The lack of treatment options for congenital (0.1%) and partial (10%) tooth anomalies highlights the need to develop innovative strategies. Over two decades of dedicated research have led to breakthroughs in the treatment of congenital and acquired tooth loss. We revealed that by inactivating USAG-1, congenital tooth agenesis can be successfully ameliorated during early tooth development and that the inactivation promotes late-stage tooth morphogenesis in double knockout mice. Furthermore, Anti- USAG-1 antibody treatment in mice is effective in tooth regeneration and can be a breakthrough in treating tooth anomalies in humans. With approximately 0.1% of the population suffering from congenital tooth agenesis and 10% of children worldwide suffering from partial tooth loss, early diagnosis will improve outcomes and the quality of life of patients. Understanding the role of pathogenic USAG-1 variants, their interacting gene partners, and their protein functions will help develop critical biomarkers. Advances in next-generation sequencing, mass spectrometry, and imaging technologies will assist in developing companion and predictive biomarkers to help identify patients who will benefit from tooth regeneration.

UNASSIGNED: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.
UNASSIGNED: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.
UNASSIGNED: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.
UNASSIGNED: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

BACKGROUND: Fluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear.
METHODS: Cell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays.
RESULTS: Low-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA.
CONCLUSIONS: NaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.

BACKGROUND: The BMP signaling pathway plays a key role in growth, differentiation and patterning during neural development. Recent work on the generation of a self-organization of three-dimensional retinal organoid (3D-retina) from human pluripotent stem cells (hPSCs) revealed that addition of recombinant human BMP4 (rhBMP4) promotes retinal differentiation in the early neural differentiation stage. For clinical application, efficient differentiation from hPSCs to retinal cells with minimal numbers of off-target non-retinal cells is desirable. We therefore aimed to further improve an efficient retinal differentiation method for future up-scaling of cell production.
METHODS: hPSCs were differentiated into 3D-retina using a modified SFEBq method. The effect of rhBMP4 with or without Checkpoint kinase 1 (Chk1) inhibitor (PD407824), a modulator of BMP signaling pathway, at day 3 was compared by characterizing the differentiating 3D-retina by the use of the hPSCs and immunohistochemical analysis.
RESULTS: The Chk1 inhibitor treatment promoted retinal differentiation from hPSCs, in combination with low-concentration rhBMP4. Addition of a Chk1 inhibitor generated a unique type of organoid with neural retina (NR) encapsulated in retinal pigment epithelium (RPE), possibly by promoting phosphorylation of SMAD1/5/9 in the cells inside the early aggregates. We confirmed that the Chk1-inhibitor-treated hPSC-3D-retina differentiated into rod and cone photoreceptor precursors and other types of retinal neurons, in long-term culture.
CONCLUSIONS: In this study, we found that combined use of rhBMP4 and a Chk1 inhibitor cooperatively promoted retinal differentiation from hPSCs. Our new retinal differentiation method is a promising option for the stable supply and up-scaling of production of 3D-retina for future cell therapy.

Bone is one of the preferential target organs of cancer metastasis. Bone metastasis is associated with various complications, of which bone pain is most common and debilitating. The cancer-associated bone pain (CABP) is induced as a consequence of increased neurogenesis, reprogramming and axonogenesis of sensory nerves (SNs) in harmony with sensitization and excitation of SNs in response to the tumor microenvironment created in bone. Importantly, CABP is associated with increased mortality, of which precise cellular and molecular mechanism remains poorly understood. Bone is densely innervated by autonomic nerves (ANs) (sympathetic and parasympathetic nerves) and SNs. Recent studies have shown that the nerves innervating the tumor microenvironment establish intimate communications with tumors, producing various stimuli for tumors to progress and disseminate. In this review, our current understanding of the role of SNs innervating bone in the pathophysiology of CABP will be overviewed. Then the hypothesis that SNs facilitate cancer progression in bone will be discussed in conjunction with our recent findings that SNs play an important role not only in the induction of CABP but also the progression of bone metastasis using a preclinical model of CABP. It is suggested that SNs are a critical component of the bone microenvironment that drives the vicious cycle between bone and cancer to progress bone metastasis. Suppression of the activity of bone-innervating SNs may have potential therapeutic effects on the progression of bone metastasis and induction of CABP.

The correct balance between collagen synthesis and degradation is essential for almost every aspect of life, from development to healthy aging, reproduction and wound healing. When this balance is compromised by external or internal stress signals, it very often leads to disease as is the case in fibrotic conditions. Fibrosis occurs in the context of defective tissue repair and is characterized by the excessive, aberrant and debilitating deposition of fibril-forming collagens. Therefore, the numerous proteins involved in the biosynthesis of fibrillar collagens represent a potential and still underexploited source of therapeutic targets to prevent fibrosis. One such target is procollagen C-proteinase enhancer-1 (PCPE-1) which has the unique ability to accelerate procollagen maturation by BMP-1/tolloid-like proteinases (BTPs) and contributes to trigger collagen fibrillogenesis, without interfering with other BTP functions or the activities of other extracellular metalloproteinases. This role is achieved through a fine-tuned mechanism of action that is close to being elucidated and offers promising perspectives for drug design. Finally, the in vivo data accumulated in recent years also confirm that PCPE-1 overexpression is a general feature and early marker of fibrosis. In this review, we describe the results which presently support the driving role of PCPE-1 in fibrosis and discuss the questions that remain to be solved to validate its use as a biomarker or therapeutic target.

Small intestine in vitro models play a crucial role in drug transport research. Although conventional 2D cell culture models, such as Caco-2 monolayer, possess many advantages, they should be interpreted with caution because they have relatively poor physiologically reproducible phenotypes and functions. With the development of 3D culture technology, pluripotent stem cells (PSCs) and adult somatic stem cells (ASCs) show remarkable self-organization characteristics, which leads to the development of intestinal organoids. Based on previous studies, this paper reviews the application of intestinal 3D organoids in drug transport mediated by P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2). The advantages and limitations of this model are also discussed. Although there are still many challenges, intestinal 3D organoid model has the potential to be an excellent tool for drug transport research.

The shortest functional domains of growth factor Bone Morphogenetic Protein 2 (BMP-2) that are dynamical implicated in osteogenesis have been investigated and well characterized. In particular, the broad C-terminal region expanding from Val63 to Arg114 as well as its shorter sequence 86-AISMLYLDEN-95 exhibited the highest osteogenic ability for regeneration and reconstruction of bone tissue. In addition, the amino acids Ser88 and Leu90 have been identified as crucial for receptor binding and osteogenic efficacy. Furthermore, the above-mentioned domains in contrary to full length BMP-2 protein signal mainly through the Smad pathway as it is evidenced by phosphorylation decrease of Extracellular-signal-Regulated Kinase (ERK1/2). Taking together, our results are significant for clinical applications regarding the generation of biomaterials and healing of orthopedic fractures.

Intratumoral heterogeneity is tightly associated with the failure of anticancer treatment modalities including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Such heterogeneity is generated in an evolutionary manner not only as a result of genetic alterations but also by the presence of cancer stem cells (CSCs). CSCs are proposed to exist at the top of a tumor cell hierarchy and are undifferentiated tumor cells that manifest enhanced tumorigenic and metastatic potential, self-renewal capacity, and therapeutic resistance. Properties that contribute to the robustness of CSCs include the abilities to withstand redox stress, to rapidly repair damaged DNA, to adapt to a hyperinflammatory or hyponutritious tumor microenvironment, and to expel anticancer drugs by the action of ATP-binding cassette transporters as well as plasticity with regard to the transition between dormant CSC and transit-amplifying progenitor cell phenotypes. In addition, CSCs manifest the phenomenon of metabolic reprogramming, which is essential for maintenance of their self-renewal potential and their ability to adapt to changes in the tumor microenvironment. Elucidation of the molecular underpinnings of these biological features of CSCs is key to the development of novel anticancer therapies. In this review, we highlight the pathological relevance of CSCs in terms of their hallmarks and identification, the properties of their niche-both in primary tumors and at potential sites of metastasis-and their resistance to oxidative stress dependent on system xc (-).

This study is evaluating the effects of ethanol and nicotine exposure during pregnancy and lactation on placenta histology and follicular atresia in the first-generation (f1) mice pups. The experimental groups were 5 groups of NMRI pregnant mice, including: control, vehicle (received normal saline) ethanol (3 g/kg/day, 20 % v/v intraperitoneally), nicotine (1 mg/kg/day, subcutaneously), and ethanol plus nicotine which received both. Pregnant animals in each group were then divided into two groups, one group for examining the placenta that was treated for 18 days and the other group for the ovary of one-day-old (PND1) and fifty-six-day-old (PND56) female offspring who were treated for 42 days (during intrauterine development and lactation). After the autopsy procedure, histopathological and morphometrical observations were done. Data revealed that the exposed mice had a significant change in the placenta morphometry and histology as well as a marked increase in the number of ovarian TUNEL positive cells on postnatal days 1 and 56. Therefore, maternal exposure to alcohol and nicotine during developmental and lactation periods could lead to changes in the placenta properties as well as an increase in the apoptotic ovarian follicles in f1 mice pups.