Induced genetic ablation of Rest leads to the alteration of stimulus-induced response of the vagal nerve.

Rest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells by preventing precocious expression of neuronal genes. In order to further investigate the function of Rest in neurons, we generated and examined mice evoking genetic ablation of Rest specifically in neural tissues by generating Rest conditional knockout mice. As the Rest knockout mice are embryonically lethal, we used a Sox1-Cre allele to excise the floxed Rest gene from the early stage of nerve cell differentiation including neural crest-derived nerve cells. Using this conditional Rest knockout Sox1-Cre; Restflox/flox mice, we have revealed the role of Rest in the parasympathetic nervous system in the stomach and heart.

Cost analysis of four types of surgeries for pelvic organ prolapse in a Japanese population.

INTRODUCTION AND HYPOTHESIS: To compare the perioperative costs analysis between laparoscopic/transvaginal and the mesh/non-mesh surgeries for pelvic organ prolapse (POP) in Japan. MATERIALS AND METHODS: From April 2013 to April 2017, 890 patients who underwent POP surgeries were enrolled in this study. Regarding transvaginal native tissue repair (TV-NTR: transvaginal hysterectomy with colpocleisis), transvaginal mesh surgery (TVM), laparoscopic native tissue repair (L-NTR: laparoscopic hysterectomy and uterosacral ligament colposuspension), and laparoscopic sacrocolpopexy (LSC), a retrospective observational study was performed. Patients' age, operation time, blood loss, perioperative complications, length of hospital stay, pre-/postoperative quality of life (QOL) scores, were reviewed from the medical records. The net income, which was calculated by using the income (the operation/anesthesia fee) and the costs (the labor and consumables costs for operation/anesthesia), was evaluated. RESULTS: The operation fees of the L-NTR ($4250) and the LSC ($4833) groups were higher than that of the TV-NTR ($2652) and the TVM ($2913) groups. The labor costs and consumables costs of operation were higher in the LSC ($1589) and the L-NTR ($1500) groups than the TV-NTR ($180) and the TVM ($178) groups. The consumables costs for anesthesia in the four groups were equal. The operation hours were significantly shorter in the TV-NTR and the TVM groups than the L-NTR and the LSC groups. CONCLUSIONS: We found that TVM operation was an economically excellent and the most efficient POP operation with shorter operation time and less consumables.

Dietary Eriodictyon angustifolium Tea Supports Prevention of Hair Graying by Reducing DNA Damage in CD34+ Hair Follicular Keratinocyte Stem Cells.

Hair follicular keratinocyte stem cells (HFKSC) which provide a functional niche for melanocyte stem cells (MSC) are the primary target of hair graying. However, little research has been done on anti-hair graying medicines targeting HFKSC. We focused on Eriodictyon angustifolium (Ea), which reduces human hair graying when applied topically. To investigate the protective effect of dietary Ea tea (EaT) on hair pigmentation, we used an acute mouse model of hair graying that mimics X-ray-induced DNA damage associated with age-related hair graying. Our results suggest that dietary EaT maintained the niche HFKSC function against X-ray-induced DNA damage and hair graying. These results indicate that dietary EaT may prevent age-related hair graying and serve as an anti-hair graying herbal medicine.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

FGF2-responsive genes in human dental pulp cells assessed using a rat spinal cord injury model.

The central nervous system in adult mammals does not heal spontaneously after spinal cord injury (SCI). However, SCI treatment has been improved recently following the development of cell transplantation therapy. We recently reported that fibroblast growth factor (FGF) 2-pretreated human dental pulp cells (hDPCs) can improve recovery in a rat model of SCI. This study aimed to investigate mechanisms underlying the curative effect of SCI enhanced via FGF2 pretreatment; we selected three hDPC lines upon screening for the presence of mesenchymal stem cell markers and of their functionality in a rat model of SCI, as assessed using the Basso, Beattie, and Bresnahan score of locomotor functional scale, electrophysiological tests, and morphological analyses. We identified FGF2-responsive genes via gene expression analyses in these lines. FGF2 treatment upregulated GABRB1, MMP1, and DRD2, which suggested to contribute to SCI or central the nervous system. In an expanded screening of additional lines, GABRB1 displayed rather unique and interesting behavior; two lines with the lowest sensitivity of GABRB1 to FGF2 treatment displayed an extremely minor effect in the SCI model. These findings provide insights into the role of FGF2-responsive genes, especially GABRB1, in recovery from SCI, using hDPCs treated with FGF2.

Flavonoids with Two OH Groups in the B-Ring Promote Pigmented Hair Regeneration.

During the process of skin regeneration following a skin injury, de novo hair follicle regeneration is initiated after wounding; however, these regenerated hairs are mostly unpigmented. The activation of epidermal melanocyte stem cells and their differentiation into regenerating hair follicles have been shown to be necessary for the pigmented hair regeneration after wounding. To determine the role of flavonoids in the regeneration of pigmented hairs, we applied the candidate flavonoids to the regenerating hair follicles after wounding and identified the flavonoid species that maximally induced pigmented hair regeneration. Flavonoids with two OH groups in the B-ring, such as sterubin, luteolin, and hydroxygenkwanin, showed promising effects in regenerating black pigmented hairs, while those with one OH group in the B-ring showed no significant change. Thus, flavonoids with two OH groups in their B-ring could be studied further as potential wound healing agents with the ability to regenerate pigmented hair.

Novel Rest functions revealed by conditional gene ablation.

Rest is a regulator of neuronal development and has been suggested to function in maintaining the pluripotent state of embryonic stem cells (ESCs); however, this remains controversial. Since Rest null mice show embryonic lethality, we herein generated conditional Rest knockout (CKO) models to investigate Rest functions in more detail. Our results revealed that Rest was not necessary for maintaining the pluripotency of ESCs and instead promoted primitive endoderm differentiation. In contrast to the repressive role of Rest in vitro, including ESCs, neural stem cells, and fibroblasts, on the expression of target neural genes, Rest CKO did not affect the in vivo development of brain tissue. However, the same Rest CKO mice showed an abnormal lens morphology after birth with augmented Notch signaling and down-regulated lens fiber regulator gene expression. The ablation of Rest during neural crest cell (NCC) development caused neonatal lethality due to swelling of the digestive tract with reductions in acetylcholinesterase activity in the myenteric plexus derived from NCCs. Furthermore, a reduced number of melanocyte precursors also derived from NCCs resulted in white spotted coat color phenotypes lacking mature melanocytes. Rest controls thousands of target genes and may have many unknown functions related to diseases.

Genetic Recombination Between Stromal and Cancer Cells Results in Highly Malignant Cells Identified by Color-Coded Imaging in a Mouse Lymphoma Model.

The tumor microenvironment (TME) promotes tumor growth and metastasis. We previously established the color-coded EL4 lymphoma TME model with red fluorescent protein (RFP) expressing EL4 implanted in transgenic C57BL/6 green fluorescent protein (GFP) mice. Color-coded imaging of the lymphoma TME suggested an important role of stromal cells in lymphoma progression and metastasis. In the present study, we used color-coded imaging of RFP-lymphoma cells and GFP stromal cells to identify yellow-fluorescent genetically recombinant cells appearing only during metastasis. The EL4-RFP lymphoma cells were injected subcutaneously in C57BL/6-GFP transgenic mice and formed subcutaneous tumors 14 days after cell transplantation. The subcutaneous tumors were harvested and transplanted to the abdominal cavity of nude mice. Metastases to the liver, perigastric lymph node, ascites, bone marrow, and primary tumor were imaged. In addition to EL4-RFP cells and GFP-host cells, genetically recombinant yellow-fluorescent cells, were observed only in the ascites and bone marrow. These results indicate genetic exchange between the stromal and cancer cells. Possible mechanisms of genetic exchange are discussed as well as its ramifications for metastasis. J. Cell. Biochem. 118: 4216-4221, 2017. © 2017 Wiley Periodicals, Inc.

Dose-dependent roles for canonical Wnt signalling in de novo crypt formation and cell cycle properties of the colonic epithelium.

There is a gradient of ß-catenin expression along the colonic crypt axis with the highest levels at the crypt bottom. In addition, colorectal cancers show a heterogeneous subcellular pattern of ß-catenin accumulation. However, it remains unclear whether different levels of Wnt signalling exert distinct roles in the colonic epithelium. Here, we investigated the dose-dependent effect of canonical Wnt activation on colonic epithelial differentiation by controlling the expression levels of stabilised ß-catenin using a doxycycline-inducible transgenic system in mice. We show that elevated levels of Wnt signalling induce the amplification of Lgr5+ cells, which is accompanied by crypt fission and a reduction in cell proliferation among progenitor cells. By contrast, lower levels of ß-catenin induction enhance cell proliferation rates of epithelial progenitors without affecting crypt fission rates. Notably, slow-cycling cells produced by ß-catenin activation exhibit activation of Notch signalling. Consistent with the interpretation that the combination of Notch and Wnt signalling maintains crypt cells in a low proliferative state, the treatment of ß-catenin-expressing mice with a Notch inhibitor turned such slow-cycling cells into actively proliferating cells. Our results indicate that the activation of the canonical Wnt signalling pathway is sufficient for de novo crypt formation, and suggest that different levels of canonical Wnt activations, in cooperation with Notch signalling, establish a hierarchy of slower-cycling stem cells and faster-cycling progenitor cells characteristic for the colonic epithelium.

White spotting phenotype induced by targeted REST disruption during neural crest specification to a melanocyte cell lineage.

Neural crest cells (NCCs) emerge from the dorsal region of the neural tube of vertebrate embryos and have the pluripotency to differentiate into both neuronal and non-neuronal lineages including melanocytes. Rest, also known as NRSF (neuro-restrictive silencer factor), is a regulator of neuronal development and function and suggested to be involved in the lineage specification of NCCs. However, further investigations of Rest gene functions in vivo have been hampered by the fact that Rest null mice show early embryonic lethality. To investigate the function of Rest in NCC development, we recently established NCC-specific Rest conditional knockout (CKO) mice and observed their neonatal death. Here, we have established viable heterozygous NCC-specific Rest CKO mice to analyze the function of Rest in an NCC-derived melanocyte cell lineage and found that the white spotting phenotype was associated with the reduction in the number of melanoblasts in the embryonic skin. The Rest deletion induced after the specification to melanocytes did not reduce the number of melanoblasts; therefore, the expression of REST during the early neural crest specification stage was necessary for the normal development of melanoblasts to cover all of the skin.

Retinal Cell Degeneration in Animal Models.

The aim of this review is to provide an overview of various retinal cell degeneration models in animal induced by chemicals (N-methyl-D-aspartate- and CoCl2-induced), autoimmune (experimental autoimmune encephalomyelitis), mechanical stress (optic nerve crush-induced, light-induced) and ischemia (transient retinal ischemia-induced). The target regions, pathology and proposed mechanism of each model are described in a comparative fashion. Animal models of retinal cell degeneration provide insight into the underlying mechanisms of the disease, and will facilitate the development of novel effective therapeutic drugs to treat retinal cell damage.

Reduced carotid baroreceptor distensibility-induced baroreflex resetting contributes to impairment of sodium regulation in rats fed a high-fat diet.

Decreased carotid arterial compliance has been reported in obese subjects and animals. Carotid baroreceptors are located at the bifurcation of the common carotid artery, and respond to distension of the arterial wall, suggesting that higher pressure is required to obtain the same distension in obese subjects and animals. A hyperosmotic NaCl solution induces circulatory volume expansion and arterial pressure (AP) increase, which reflexively augment renal excretion. Thus, we hypothesized that sodium regulation via the baroreflex might be impaired in response to chronic hyperosmotic NaCl infusion in rats fed a high-fat diet. To examine this hypothesis, we used rats fed a high-fat (Fat) or normal (NFD) diet, and measured mean AP, water and sodium balance, and renal function in response to chronic infusion of hyperosmotic NaCl solution via a venous catheter. Furthermore, we examined arterial baroreflex characteristics with static open-loop analysis and distensibility of the common carotid artery. Significant positive water and sodium balance was observed on the 1st day of 9% NaCl infusion; however, this disappeared by the 2nd day in Fat rats. Mean AP was significantly higher during 9% NaCl infusion in Fat rats compared with NFD rats. In the open-loop analysis of carotid sinus baroreflex, a rightward shift of the neural arc was observed in Fat rats compared with NFD rats. Furthermore, distensibility of the common carotid artery was significantly reduced in Fat rats. These results indicate that a reduced baroreceptor distensibility-induced rightward shift of the neural arc might contribute to impairment of sodium regulation in Fat rats.

Genetic ablation of Rest leads to in vitro-specific derepression of neuronal genes during neurogenesis.

Rest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. However, the function of Rest during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In the present study, we have generated Rest conditional knockout mice, which allow the effect of genetic ablation of Rest during embryonic neurogenesis to be examined in vivo. We show that Rest plays a role in suppressing the expression of neuronal genes in cultured neuronal cells in vitro, as well as in non-neuronal cells outside of the central nervous system, but that it is dispensable for embryonic neurogenesis in vivo. Our findings highlight the significance of extrinsic signals for the proper intrinsic regulation of neuronal gene expression levels in the specification of cell fate during embryonic neurogenesis in vivo.

Neonatal lethality of neural crest cell-specific Rest knockout mice is associated with gastrointestinal distension caused by aberrations of myenteric plexus.

RE1-silencing transcription factor (REST), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes. Rest null mice have embryonic lethality which prevents further investigations of the functions of the Rest gene in vivo. We studied neonatal but not embryonic lethality that was characterized by gastrointestinal tract dilation in the neural crest cell (NCC)-specific Rest conditional knockout (CKO) mice. While no histological abnormalities except the thinning of the digestive tract as a consequence of the gas accumulation were found in the digestive tract of the mutant mice, they do not have proper gastric retention after oral dye administration and the reduction of acetylcholinesterase (AChE) activity in NCC-derived myenteric plexus in the stomach was detected. High CO2 concentration in the dilated digestive tract of the Rest CKO mice indicates a failure of gut function by underdeveloped cholinergic transmission in the enteric nervous system. The observed gastrointestinal distension phenotype provides a model for understanding the genetic and molecular basis of NCC defects in humans.

Hair follicle melanocyte precursors are awoken by ultraviolet radiation via a cell extrinsic mechanism.

Melanocyte stem cells (MCSCs) in the upper portion of the hair follicle periodically supply melanocytes (MCs) that migrate downward into the hair bulb during anagen, the growth phase of the hair cycle. However MCs can also migrate upwards. We previously observed an increase in epidermal MC density in the mouse epidermis after a single ultraviolet radiation (UVR) exposure in neonatal, but not adult mice. To better understand MCSC activation by UVR we methodically studied the response of MCs to narrow band UVB (since UVA does not invoke this response) exposure in neonatal mice, and in adults at different stages of the hair cycle. We found that a single exposure of adult mice did not induce activation of MCSCs, in any stage of the hair cycle. When adult mice MCSCs were isolated in telogen, multiple UVB exposures resulted in their activation and production of daughter cells, which migrated upwards to the epidermis. Importantly, the MCSCs produced new progeny without themselves having incurred DNA damage after UVB exposure. This, together with examination of MC localisation in the skin of mice overexpressing stem cell factor in their keratinocytes, leads us to conclude that MCSC activation by UVB is driven via paracrine production of either SCF and/or other keratinocyte cytokines. We re-examined the increase in epidermal MC density in neonatal mouse skin. This effect was much more profound after only a single exposure than that of even multiple exposures to adult skin, and we show that in this setting also, the epidermal MCs mostly derive from activation of MC precursors in the upper hair follicle, and most likely via a cell extrinsic mechanism. Hence, although adaptive changes in the skin induced by repetitive UVB exposures are necessary in adult mice, in both the adult and neonatal context the division and migration upwards of follicular MCSCs is the major mode by which epidermal MC numbers increase after UVR exposure.

[Health effects of interventions to promote physical activity in survivors of the 2011 Great East Japan Earthquake. A longitudinal study].

OBJECTIVES: Interventions that promote physical activity to prevent psychological distress and disuse syndromes were carried out in disaster-stricken areas. However, the effect of these interventions to promote physical activity in disaster-stricken areas has not yet been fully clarified. The purpose of this study was to examine the health effects of promoting physical activity in a disaster-stricken area. METHODS: We conducted an exercise intervention as part of a health survey project among residents of Ishinomaki-city, Miyagi, Japan in 2012. To determine if changes in health condition differed between intervention participants and nonparticipants, health condition data from 81 participants were compared with data from 81 nonparticipants selected by propensity score matching. Factors including sex, age, original address (pre-quake), and six outcome variables (psychological distress [K6 score], subjective health status, sleep duration, sleep quality, frequency of outings, and time spent walking) were used for matching. A linear mixed model was used for statistical analysis. RESULTS: There were no significant differences in K6 score between participants and nonparticipants (P=0.913). Significant improvements were observed in subjective health status (P=0.011) and outing frequency (P=0.002), but not in other outcome variables. CONCLUSION: Subjective health status and outing frequency were significantly improved among participants of the exercise intervention. Exercise intervention may be an effective public health strategy in disaster-stricken areas.

The stemness of neural crest cells and their derivatives.

Neural crest cells (NCCs) are unique to vertebrates and emerge from the border of the neural plate and subsequently migrate extensively throughout the embryo after which they differentiate into many types of cells. This multipotency is the main reason why NCCs are regarded as a versatile tool for stem cell biology and have been gathering attention for their potential use in stem cell based therapy. Multiple sets of networks comprised of signaling molecules and transcription factors regulate every developmental phase of NCCs, including maintenance of their multipotency. Pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem (iPS) cells, facilitate the induction of NCCs in combination with sophisticated culture systems used for neural stem cells, although at present, clinical experiments for NCC-based cell therapy need to be improved. Unexpectedly, the multipotency of NCCs is maintained after they reach the target tissues as tissue neural crest stem cells (NCSCs) that may contribute to the establishment of NCC-derived multipotential stem cells. In addition, under specific culture conditions, fate-restricted unipotent descendants of NCCs, such as melanoblasts, show multipotency to differentiate into melanocytes, neurons, and glia cells. These properties contribute to the additional versatility of NCCs for therapeutic application and to better understand NCC development.

Generation of iPSCs Using Sendai Virus Vectors.

Induced pluripotent stem cells (iPSCs) are in vitro-derived cells capable of giving rise to several different cell types. The generation of iPSCs holds great promise for regenerative medicine and drug discovery research because it allows mature cells to be reprogrammed into a state of pluripotency. These highly versatile cells can then be induced to produce a variety of cell lineages and tissues by activating specific regulatory genes that drive their differentiation along distinct lineages. The great potential of these cells was recognized by Shinya Yamanaka who was awarded the 2012 Nobel Prize for the discovery of iPSCs. Following their discovery, various methods have now been developed for generating iPSCs. Here, we describe a method for deriving iPSCs from human dental pulp using Sendai virus vectors.

Postnatal Expression of Kitl Affects Pigmentation of the Epidermis.

KITL signaling is important for melanocyte development in mammals; however, its function in the melanocyte stem cells in adult skin is not well-understood. In this study, we have generated genetically modified mice that express a Kitl transgene under the control of a doxycycline-inducible promoter to investigate the impact of its overexpression in embryo, young postnatal, and adult skin with intact hair follicles. We report that overexpression of KITL influences the proliferation and differentiation of melanocytes as well as the self-renewal capacity of resident melanocyte stem cells within the follicular niche. Notably, activation of Kit-KITL signaling induced the migration of melanocytes from hair follicles to the epidermis. In addition, we demonstrate that a single pulse of Kitl transgene expression in postnatal mice results in long-lasting effects on melanocyte stem cells and their differentiated progeny as pigmented skin cells that persist through adulthood. Our findings indicate that regulation of KITL signaling in melanocyte lineage is crucial for melanocyte stem cell homeostasis and melanocyte cell differentiation in postnatal and adult mice.

Dual origin of melanocytes defined by Sox1 expression and their region-specific distribution in mammalian skin.

Melanocytes are pigment-producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1(+) dorsal neuroepithelial cells but are derived from Sox1(-) cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1(-) cells clearly segregate from cells that originated from Sox1(+) dorsal neuroepithelial cell-derived NCCs. The possible derivation of Sox1(-) cells from epidermal cells also strengthens their non-neuroepithelial origin.