these species at the 100 and 200 band levels. Comparing the banding patterns between these species, it was suggested that at least 10 times the number of Robertsonian fusions occurred in T. osimensis chromosomes. However, no karyotypic differences were observed between sexes in each species. To detect the sex-specific chromosomal region of these X0 species we applied the comparative genomic hybridization (CGH) method. Although the male- and female-derived gains and losses were detected in several chromosome regions, all of them were located in the heterochromatic and/or telomeric regions. This result suggested that the differences detected by CGH might be caused by the polymorphism on the copy numbers of repeated sequences in the heterochromatic and telomeric regions. Our result indicated that the sex-specific region, where the key to sex determination lies, is very minute in X0 species of Tokudaia.

Objective: This study aims to reveal the morphological, histological, and immunohistochemical mechanism of pannus formation using resected pannus tissue from patients with prosthetic valve dysfunction., Method: Eleven patients with prosthetic valve (St Jude Medical valve) dysfunction in the aortic position who underwent reoperation were studied. We used specimens of resected pannus for histological staining (hematoxylin and eosin, Grocott's, azan, elastica van Gieson) and immunohistochemical staining (transforming growth factor-beta, transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, epithelial membrane antigen, CD34, factor VIII, CD68KP1, matrix metalloproteinase-1, matrix metalloproteinase-3, and matrix metalloproteinase-9)., Results: Pannus without thrombus was observed at the periannulus of the left ventricular septal side; it extended into the pivot guard, interfering with the movement of the straight edge of the leaflet. The histological staining demonstrated that the specimens were mainly constituted with collagen and elastic fibrous tissue accompanied by endothelial cells, chronic inflammatory cells infiltration, and myofibroblasts. The immunohistochemical findings showed significant expression of transforming growth factor-beta, transforming growth factor-beta receptor 1, CD34, and factor VIII in the endothelial cells of the lumen layer; strong transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, and epithelial membrane antigen in the myofibroblasts of the media layer; and transforming growth factor-beta, transforming growth factor-beta receptor 1, and CD68KP1 in macrophages of the stump lesion., Conclusions: Pannus appeared to originate in the neointima in the periannulus of the left ventricular septum. The structure of the pannus consisted of myofibroblasts and an extracellular matrix such as collagen fiber. The pannus formation after prosthetic valve replacement may be associated with a process of periannular tissue healing via the expression of transforming growth factor-beta.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.

Menkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations to the Menkes (MNK) gene. We identified three novel mutations of the MNK gene in three unrelated Japanese patients with classical Menkes disease by analyzing reverse-transcriptase polymerase chain reaction products and genomic DNA of the MNK gene. Firstly, an insertional mutation was found, 1173 ins A, which led to a premature termination and resulted in a very immature Menkes protein. Secondly, we found a point mutation, T2763G, resulting in a leucine-to-arginine conversion, which we predicted would cause a change in the secondary structure of the Menkes protein. Finally, we identified a splicing mutation, 2317 + 5G > C, which resulted in the skipping of both exons 8 and 9 or exon 9 only, and led to a truncation of the protein. Each of these mutations is hypothesized to destroy copper-ATPase-mediated copper transport. We propose that each of these mutations in the MNK gene plays a causative role in the disease.

Utilizing PCR and PCR-SSCP analysis we investigated the prevalence of glucocerebrosidase gene mutations in 47 unrelated Japanese patients with Gaucher disease. Sixty alleles (63.8%) and 20 alleles (21.3%) were identified by analysis for common mutations and PCR-SSCP analysis, respectively. The L444P and F213I mutations were common, accounting for 41 alleles (43.6%) and 14 alleles (14.9%). R496C, D409H, S366G and 1447-1466 del ins TG mutations were identified in 5, 3, 3 and 3 alleles, respectively. The other mutations were unique. In spite of vigorous screening, 14 alleles (14.9%) could not be identified. Four novel mutations were identified by PCR-SSCP analysis: G189V, S366G, K413Q and R433G. These data indicate that besides the L444P mutation no other frequent mutation is present and there is broad heterogeneity of the glucocerebrosidase gene mutations in Japanese patients with Gaucher disease.

Human skin samples were obtained from the normal peripheral portion of specimens removed from persons with various cutaneous and systemic diseases. A portion of each specimen was embedded in paraffin and another part in water-soluble embedding medium, and some was frozen in liquid nitrogen for light microscopy and histochemistry. Some specimens were also investigated by electron microscopy. In 31 of 32 specimens, cells containing brown pigment were observed in the superficial dermis. Because both acid phosphatase and Masson-Fontana staining were positive and the 3,4-dihydroxyphenylalanine reaction negative, the cells were considered to be melanophages. Electron microscopic examination revealed that these cells contained melanosome-laden phagosomes. Some fibroblastlike cells were also observed with intracellular single or multiple melanosomes. This study documents the occurrence of melanophages in the normal skin of Japanese subjects.

A new point mutation within exon 5 of beta-hexosaminidase alpha subunit gene (guanine509----adenine; arginine170----glutamine) has been identified as being responsible for the typical clinical and enzymological phenotype of infantile Tay-Sachs disease in a Japanese infant. Expression of the mutant enzyme protein in the COS I cell system indicated that it is catalytically inactive and also is unstable. The patient is a compound heterozygote, and the exact abnormality in the other allele could not be identified except that it is not any of the other nine known mutations of the beta-hexosaminidase alpha. The data collectively suggest that the other allele is not producing stable messenger RNA (mRNA). The rapidly increasing number of mutations responsible for clinical and enzymological phenotypes and the very large number of statistically possible combinations among them for compound heterozygosity pose a serious pragmatic problem for classification and nomenclature of this group of rare genetic disorders.

The G-banded karyotype of the Iriomote cat, Felis iriomotensis, was found to be indistinguishable from that of F. bengalensis, F. viverrinus, and F. concolor. The karyotype of these four cats is, however, distinguishable from karyotypes of all other species of cats whose chromosomes have been studied. Silver-staining showed nucleolar organizing regions on the satellited stalks of the E1 chromosomes. Based on morphological and cytogenetic parameters F. iriomotensis is probably most closely related to F. bengalensis, the only other wild felid native to Japan.