記述言語：英語   掲載種別：研究論文（学術雑誌）  

Progesterone (P4) exerts long-term neuroprotective effects in animal models of stroke, and P4 receptors play a crucial role in this neuroprotection. However, it currently remains unclear whether the activation of P4 receptors alone is sufficient to exert long-term neuroprotection because P4 exhibits other steroidogenic and GABAergic activities via several of its metabolites. Nestorone is a potent selective P4 receptor agonist without other steroidogenic and GABAergic activities. Therefore, we examined the effects of nestorone in adult male rats subjected to transient middle cerebral artery occlusion (MCAO). The dose-response relationship of nestorone showed that the 6-h post-ischemic administration of 10 μg/kg nestorone resulted in greater reductions in infarct sizes 48 h after MCAO than the other two doses tested (5 and 80 μg/kg), and this dose of nestorone significantly decreased astrocyte activation in the peri-infarct cortical region. Moreover, 10 μg/kg nestorone significantly prevented functional impairments on the 28th and 29th days and slightly reduced infarct size on the 30th day after MCAO. The present results suggest that the activation of P4 receptors alone is sufficient to exert neuroprotection against transient cerebral ischemia in adult male rats; therefore, nestorone is a promising agent in post-stroke treatment due to its potent progestational effects without other steroid-related activities.

DOI： 10.1016/j.brainres.2018.09.022

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Progesterone (P4) exerts potent neuroprotection both in young and aged animal models of stroke. The neuroprotection is likely to be mediated by allopregnanolone (ALLO) metabolized from P4 by 5α-reductase, since the neuroprotection is attenuated by the 5α-reductase inhibitor finasteride, which was done only with young animals though. Thus, we do not know the contribution of ALLO to the P4-induced neuroprotection in aged animals. We examined effects of finasteride on the P4-induced neuroprotection in aged (16-18-month-old) male rats subjected to transient focal cerebral ischemia. Transient focal cerebral ischemia was induced by left middle cerebral artery occlusion (MCAO) and occlusion of the bilateral common carotid arteries. MCAO rats were given an 8 mg/kg P4 6 h after MCAO followed by the same treatment once a day for successive 3 days. Finasteride, a 5α-reductase inhibitor, at 20 mg/kg was intraperitoneally injected 30 min prior to the P4-injections. P4 markedly reduced neuronal damage 72 h after MCAO, and the P4-induced neuroprotection was apparently suppressed by finasteride in the aged animals. However, post-ischemic administration of finasteride alone (20 mg/kg) significantly prevented neuronal damage and the impairment of Rotarod performance after MCAO in aged male rats, but not in young ones. The androgen receptor antagonist flutamide markedly suppressed the neuroprotection of finasteride in the cerebral cortex, but not in the striatum, suggesting the androgen receptor-dependent mechanism of the finasteride-induced neuroprotection in the cerebral cortex. Our findings suggested, for the first time, the potential of finasteride as a therapeutic agent in post-ischemic treatment of strokes in aged population.

DOI： 10.1007/s12975-018-0624-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Progesterone hormone (P4) is a promising agent against strokes because post-ischemic administration of P4 exerts neuroprotective effects in several young and aged animal models of stroke. However, in contrast to a majority of the studies using male animals, female animals remain underrepresented. In addition, we do not know whether the same administration way of P4 is effective in both male and female animals because there are gender different responses to steroid hormones and stroke. In this study, we thus evaluated long-term histological and functional outcomes in the same treatment with P4 in both 18-month old male and age-matched female rats subjected to transient middle cerebral artery occlusion (MCAO). MCAO aged male and female rats were given a subcutaneous injection of P4 (4 mg/kg) 6 h after MCAO followed by once daily for successive 7 days. The post-ischemic administration of P4 significantly improved the impairments of spatial working memory and motor coordination 28-29 days after MCAO in both aged male and age-matched female rats. However, the P4 administration slightly but not significantly reduced infarct sizes 30 days after MCAO in aged female rats, in contrast to significant better histological outcome in P4-treated aged males. On the other hand, these histological and behavioral analyses showed no adverse effects of P4 in aged rats of both sexes. Collectively, our study provides preclinical evidence to prompt further preclinical studies for post-stroke treatment with P4 and the translation of its clinical trials in old stroke patients of both sexes.

DOI： 10.1016/j.exger.2018.09.012

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chromosome instability, frequently found in cancer cells, is caused by a deficiency in cell division, including centrosomal amplification and cytokinesis failure, and can result in abnormal chromosome content or aneuploidy. The small GTPase pathways have been implicated as important processes in cell division. We found that knockdown of a tumor suppressor protein Kank1 increases the number of cells with a micronucleus or bi-/multi-nuclei, which was likely caused by centrosomal amplification. Kank1 interacts with Daam1, known to bind to and activate a small GTPase, RhoA, in actin assembly. Knockdown of Kank1 or overexpression of Daam1, respectively, hyperactivates RhoA, potentially leading to the modulation of the activity of Aurora-A, a key regulator of centrosomal functions, eventually resulting in centrosomal amplification. Kank1 is also associated with contractile ring formation in collaboration with RhoA, and its deficiency results in the interruption of normal daughter cell separation, generating multinucleate cells. Such abnormal segregation of chromosomes may cause further chromosomal instability and abnormal gene functions, leading to tumorigenesis. Thus, Kank1 plays a crucial role in regulating the activity of RhoA through retrieving excess Daam1 and balancing the activities of RhoA and its effectors.

DOI： 10.1016/j.yexcr.2017.03.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

New fluorescent Fluolid dyes have advantages over others such as stability against heat, dryness, and excess light. Here, we performed simultaneous immunostaining of renal tumors, clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, acquired cystic disease-associated RCC (ACD-RCC), and renal angiomyolipoma (AML), with primary antibodies against Kank1, cytokeratin 7 (CK7), and CD10, which were detected with secondary antibodies labeled with Fluolid-Orange, Fluolid-Green, and Alexa Fluor 647, respectively. Kank1 was stained in normal renal tubules, papillary RCC, and ACD-RCC, and weakly or negatively in all other tumors. CK7 was positive in normal renal tubules, papillary RCC, and ACD-RCC. In contrast, CD10 was expressed in renal tubules and clear cell RCC, papillary RCC, AML, and AC-RCC, and weakly in chromophobe RCC. These results may contribute to differentiating renal tumors and subtypes of RCCs. We also examined the stability of fluorescence and found that fluorescent images of Fluolid dyes were identical between a tissue section and the same section after it was stored for almost three years at room temperature. This indicates that tissue sections can be stored at room temperature for a relatively long time after they are stained with multiple fluorescent markers, which could open a door for pathological diagnostics.

DOI： 10.1155/2014/437871

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

DOI： 10.1038/nature12271

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The usefulness of Fluolid-Orange, a novel fluorescent dye, for DNA microarray and immunological assays has been examined. Fluolid-Orange-labeled probes (DNA and IgG) were stable as examined by laser-photo-bleaching and under heat and dry conditions. Statistical analyses were performed to evaluate the reproducibility of the microarray assay, while stage-specific immunostaining of marker proteins, Kank1 and calretinin, was performed for renal cancers, both giving satisfactory results. The stability of the dye should provide advantages for storing fluorescently labeled probes and re-examining the specimens later in genetic and pathological diagnostics.

DOI： 10.1007/s10529-011-0646-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34(-/low)c-Kit(+)Sca-1(+)lineage(-) HSCs (CD34(-)KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

DOI： 10.1002/stem.42

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone morphogenetic protein (BMP) signaling is essential for normal development of the gastrointestinal (GI) tract. BMPs also play multiple roles in vascular smooth muscle cells; however, the BMP signaling in the development of the GI musculature remains to be clarified. We investigated the expression of BMPs and their receptors in mouse embryonic GI tracts by immunohistochemistry and in situ hybridization. We demonstrated that BMP2, BMP receptor Ib and BMP receptor II were expressed in the smooth muscle progenitors from E12 to E13 for the first time. BMP signaling on smooth muscle differentiation was examined by implantation of agarose beads soaked with BMPs in the in vitro developmental model that is gut-like structures from mouse embryonic stem (ES) cells. BMP2 rather than BMP4 beads enhanced smooth muscle differentiation, and increased gut-like structures showing spontaneous contractions and expressing intensive alpha-smooth muscle actin immunoreactivity. This increase was confirmed by up-regulation of SM22 mRNA shown by real-time PCR. By addition of noggin beads or noggin to the medium at BMP2 bead implantation, the ratio of contractive gut-like structures decreased. Implantation of BMP2 beads at EB7 (EB--embryoid bodies) (corresponding to E12 or E13 of mouse embryo) showed the highest effects and up-regulation of transcription factors msx-1 after 24h. This increase was blocked by noggin, and msx-1 decreased to almost the control level after 60 h. BMP2 beads at EB7 increased platelet-derived growth factor-A (PDGF-A) in the differentiating smooth muscle cells. We have recently reported that PDGF-A is expressed in the developing inner circular smooth muscle and is crucial for the longitudinal smooth muscle differentiation. Taken together, BMP signaling was expressed for a short window in the smooth muscle progenitors and the signal, especially BMP2, plays an essential role in smooth muscle differentiation in cooperation with PDGF signaling.

DOI： 10.1016/j.diff.2008.10.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+)CD41(+)CD45(-) phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

DOI： 10.1371/journal.pone.0004820

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Actin polymerization is crucial in throm-bopoiesis, platelet adhesion, and mega-karyocyte (MK) and platelet spreading. The Wiskott-Aldrich syndrome protein (WASp) homolog WAVE functions downstream of Rac and plays a pivotal role in lamellipodia formation. While MKs and platelets principally express WAVE1 and WAVE2, which are associated with Abi1, the physiologic significance of WAVE isoforms remains undefined. We generated WAVE2(-/-) embryonic stem (ES) cells because WAVE2-null mice die by embryonic day (E) 12.5. We found that while WAVE2(-/-) ES cells differentiated into immature MKs on OP9 stroma, they were severely impaired in terminal differentiation and in platelet production. WAVE2(-/-) MKs exhibited a defect in peripheral lamellipodia on fibrinogen even with phorbol 12-myristate 13-acetate (PMA) costimulation, indicating a requirement of WAVE2 for integrin alpha(IIb)beta(3)-mediated full spreading. MKs in which expression of Abi1 was reduced by small interfering RNA (siRNA) exhibited striking similarity to WAVE2(-/-) MKs in maturation and spreading. Interestingly, the knockdown of IRSp53, a Rac effector that preferentially binds to WAVE2, impaired the development of lamellipodia without affecting proplatelet production. In contrast, thrombopoiesis in vivo and platelet spreading on fibrinogen in vitro were intact in WAVE1-null mice. These observations clarify indispensable roles for the WAVE2/Abi1 complex in alpha(IIb)beta(3)-mediated lamellipodia by MKs and platelets through Rac and IRSp53, and additionally in thrombopoiesis independent of Rac and IRSp53.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recently, we reported the formation of gut-like structures from mouse ESCs in vitro. To determine whether ESCs provide an in vitro model of gastrointestinal (GI) tracts and their organogenesis, we investigated the morphological features, formation process, cellular development, and regional location within the GI tract by immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction. We also examined the developmental potential by transplantation into kidney capsules. The results demonstrated that Id2-expressing epithelium developed first, alpha-smooth muscle actin appeared around the periphery, and finally, the gut-like structures were formed into a three-layer organ with well-differentiated epithelium. A connective tissue layer and musculature with interstitial cells of Cajal developed, similar to organogenesis of the embryonic gut. Enteric neurons appeared underdeveloped, and blood vessels were absent. Many structures expressed intestinal markers Cdx2 and 5-hydroxytryptamine but not the stomach marker H(+)/K(+) ATPase. Transplants obtained blood vessels and extrinsic nerve growth from the host to prolong life, and even grafts of premature structures did not form teratoma. In conclusion, gut-like structures were provided with prototypical tissue components of the GI tract and are inherent in the intestine rather than the stomach. The formation process was basically same as in gut organogenesis. They maintain their developmental potential after transplantation. Therefore, gut-like structures provide a unique and useful in vitro system for development and stem cell studies of the GI tract, including transplantation experiments.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Vasculogenesis and hematopoiesis are thought to arise in hemangioblasts, the common progenitors of cells in vessels and in blood. This scheme was challenged by kinetic analysis of vascular endothelial and hematopoietic progenitors in early gastrulating mouse embryos. The OP-9 co-culture system with a combination of cytokines permitted the detection of endothelial progenitors, as well as stroma-dependent hematopoietic progenitors. Endothelial progenitors were detected as early as embryonic day (E) 5.50, after which time their numbers increased. Stroma-dependent hematopoietic progenitors were detected at E6.75, the time point when hemangioblasts reportedly emerge. Colony-forming units in culture (CFU-c), most likely generated from stroma-dependent hematopoietic progenitors via contact with the microenvironment, were detected at E7.50, concomitant with the onset of primitive hematopoiesis in the yolk sac. The presence of nucleated erythrocytes and the expression of an embryonic-type globin in erythroid colonies derived from stroma-dependent hematopoietic progenitors and from CFU-c support the notion that these progenitors coordinately establish primitive hematopoiesis. Using Oct3/4 promoter-driven GFP transgenic mice, early endothelial progenitors, stroma-dependent hematopoietic progenitors, and CFU-c were all shown to express the Oct3/4 transcription factor. Among Oct3/4-positive cells, both endothelial and hematopoietic progenitors were present in the CD31-positive fraction, leaving a subset of endothelial progenitors in the CD31-negative fraction. These data imply that Oct3/4-positive mesoderm gives rise to CD31-negative angioblasts, CD31-positive angiboblasts and CD31-positive hemangioblasts. We propose a distinct developmental pathway in which the angioblast lineage directly diverges from mesoderm prior to and independent of hemangioblast development.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The growth of cells in vitro can provide useful models for investigating their behaviour and improving our understanding of their function in vivo. Although the developmental regulation of enamel matrix formation has been comprehensively analysed, the detailed cellular characteristics of ameloblasts remain unclear because of the lack of a system of long-term in vitro culture. Therefore, the establishment of odontogenic epithelial cell lines has taken on a new significance. Here, we report on a novel porcine odontogenic epithelial cell-culture system, which has permitted serial culture of these cells. Epithelial cells were harvested from third molar tooth buds in the fresh mandibles of 6-month-old pigs, and seeded on dishes in D-MEM containing 10% FBS. Before the cells reached confluence, the medium was changed to LHC-9 to select the epithelial cells. When trypsinized epithelial cells were plated together with 3T3-J2 cells as a feeder layer, the epithelial cells grew from single cells into colonies. The colonies then expanded and became confluent, and could be sub-cultured for up to 20 passages. The long-term culture cells expressed mRNA for amelogenin and ameloblastin, as well as enamelysin (MMP-20), which is a tissue-specific gene product unique to ameloblasts. These results show that the system is capable of sustaining the multiplication of odontogenic epithelial cells with the characteristics of ameloblasts.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mouse embryonic stem (ES) cells are pluripotent and retain the potential to form an organ similar to the gut showing spontaneous contractions in vitro. The morphological features of these structures and their formation, as assessed using the hanging drop method to produce embryoid bodies (EBs), seem to be similar to those in vivo. To determine whether the same molecular mechanisms are involved in the formation process, the expression pattern of transcription factors regulating endoderm and gut development in the mouse embryo was examined by in situ hybridization and compared with in vivo expression. Expression of gene products was also examined by immunohistochemistry, and expression colocalization was analyzed with double staining. The results showed that all factors examined, that is, Sox17, Id2, HNF3beta/Foxa2, and GATA4, were expressed in both EBs and gut-like structures. Moreover, their expression patterns were similar to those in the mouse embryo. EBs after the hanging drop period and before outgrowth already expressed all factors that were colocalized with each other in EB epithelial structures. These findings suggest that the origin of the gut-like structure is determined during the hanging drop period and that the gut-like structure is formed as the epithelial structure in EBs during the hanging drop period. They also indicate that the in vitro system using mouse ES cells mimics in vivo development and should prove useful in the study of molecular mechanisms for endoderm and gut development.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: We previously reported a method for the development of tissue-engineered tooth. However, 1 drawback of the procedure was the inability to determine whether the tooth would function when transplanted in the jaw because it was formed in the omentum of the abdomen. Therefore, the present study was designed to evaluate whether transplantation of dissociated odontogenic cells could induce tissue-engineered odontogenesis in the canine jaw. MATERIALS AND METHODS: Cells were harvested from canine first molar tooth buds and the resulting heterogeneous cell population was seeded on a biodegradable polymer. These constructs were then transplanted into the same sockets after extracting the tooth buds. After transplantation, we evaluated the transplanted constructs using dental x-ray, micro-computed tomography, histology, and immunohistochemistry. RESULTS: After 24 weeks, micro-x-ray computed tomography showed regenerated hard tissues in the jaw, and hematoxylin and eosin staining showed tubular dentin and bone. In the regenerated tissue, osteopontin, osteonectin, and osteocalcin antibodies stained the dentinal matrix. However, enamel tissue and dental-root formation were not observed. CONCLUSION: These data show for the first time the formation of dentin and bone from dissociated odontogenic cells in the canine jaw.

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記述言語：日本語   出版者・発行元：(株)メディカルレビュー社  

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その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2005&ichushi_jid=J03810&link_issn=&doc_id=20050209210010&doc_link_id=%2Fai1regeb%2F2005%2F000401%2F011%2F0085-0091%26dl%3D0&url=http%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fai1regeb%2F2005%2F000401%2F011%2F0085-0091%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

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記述言語：日本語   出版者・発行元：(NPO)日本口腔科学会  

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記述言語：日本語   出版者・発行元：(一社)日本炎症・再生医学会  

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