Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.

DOI： 10.1073/pnas.2016850117

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Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.2016850117

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The Cre-loxP recombination system is widely used to generate genetically modified mice for biomedical research. Recently, a highly efficient photoactivatable Cre (PA-Cre) based on reassembly of split Cre fragments has been established. This technology enables efficient DNA recombination that is activated upon blue light illumination with spatiotemporal precision. In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.

DOI： 10.1016/j.bbrc.2020.03.015

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BACKGROUND: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. METHODS: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. RESULTS: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. CONCLUSION: Targeting the PRRX1-TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

DOI： 10.1038/s41416-024-02632-8

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Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

DOI： 10.1016/j.bbrc.2023.149146

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With the advancement of tissue engineering technologies, implantable materials have been developed for use in facial plastic surgery, including auriculoplasty and rhinoplasty. Tissue-engineered cartilage comprising only cells and cell-produced extracellular matrix is considered valuable as there is no need to consider problems associated with the absorption of scaffold materials and the generation of immune responses. However, it is exceedingly difficult to produce large-sized complex shapes of cartilage without the use of scaffolds. In this study, we describe the production of shape-designable cartilage using a novel cell self-aggregation technique (CAT) and chondroprogenitor cells derived from human induced pluripotent stem cells as the source. The method described does not require special equipment such as bio-3D printers, and the produced tissue can be induced into well-matured cartilage with abundant cartilage matrix in vitro. Using CAT, we were able to generate cartilage in the form of rings or tubes with adjustable inner diameter and curvature, over a range of several centimeters, without the use of scaffolds. The in vitro fabrication of shape-designable cartilage using CAT will undoubtedly contribute to developments in the field of facial plastic surgery.&#xD.

DOI： 10.1088/1748-605X/ad02d1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Partial or whole regeneration of the uterine endometrium using extracellular matrix (ECM)-based scaffolds is a therapeutic strategy for uterine infertility due to functional and/or structural endometrial defects. Here, we examined whether the entire endometrium can be regenerated circumferentially using an acellular ECM scaffold (decellularized endometrial scaffold, DES) prepared from rat endometrium. We placed a silicone tube alone to prevent adhesions or a DES loaded with a silicone tube into a recipient uterus in which the endometrium had been surgically removed circumferentially. Histological and immunofluorescent analyses of the uteri one month after tube placement revealed more abundant regenerated endometrial stroma in the uterine horns treated with tube-loaded DES compared to those treated with a tube alone. Luminal and glandular epithelia, however, were not fully recapitulated. These results suggest that DES can enhance the regeneration of endometrial stroma but additional intervention(s) are needed to induce epithelization. Furthermore, the prevention of adhesions alone allowed the endometrial stroma to regenerate circumferentially even without a DES, but to a lesser degree than that with a DES. The use of a DES together with the prevention of adhesions may be beneficial for efficient endometrial regeneration in the uterus that is largely deficient of endometrium.

DOI： 10.3390/ijms24087605

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX⁺ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato⁺ cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato⁺ cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.

DOI： 10.1186/s13287-022-03203-5

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Other Link： https://link.springer.com/article/10.1186/s13287-022-03203-5/fulltext.html

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Objective: To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). Design: Experimental study. Setting: Academic research laboratory. Patient(s): Women undergoing hysterectomy for leiomyoma. Intervention(s): CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. Main Outcome Measure(s): Cell proliferation, collagen production, and in vivo tumorigenicity of USMCs with vs. without the MED12 mutation. Result(s): Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC in vitro, and this production was likely enhanced by EP and TGFB3. Conclusion(s): The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrial stem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.

DOI： 10.1016/j.xfss.2022.04.002

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Optogenetics, a technology to manipulate biological phenomena thorough light, has attracted much attention in neuroscience. Recently, the Magnet System, a photo-inducible protein dimerization system which can control the intracellular behavior of various biomolecules with high accuracy using light was developed. Furthermore, photoactivation systems for controlling biological phenomena are being developed by combining this technique with genome-editing technology (CRISPR/Cas9 System) or DNA recombination technology (Cre-loxP system). Herein, we review the history of optogenetics and the latest Magnet System technology and introduce our recently developed photoactivatable Cre knock-in mice with temporal-, spatial-, and cell-specific accuracy.

DOI： 10.18926/AMO/63202

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Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

DOI： 10.1002/ijc.33713

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Paired related homeobox 1 (PRRX1) is a marker of limb bud mesenchymal cells, and deficiency of p53 or Rb in Prrx1-positive cells induces osteosarcoma in several mouse models. However, the regulatory roles of PRRX1 in human osteosarcoma have not been defined. In this study, we performed PRRX1 immunostaining on 35 human osteosarcoma specimens to assess the correlation between PRRX1 level and overall survival. In patients with osteosarcoma, the expression level of PRRX1 positively correlated with poor prognosis or the ratio of lung metastasis. Additionally, we found PRRX1 expression on in 143B cells, a human osteosarcoma line with a high metastatic capacity. Downregulation of PRRX1 not only suppressed proliferation and invasion but also increased the sensitivity to cisplatin and doxorubicin. When 143B cells were subcutaneously transplanted into nude mice, PRRX1 knockdown decreased tumor sizes and rates of lung metastasis. Interestingly, forskolin, a chemical compound identified by Connectivity Map analysis using RNA expression signatures during PRRX1 knockdown, decreased tumor proliferation and cell migration to the same degree as PRRX1 knockdown. These results demonstrate that PRRX1 promotes tumor malignancy in human osteosarcoma.

DOI： 10.1016/j.tranon.2020.100960

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<h4>Introduction</h4>P2Y14, one of the P2Y purinergic G-protein coupled receptors, is expressed in a variety of cells and tissues. Its ligand, UDP-glucose (UDPG), is released from damaged and stress-stimulated cells and acts as a danger signal via P2Y14. Thus, P2Y14 plays an important role in immunological defense systems. Here, we aimed to elucidate the expression, localization, and role of P2Y14 in human trophoblasts and the placenta.<h4>Methods</h4>Human chorionic villus and placental tissues were subjected to immunostaining for P2Y14 protein and an extravillous trophoblast (EVT) marker, HLA-G. We examined the expression of P2Y14 and the effect of UDPG on cell proliferation and invasion in an EVT cell line, HTR-8/SVneo, using an MTS assay and a Transwell assay, respectively. We tested the effect of UDPG on cell invasion in P2Y14-underexpressing HTR-8/SVneo clones established by the lentiviral introduction of shRNA for P2RY14 mRNA.<h4>Results</h4>Immunostaining revealed that P2Y14 was exclusively expressed by EVTs. P2RY14 mRNA and P2Y14 protein were expressed in HTR-8/SVneo cells. UDPG did not affect cell proliferation but it did enhance invasion. Inhibition of P2Y14 and decreasing the expression of P2Y14 suppressed UDPG-mediated invasive activity.<h4>Conclusions</h4>These results showed that EVT selectively expressed P2Y14 and that P2Y14 was positively involved in UDPG-enhanced EVT invasion. It suggests the possible existence of a danger signal-mediated physiological system at the fetomaternal interface where UDPG released from maternal tissues through destruction by EVT invasion may accelerate EVT invasion, allowing EVTs to undergo successful placentation and vascular remodeling.

DOI： 10.1016/j.placenta.2020.09.061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

DOI： 10.1093/biolre/ioz004

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A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P &lt; 0.05). Despite the changes in tiss

DOI： 10.1093/biolre/ioz004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

BHLHE40 and BHLHE41 (BHLHE40/41) are basic helix-loop-helix type transcription factors that play key roles in multiple cell behaviors. BHLHE40/41 were recently shown to be involved in an epithelial-to-mesenchymal transition (EMT). However, the precise mechanism of EMT control by BHLHE40/41 remains unclear. In the present study, we demonstrated that BHLHE40/41 expression was controlled in a pathological stage-dependent manner in human endometrial cancer (HEC). Our<italic>in vitro</italic>assays showed that BHLHE40/41 suppressed tumor cell invasion. BHLHE40/41 also suppressed the transcription of the EMT effectors<italic>SNAI1</italic>,<italic>SNAI2</italic>, and<italic>TWIST1</italic>. We identified the critical promoter regions of<italic>TWIST1</italic>for its basal transcriptional activity. We elucidated that the transcription factor SP1 was involved in the basal transcriptional activity of<italic>TWIST1</italic>and that BHLHE40/41 competed with SP1 for DNA binding to regulate gene transcription. This study is the first to report the detailed functions of BHLHE40 and BHLHE41 in the suppression of EMT effectors<italic>in vitro</italic>. Our results suggest that BHLHE40/41 suppress tumor cell invasion by inhibiting EMT in tumor cells. We propose that BHLHE40/41 are promising markers to predict the aggressiveness of each HEC case and that molecular targeting strategies involving BHLHE40/41 and SP1 may effectively regulate HEC progression.

DOI： 10.1128/mcb.00678-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca(2+)-permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT-PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild-type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up-regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat-induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.

DOI： 10.1096/fj.14-251314

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.14-251314

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Dioxins are persistent environmental pollutants that cause multiple adverse health effects in humans, mainly through binding to the ligand-activated transcription factor, aryl hydrocarbon receptor (AhR). Genetic variation in AhR may modulate the susceptibility to dioxins. In this study, we aimed to evaluate the effects of the single nucleotide polymorphism (SNP) -130 C/T in the AhR promoter on dioxin-inducible gene transcription, and to investigate interleukin-24 (IL-24) and interleukin-1β (IL-1β) as proxies for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Using primary human chorionic stromal cells, we found that cells with the TT genotype showed higher AhR mRNA and protein levels than did those of the CC genotype. Microarray was carried out to analyze the gene expression profiles of cells (CC and TT genotype) after exposing the cells to TCDD. Several genes associated with human disorders were more highly up-regulated in cells of the TT genotype. Higher up-regulation of IL-24 and IL-1β mRNA in cells with the TT genotype was observed. Furthermore, blood samples from 64 Yusho patients who were accidentally exposed to high concentrations of dioxins were analyzed for the genotype, dioxins concentrations and serum levels of IL-24 and IL-1β. We observed higher serum IL-24 levels and lower serum IL-1β levels in Yusho patients with the TT genotype than in those with the CC genotype. AhR SNP -130 C/T affects serum IL-24 and IL-1β levels, independently of serum dioxins concentrations in Yusho patients. Our observations demonstrate that SNP -130 C/T modulates AhR expression and expression levels of IL-24 and IL-1β, and suggest an association of AhR SNP -130 C/T with the susceptibility to dioxins.

DOI： 10.1016/j.toxlet.2014.11.025

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A total of 297 samples of hydropic villi were classified according to DNA polymorphisms as androgenetic moles, dispermic triploids, or biparental diploids. A subset of 267 appropriate samples was included in the study. Most of the macroscopically diagnosed complete mole cases were genetically androgenetic in origin. The partial mole cases consisted of 30 androgenetic moles and 12 dispermic triploids. For the 59 cases macroscopically categorized as hydropic abortion, the genetic analysis revealed 38 androgenetic moles, seven dispermic triploids and 14 biparental diploids. These results showed that a new diagnostic method was required for the management of patients with hydropic villi. We identified the TSSC imprint gene of which expression was shown in normal and partial mole villi but was silenced in complete mole villi. Immunohistochemistry using the TSSC3 antibody demonstrated its efficacy as the differential diagnostic method. TSSC3 play an important role in the differentiation from trophoblast stem cells to progenitors and/or labyrinth trophoblast through the TSSC3/PI3K/Akt/Mash2 signaling pathway.

DOI： 10.1111/jog.12100

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In orthodontic tooth movement (OTM), we should be concerned about external root resorption (ERR) as an undesirable iatrogenic problem, but its mechanisms are not fully understood. Since our previous epidemiologic studies found that patients with allergic diseases showed higher rates of ERR during orthodontic treatment, we explored the possible effect of allergic sensitization on ERR. In ovalbumin (OVA)-sensitized Brown-Norway rats, the amounts of ERR and OTM were greater than those in animals subjected to orthodontic force alone. The expression levels of RANKL and pro-inflammatory cytokines were increased in the periodontal tissues of sensitized rats with OTM, compared with control rats. Furthermore, leukotriene B4 (LTB4), a potent lipid mediator of allergic inflammation, and enzymes of the 5-lipoxygenase pathway, the biosynthetic pathway of leukotrienes, were also up-regulated. We found that low doses of aspirin suppressed ERR in allergen-sensitized rats, as well as the expressions of RANKL, pro-inflammatory cytokines, and LTB4. The present findings indicate that allergen sensitization has adverse effects on ERR under OTM, and that aspirin is a potential therapeutic agent for combating ERR. © International &amp
American Associations for Dental Research.

DOI： 10.1177/0022034513488787

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Animal Reproduction  

Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H₂O₂) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H₂O₂ in substitution for X/XO. We assessed the effects of H₂O₂ on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H₂O₂. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H₂O₂ also decreased the relative cell number. Pretreatment with H₂O₂ significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H₂O₂ also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.

DOI： 10.1262/jrd.2012-053

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Involvement of the aryl hydrocarbon receptor (AHR) in carcinogenesis has been suggested in many studies. Upregulation of AHR has been reported in some cancer species, and an association between single-nucleotide polymorphisms (SNPs) of AHR and cancer risk or cancer development has also been reported. This evidence suggests the involvement of some specific SNPs in AHR transcriptional regulation in the process of carcinogenesis or cancer development, but there have been no studies to elucidate the mechanism involved. In this study, we identified the transcription factor Nuclear Factor 1-C (NF1C) as a candidate to regulate AHR transcription in a polymorphism-dependent manner. SNP rs10249788 was included in a consensus binding site for NF1C. Our results suggested that NF1C preferred the C allele to the T allele at rs10249788 for binding. Forced expression of NF1C suppressed the activity of the AHR promoter with C at rs10249788 stronger than that with T. Moreover, expression analysis of human uterine endometrial cancer (HEC) specimens showed greater upregulation of AHR and downregulation of NF1C than those of normal endometrium specimens. Sequence analysis showed HEC patients at advanced stages tended to possess T/T alleles more frequently than healthy women. We also demonstrated that NF1C suppressed proliferation, motility and invasion of HEC cells. This function was at least partially mediated by AHR. This study is the first to report that a polymorphism on the AHR regulatory region affected transcriptional regulation of the AHR gene in vitro. Because NF1C is a tumor suppressor, our new insights into AHR deregulation and its polymorphisms could reveal novel mechanisms of genetic susceptibility to cancer.

DOI： 10.1038/onc.2012.509

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Other Link： http://www.nature.com/articles/onc2012509

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Tssc3 is a maternally expressed/paternally silenced imprinted gene. Recent evidence suggests that the loss of TSSC3 results in placental overgrowth in mice. These findings showed that the TSSC3 gene functions as a negative regulator of placental growth. In this study, we describe the function of TSSC3 and its signaling pathway in mouse trophoblast stem (TS) cell differentiation. First of all, we tested Tssc3 expression levels in TS cells. TS cells expressed Tssc3, and its expression level was the highest from day 1 to 4 but was down-regulated at day 5 after the induction of differentiation. Overexpression of TSSC3 in TS cells up-regulated Gcm1 and Mash2, which are marker genes of mouse trophoblast differentiation. Down-regulation of TSSC3 by siRNA enhanced Pl1 and Tpbpa expression in TS cells cultured under stem cell conditions, suggesting the contribution of TSSC3 to the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts. TSSC3 activated the PI3K/AKT pathway through binding with phosphatidylinositol phosphate lipids and enhanced the activity of a promoter containing an E-box structure, which is the binding sequence of the Mash2 downstream target gene promoter. PI3K inhibitor suppressed the promoter activity induced by TSSC3. TSSC3 induced Sp1 translocation from cytoplasm to nucleus through the PI3K/AKT pathway. Nuclear Sp1 activated the Mash2 transcription by Sp1 binding with a consensus Sp1-binding motif. This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway.

DOI： 10.1074/jbc.m112.388777

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Low level, antenatal exposure to dioxins is associated with low birth weight, which in turn is associated with long-term sequelae. We exposed the human extravillous cytotrophoblast (EVT) lines HTR-8/SV40 and TCLI to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and assessed cell growth, invasion, and differentiation. TCDD had no effect on cell proliferation, invasion, or tube formation in Matrigel. The EVT-derived cells expressed a functional aryl hydrocarbon receptor protein; however, TCDD exposure did not alter expression levels of proteins involved in EVT differentiation in early pregnancy, including hypoxia-inducible factor IA (HIFIA), vascular endothelial growth factor (VEGF), Integrin AI, A6, and AVB3. These results suggest that the reduction in fetal weight induced by dioxin is not the result of vascular remodeling via EVT dysfunction.

DOI： 10.1177/0960327111424305

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Recently, numerous studies have identified that immature cell populations including stem cells and progenitor cells can be found among "side-population" (SP) cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP remained to be reported. In this study, HTR-8/SVneo cells and human primary villous cytotrophoblasts (vCTBs) were stained with Hoechst 33342 and SP and non-SP (NSP) fractions were isolated using a cell sorter. A small population of SP cells was identified in HTR-8/SVneo cells and in vCTBs. SP cells expressed several vCTB-specific markers and failed to express syncytiotrophoblast (STB) or extravillous cytotrophopblast (EVT)-specific differentiation markers. SP cells formed colonies and proliferated on mouse embryonic fibroblast (MEF) feeder cells or in MEF conditioned medium supplemented with heparin/FGF2, and they also showed long-term repopulating property. SP cells could differentiate into both STB and EVT cell lineages and expressed several differentiation markers. Microarray analysis revealed that IL7R and IL1R2 were exclusively expressed in SP cells and not in NSP cells. vCTB cells sorted as positive for both IL7R and IL1R2 failed to express trophoblast differentiation markers and spontaneously differentiated into both STB and EVT in basal medium. These features shown by the SP cells suggested that IL7R and IL1R2 are available as markers to detect the SP cells and that vCTB progenitor cells and trophoblast stem cells were involved in the SP cell population.

DOI： 10.1371/journal.pone.0021990

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for Cancer Research (AACR)  

We previously isolated side-population (SP) cells from a human endometrial cancer cell line, Hec1, and determined that Hec1-SP cells have cancer stem-like cell features. In this study, we isolated SP cells and non-SP (NSP) cells derived from a rat endometrial cell line expressing human [(12)Val] KRAS (RK12V cells) and determined the SP phenotype. RK12V-SP cells showed self-renewal capacity, the potential to develop into stromal cells, reduced expression levels of differentiation markers, long-term proliferating capacity in cultures, and enhanced tumorigenicity, indicating that RK12V-SP cells have cancer stem-like cell features. RK12V-SP cells also display higher resistance to conventional chemotherapeutic drugs. In contrast, treatment with a histone deacetylases (HDAC) inhibitor, sodium butyrate (NaB), reduced self-renewal capacity and completely suppressed colony formation of RK12V-SP cells in a soft agar. The levels of intracellular reactive oxygen species (ROS) and the number of γH2AX foci were increased by NaB treatment of both RK12V-SP cells and RK12V-NSP cells. The expression levels of γH2AX, p21, p27, and phospho-p38 mitogen-activated protein kinase were enhanced in RK12V-SP cells compared with RK12V-NSP cells. These results imply that treatment with NaB induced production of intracellular ROS and DNA damage in both RK12V-SP and RK12V-NSP cells. Following NaB treatment, DNA damage response signals were enhanced more in RK12V-SP cells than in RK12V-NSP cells. This is the first article on an inhibitory effect of NaB on proliferation of endometrial cancer stem-like cells. HDAC inhibitors may represent an attractive antitumor therapy based upon their inhibitory effects on cancer stem-like cells.

DOI： 10.1158/1535-7163.mct-10-1062

DOI： 10.1158/1535-7163.MCT-10-1062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Cancer stem-like cell subpopulations, referred to as "side-population" (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, alpha-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into alpha-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.

DOI： 10.2353/ajpath.2010.090056

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

DOI： 10.1007/s00418-009-0616-y

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Other Link： http://link.springer.com/article/10.1007/s00418-009-0616-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

HOPX (homeodomain only protein X) is a newly identified homeobox gene whose loss of expression has been reported for several types of neoplasm. Although we found most human uterine endometrial cancers (HEC) defective in HOPX expression, genetic mutations in the HOPX gene were undetectable. As is the case with several tumor suppressor genes, the promoter region of HOPX is densely methylated in HEC tissue samples obtained by laser capture microdissection. HOPX mRNA and protein levels were reduced in the majority of samples, and this correlated with hypermethylation of the HOPX promoter. Forced expression of HOPX resulted in a partial block in cell proliferation, in vivo tumorigenicity and c-fos gene expression in HEC and MCF7 cells in response to 17beta-estradiol (E(2)) stimulation. Analysis of the serum response element (SRE) of c-fos gene promoter showed that the effect of HOPX expression is associated with inhibition of E(2)-induced c-fos activation through the serum response factor (SRF) motif. Knockdown of HOPX in immortalized human endometrial cells resulted in accelerated proliferation. Our study indicates that transcriptional silencing of HOPX results from hypermethylation of the HOPpromoter, which leads to HEC development.

DOI： 10.1002/ijc.24217

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Language：Japanese   Publisher：(公社)日本整形外科学会  

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Language：Japanese   Publisher：日本レックリングハウゼン病学会  

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Language：Japanese   Publisher：(一社)日本移植学会  

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Language：Japanese   Publisher：日本骨形態計測学会  

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Language：Japanese   Publisher：Japanese Pharmacological Society  

[Background] To achieve the disease modeling of skeletal dysplasia, human induced pluripotent stem cells (iPSCs) will be useful but the induction method for limb bud mesenchymal (LBM) cells, which can give rise to most of the future limb elements - bone, cartilage and tendon/ligament, has not been established. In this study, we developed the induction and expansion protocol of LBM cells from human iPSCs, and a high-throughput drug screening system using human LBM cells differentiated from iPSC which is derived from patients with skeletal dysplasia.

[Method] Paired-related homeobox 1 (PRRX1) serves as a marker for the LBM. To assess the induction efficiency of PRRX1 positive cells during each differentiation step, we established PRRX1-tdTomato reporter human iPSC line. Lateral plate mesoderm cells (LPM) derived from PRRX1-tdTomato reporter were used to establish the LBM-inducing method that recapitulates human developmental process. The chondrogenic capacity of expandable LBM (ExpLBM) cells was assessed using our two- or three- dimensional chondrogenic induction method (2DCI or 3DCI). In addition, ExpLBM cells differentiated from iPSCs derived from patients with type II collagenopathy (COL2pathy), one of the skeletal dysplasia arising from mutations in COL2A1, were used to develop high-throughput screening system.

[Results] By activating WNT signaling and inhibiting BMP/TGFb/headge hog signaling pathways, almost all LPM cells could be induced to PRRX1 positive LBM cells. Interestingly, we found the defined culture method that can not only stably expand LBM cells (ExpLBM) but also maintain their PRRX1 expression. ExpLBM formed Alcian Blue positive nodules under 2DCI condition and Safranin O positive cartilaginous particles under 3DCI condition. 2DCI-based high-throughput screening system found that several chemicals improved the chondrogenic capacity of ExpLBM derived from COL2pathy patient.

[Conclusion] ExpLBM cells will be a potential tool to study human bone development, cartilage regeneration and drug discovery using disease-specific human iPSCs.

DOI： 10.1254/jpssuppl.95.0_2-o-087

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：(公社)日本生物工学会  

DOI： 10.34565/seibutsukogaku.99.1_34

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2021&ichushi_jid=J02754&link_issn=&doc_id=20210127160005&doc_link_id=10.34565%2Fseibutsukogaku.99.1_34&url=https%3A%2F%2Fdoi.org%2F10.34565%2Fseibutsukogaku.99.1_34&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

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DOI： 10.14952/seikagaku.2021.930414

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Language：Japanese   Publisher：日本生殖内分泌学会  

光応答性CRISPR/Cas9システムが実際にCas9として機能するのか、マウス胚着床を制御することが可能かについて検討した。子宮体癌細胞株Ishikawa(IK)細胞に上皮マーカーであるEpCAMプロモーターEp-Prを挿入したLuciferase誘導細胞株としてIK-Ep-Pr細胞を作製し、このEpCAMプロモーターに対し特異的なsgRNA(sgProm)と対照群のコントロールsgRNA(sgCtrl)を作製した。腫瘍内および腹腔内両者ともにLuciferase活性が抑制されていることが確認され、in vitroおよびin vivoともに光Cas9が機能することが明らかとなった。さらに、光Cas9で制御される胚着床モデルの作製を行った。4.5日目のマウス子宮を調べた結果、妊娠コントロール群で強いleukemia inhibitory factor(LIF)発現が認められたが、LED照射を行ったノックアウト群ではLIFの発現が減少していた。7.5日目のマウス子宮内胎児数では、妊娠コントロール群に比較してノックアウト群ではその数が有意に減少していた。光Cas9を用いてin vivo胚着床モデルの作製が可能となった。

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光応答性CRISPR/Cas9システムが実際にCas9として機能するのか、マウス胚着床を制御することが可能かについて検討した。子宮体癌細胞株Ishikawa(IK)細胞に上皮マーカーであるEpCAMプロモーターEp-Prを挿入したLuciferase誘導細胞株としてIK-Ep-Pr細胞を作製し、このEpCAMプロモーターに対し特異的なsgRNA(sgProm)と対照群のコントロールsgRNA(sgCtrl)を作製した。腫瘍内および腹腔内両者ともにLuciferase活性が抑制されていることが確認され、in vitroおよびin vivoともに光Cas9が機能することが明らかとなった。さらに、光Cas9で制御される胚着床モデルの作製を行った。4.5日目のマウス子宮を調べた結果、妊娠コントロール群で強いleukemia inhibitory factor(LIF)発現が認められたが、LED照射を行ったノックアウト群ではLIFの発現が減少していた。7.5日目のマウス子宮内胎児数では、妊娠コントロール群に比較してノックアウト群ではその数が有意に減少していた。光Cas9を用いてin vivo胚着床モデルの作製が可能となった。

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO LTD  

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