Conditional heterozygous loss of Kit receptor tyrosine kinase in neural crest cell lineage is associated with midline cleft lip and bifid nose deformity.

OBJECTIVES: The receptor tyrosine kinase Kit is expressed in cells derived from the trunk neural crest (NC), such as melanocytes; however, its role in cranial NC cell development is not fully understood. METHODS: We investigated the effects of the heterozygous loss of Kit in NC cells during embryonic development by mating Kit2lox/+ mice with Wnt1-Cre mice to produce Wnt1-Cre; Kit2lox/+ embryos. In addition, Wnt1-Cre mice were mated with Rosa26R-yellow fluorescent protein (YFP) mice to visualize the tissue regions expressing Cre recombinase. Histological studies of the craniofacial regions of these mice were performed using samples from embryonic day (E) 12.5 and postnatal day (P) 1. Cellular apoptosis and proliferation were both analyzed through the immunostaining of tissue sections collected on E13.5 and E14.5 using anti-cleaved caspase 3 (CC3) to detect apoptosis and anti-Ki67 to detect proliferation. Cells from YFP-positive tissue regions of the facial areas of Wnt1-Cre; Kit+/+; Rosa26R-YFP embryos and Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos collected on E12.5 and E15.5 were cultured and evaluated for cell proliferation. RESULTS: Compared with control littermates, Wnt1-Cre; Kit2lox/+ embryos exhibited midline cleft lip and bifid nose deformities. Substantial early (P1) postnatal lethality was observed in Wnt1-Cre; Kit2lox/+ mice, with none surviving to 3 weeks of age. YFP-positive cells from the maxillary regions of Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos exhibited defective cell growth and self-renewal in vitro. CONCLUSION: Conditional heterozygous loss of Kit in Wnt1-Cre; Kit2lox/+ embryos is associated with craniofacial dysplasia and exhibit defective NC development in vitro and in vivo.

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.

Are dark-eyed dogs favoured by humans? Domestication as a potential driver of iris colour difference between dogs and wolves.

Comparative studies have shown that the eye morphology of primates has been shaped by a variety of selection pressures (e.g. communication, environmental factors). To comprehensively elucidate the complex links between ocular morphology and its evolutionary drive, attention should be paid to other phylogenetic groups. Here, we address a new question regarding the evolution of eye colour patterns in the oldest domesticated animal, namely, the domestic dog (Canis familiaris). In this study, we conducted an image analysis of dogs and their closest relatives, grey wolves (Canis lupus), to compare the colours of their irises, with the aim of assessing whether eye colours of dogs affect how humans perceived dogs. We found that the irises of dogs were significantly darker than those of wolves. We also found that facial images of dark-eyed dogs were perceived as more friendly and immature, potentially eliciting caregiving responses from humans. Our findings are consistent with our expectation that humans favour dark-eyed dogs over light-eyed ones and provide an updated hypothesis that dogs with dark eyes may have evolved by acquiring a facial trait that sends a non-threatening gaze signal to humans.

Compensatory gene expression potentially rescues impaired brain development in Kit mutant mice.

While loss-of-function mutations in the murine dominant white spotting/Kit (W) locus affect a diverse array of cell lineages and organs, the brain, organ with the highest expression show the least number of defective phenotypes. We performed transcriptome analysis of the brains of KitW embryos and found prominent gene expression changes specifically in the E12.5 KitW/W homozygous mutant. Although other potentially effective changes in gene expression were observed, uniform downregulation of ribosomal protein genes and oxidative phosphorylation pathway genes specifically observed in the E12.5 brain may comprise a genetic compensation system exerting protective metabolic effects against the deleterious effect of KitW/W mutation in the developing brain.

Protective Effect of Hydroxygenkwanin against Hair Graying Induced by X-Ray Irradiation and Repetitive Plucking.

Hair graying in mice is caused by various injuries such as X-ray radiation and repeated plucking that ultimately damage melanocytes and their stem cells (melanocyte stem cells). In X-ray‒induced hair graying, injuries first manifest as a loss-of-niche function of hair follicular keratinocyte stem cells to maintain melanocyte stem cells. Thus, we hypothesized that hair follicular keratinocyte stem cells could be a practical target to prevent hair graying. In this study, we investigated the in vivo effect of the flavonoid hydroxygenkwanin, which has been shown to exert the best protection on human epidermal keratinocytes against in vitro X-ray‒induced cytological effects, using X-ray‒induced and repeated hair plucking‒induced hair graying mice models. We found that hydroxygenkwanin exerted a remarkable effect in preventing hair graying; however, when receptor Y kinase Kit-mutant mice were used, no prevention effect was observed. Therefore, we propose that Kit signaling might be involved in the hydroxygenkwanin-induced protective effect against hair graying.

Induction of iPSC-derived Prg4-positive cells with characteristics of superficial zone chondrocytes and fibroblast-like synovial cells.

BACKGROUND: Lubricin, a proteoglycan encoded by the PRG4 gene, is synthesised by superficial zone (SFZ) chondrocytes and synovial cells. It reduces friction between joints and allows smooth sliding of tendons. Although lubricin has been shown to be effective against osteoarthritis and synovitis in animals, its clinical application remains untested. In this study, we aimed to induce lubricin-expressing cells from pluripotent stem cells (iPSCs) and applied them locally via cell transplantation. METHODS: To generate iPSCs, OCT3/4, SOX2, KLF4, and L-MYC were transduced into fibroblasts derived from Prg4-mRFP1 transgenic mice. We established a protocol for the differentiation of iPSC-derived Prg4-mRFP1-positive cells and characterised their mRNA expression profile. Finally, we injected Prg4-mRFP1-positive cells into the paratenon, surrounding the Achilles tendons and knee joints of severe combined immunodeficient mice and assessed lubricin expression. RESULT: Wnt3a, activin A, TGF-ß1, and bFGF were applied to induce the differentiation of iPSC-derived Prg4-mRFP1-positive cells. Markers related to SFZ chondrocytes and fibroblast-like synovial cells (FLSs) were expressed during differentiation. RNA-sequencing indicated that iPSC-derived Prg4-mRFP1-positive cells manifested expression profiles typical of SFZ chondrocytes and FLSs. Transplanted iPSC-derived Prg4-mRFP1-positive cells survived around the Achilles tendons and in knee joints. CONCLUSIONS: The present study describes a protocol for the differentiation of iPSC-derived Prg4-positive cells with characteristics of SFZ chondrocytes and FLSs. Transplantation of lubricin-expressing cells offers promise as a therapy against arthritis and synovitis.

Exosome Transfer Between Pancreatic-cancer Cells Is Associated With Metastasis in a Nude-mouse Model.

BACKGROUND/AIM: Cancer-derived exosomes play an important role in metastasis. In the present study, we determined whether exosome transfer between cancer cells is associated with metastasis in a mouse model. MATERIALS AND METHODS: AsPC-1 human pancreatic-cancer cells expressing red fluorescent protein (RFP) and AsPC-1 human pancreatic-cancer cells transduced by exosome-specific pCT-CD63-green fluorescent protein (GFP), were co-injected into the spleen of nude mice. RESULTS: Both pancreatic-cancer cell lines grew in the spleen and metastasized to the liver, peritoneum, and lungs, as shown by color-coded imaging. The ratio of GFP-expressing exosomes incorporated in RFP-labeled AsPC-1 cells was statistically-significantly higher in the liver, lung, and peritoneal metastases than in the spleen. CONCLUSION: Exosome transfer between cancer cells is associated with metastasis. Exosome transfer may play a role in increasing the metastatic capability of the recipient cells.

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Inhibition of FGF10-ERK signal activation suppresses intraductal papillary neoplasm of the bile duct and its associated carcinomas.

Evidence regarding intraductal papillary neoplasm of the bile duct (IPNB) as a type of precancerous lesion of cholangiocarcinoma is limited. Moreover, a reproducible in vivo model is lacking, and IPNB pathogenesis remains unclear. Here, we use a doxycycline-inducible tetracycline (Tet)-on mice model to control fibroblast growth factor 10 (FGF10) expression, which regulates branching and tubule formation. FGF10-induced IPNB mimics the multifocal and divergent human IPNB phenotypes via the FGF10-FGF receptor 2 (FGFR2)-RAS-extracellular-signal-regulated kinase (ERK) signaling pathway. A paracrine/autocrine growth factor is sufficient to initiate and maintain IPNB originating from the peribiliary glands, including biliary stem/progenitor cells. With KrasG12D, p53, or p16 mutations or both, Fgf10-induced IPNB shows stepwise carcinogenesis, causing associated invasive carcinoma. Fgf10-induced papillary changes and progression are suppressed by the inhibition of the FGF10-FGFR2-RAS-ERK signaling pathway, demonstrating that the signal is a therapeutic target for IPNB and associated carcinoma.

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.

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.

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.

Induced pluripotent stem cell-derived tenocyte-like cells promote the regeneration of injured tendons in mice.

Tendons are dense fibrous structures that attach muscles to bones. Healing of tendon injuries is a clinical challenge owing to poor regenerative potential and scarring. Here, we created reporter mice that express EGFP, driven by the promoter of the tendon-specific Scleraxis (Scx) transcription-factor gene; we then generated induced pluripotent stem cells (iPSCs) from these mice. Utilising these fluorescently labelled iPSCs, we developed a tenogenic differentiation protocol. The iPSC-derived EGFP-positive cells exhibited elevated expression of tendon-specific genes, including Scx, Mohawk, Tenomodulin, and Fibromodulin, indicating that they have tenocyte-like properties. Finally, we demonstrated that these cells promoted tendon regeneration in mice after transplantation into injured tendons reducing scar formation via paracrine effect. Our data demonstrate that the tenogenic differentiation protocol successfully provided functional cells from iPSCs. We propose that pluripotent stem cell-based therapy using this protocol will provide an effective therapeutic approach for tendon injuries.

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.

Characterization of a BAC transgenic mouse expressing Krt19-driven iCre recombinase in its digestive organs.

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, ß-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.

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.

Biochemical and Morphological Characterization of a Guanine Nucleotide Exchange Factor ARHGEF9 in Mouse Tissues.

ARHGEF9, also known as Collybistin, a guanine nucleotide exchange factor for Rho family GTPases, is thought to play an essential role in the mammalian brain. In this study, we prepared a specific polyclonal antibody against ARHGEF9, anti-ARHGEF9, and carried out expression analyses with mouse tissues especially brain. Western blotting analyses demonstrated tissue-dependent expression profiles of ARHGEF9 in the young adult mouse, and strongly suggested a role during brain development. Immunohistochemical analyses revealed developmental stage-dependent expression profiles of ARHGEF9 in cerebral cortex, hippocampus and cerebellum. ARHGEF9 exhibited partial localization at dendritic spines in cultured hippocampal neurons. From the obtained results, anti-ARHGEF9 was found to be a useful tool for biochemical and cell biological analyses of ARHGEF9.

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.