INTRODUCTION:Human eyelid development begins during the embryonic period. Although the general outline of early eyelid development has been described, many structures and developmental phenomena remain insufficiently investigated or controversial mainly due to the scarcity of human fetal specimens. The purpose of this study is to clarify these unresolved issues and to determine the precise developmental timing of each eyelid structure. METHODS:Fifty-six embryos (Carnegie stage (CS) 18-23) and six fetuses (early 9th week of development) from the Kyoto Collection were included in this study. The anatomical structures of the eyelid were observed using serial histological sections and external photographs, and the findings were described according to each developmental stage. RESULTS:The medial and lateral canthi formed between CS19 and 21. Mesenchymal condensations corresponding to the prospective upper and lower eyelid retractor regions were first observed at CS18 and acquired a sheet-like configuration by the 9th week of development. Mesenchymal condensation at the future location of the medial palpebral ligament was first observed at CS20. The orbicularis oculi muscle primordium emerged at the 9th week. Eyelid fusion initiated on the nasal side at CS22. It then progressed from both the nasal and temporal sides towards the center, until completion at the 9th week. The lacrimal canaliculi first appeared at CS19. CONCLUSIONS:Using the rich specimens of the Kyoto collection, we newly identified the developmental timing of the prospective lower eyelid retractor complex and medial palpebral ligament regions, as well as the initiation of eyelid fusion on the nasal side. Furthermore, we established a detailed timeline of early human eyelid morphogenesis. Our findings may guide future research on human eyelid development, anatomy, and the mechanisms underlying congenital eyelid disorders.
PURPOSE:To propose that progressive neuronal and satellite glial cell death within the pterygopalatine ganglion (PPG) is an underrecognized upstream mechanism in chronic aqueous-deficient dry eye, with particular relevance to diabetes mellitus, and to examine whether this ganglionic lesion also unifies broader secretory and cerebrovascular abnormalities into a single "diabetic secretory denervation syndrome". METHODS:A narrative, hypothesis-driven synthesis was performed across ocular surface disease, autonomic neuroscience, diabetic neuropathy, regulated cell death, surgical denervation models, and cerebrovascular physiology. PubMed was searched using both "pterygopalatine ganglion" and "sphenopalatine ganglion" as synonyms, combined with terms for cell death, diabetes, vidian neurectomy, and cerebrovascular function. Reference lists were hand-searched. RESULTS:The PPG is the sole parasympathetic relay for lacrimal, nasal, palatal, and conjunctival secretion, and a major source of vasodilator drive to the anterior cerebral circulation. Surgical interruption of this pathway reliably causes aqueous-deficient dry eye. In streptozotocin-induced diabetic rats, nitrergic PPG neurons undergo TUNEL-confirmed apoptosis during an irreversible second phase of diabetic nitrergic neuropathy, driven by the synergistic action of advanced glycation end products and endogenous nitric oxide through a caspase-3-dependent mechanism. Additional cell-death modalities (necroptosis, pyroptosis, ferroptosis, and satellite glial cell apoptosis) are predicted by analogy with other autonomic ganglia under equivalent metabolic stress. Its degeneration is therefore predicted to cause coordinated multi-territory secretory failure rather than isolated dry eye. CONCLUSION:PPG neurodegeneration has been directly demonstrated in the diabetic rodent ganglion and mechanistically anchored in AGE-RAGE (advanced glycation end products and their receptor, RAGE)-mediated apoptosis. Extending this experimentally grounded finding, we hypothesise that these deficits (diabetic dry eye, impaired nasal mucociliary clearance, palatine xerostomia, goblet cell depletion, and reduced cerebral perfusion) may share a common upstream ganglionic lesion, a unifying proposal that remains to be tested directly. The hypothesis generates testable predictions and reframes therapy toward neuroprotection of the PPG.
INTRODUCTION:The chicken chorioallantoic membrane (CAM) is a widely used in vivo model for studying angiogenesis and tumor growth in accordance with the 3Rs principles. However, its multilayered vascular organization complicates quantitative analysis, because overlapping superficial and deeper vascular compartments are difficult to distinguish in conventional two-dimensional imaging. Although artificial intelligence (AI)-based methods improve vascular quantification, many existing workflows require extensive preprocessing or user-side model training. METHODS:We established an AI-assisted protocol that restricts vascular quantification to the superficial CAM capillary plexus, the vascular compartment most responsive to angiogenic and anti-angiogenic stimuli. Functional separation of this layer was achieved by intra-CAM injection of commercially available bovine whipped cream (minimum 30% fat), creating a diffuse white background that optically masks deeper vessels without altering vascular morphology. A U-Net-based model trained on manually annotated vessel masks was used for automated two-dimensional quantification of vessel area, length, branching points, and thickness. RESULTS:The segmentation model achieved a Dice similarity coefficient of 0.831. Application at embryonic days 11 and 15 revealed remodeling of the superficial CAM vasculature, including increased branching and changes in perfusion area. The workflow reduced preprocessing complexity and enabled standardized analysis using a pre-trained segmentation model. CONCLUSION:This method combines experimental layer isolation with automated vessel segmentation to enable two-dimensional quantification of superficial CAM vasculature without requiring custom model training. It supports practical and reproducible CAM angiogenesis studies by providing a standardized analysis workflow.
Introduction: Aged dermal fibroblasts exhibit reduced migration, proliferation, and extracellular matrix production, alongside upregulation of pro-inflammatory signaling that disrupts the proliferative, granulation, and remodeling phases of wound healing. Despite these age-related impairments, tissue engineering biomaterials are rarely evaluated using age-specific fibroblasts. Current in vitro skin aging models commonly rely on acute stressors, such as oxidative stress or DNA damage, which only partially recapitulate chronological aging. Alternatively, fibroblasts are artificially aged through extensive passaging, which may not reflect physiological aging. This work established a system for accurately representing chronological aging of mouse fibroblasts in vitro by isolating and evaluating fibroblasts from age-specific mice. While senescence in human fibroblasts results from telomere shortening, primary mouse fibroblasts senesce by oxidative damage in atmospheric O2, requiring careful culture conditions. Methods: We demonstrate that physiological O2 levels (3%) abrogate the detrimental effects of 20% O2 by comparing cell proliferation, senescence markers, and morphology of explant-derived fibroblasts. Next, fibroblasts from young (5 weeks), middle-aged (47 weeks), and old (90 weeks) mice were cultured (3% O2) and assessed for age-dependent changes in their proliferation, migration, immune signaling, collagen production, and metabolic activity in a 3D collagen matrix. Results: Our data demonstrate the impaired metabolic activity and migratory ability of old fibroblasts, as well as an overall amplification of pro-inflammatory proteins (e.g., MCP1 and IL-1α), which shifted tissue repair macrophages (“M2”) toward a pro-inflammatory (“M1”) phenotype. In 3D collagen-glycosaminoglycan scaffolds, old fibroblasts also showed significantly reduced metabolic activity compared to young and middle-aged cells. Conclusion: Together, these findings show that primary mouse fibroblasts can retain chronological age-related characteristics when cultured under physiological conditions in vitro, making them relevant in models of skin aging. Studying aging using these robust in vitro methods will be essential for improving the design and translational relevance of biomaterials and therapeutic strategies for wound healing and skin tissue engineering.
Background: The 26S proteasome degradation system is a central regulator of protein homeostasis and immune function, integrating antigen processing, inflammatory signaling, and cell fate decisions. Dysregulation of this system has profound consequences for immune tolerance, immune evasion, and immune-mediated pathology. Summary: In this review, we examine the roles of the constitutive 26S proteasome and the inducible immunoproteasome in shaping cellular immunological characteristics, with a particular focus on mesenchymal stem cells and cancer cells, two cell types that exhibit context-dependent immune privilege. We discuss how proteasome dynamics regulate antigen presentation, cytokine production, and the expression of immune checkpoint and immunomodulatory molecules under physiological and stress conditions such as hypoxia and metabolic imbalance. We also summarize evidence linking altered proteasome function to the pathogenesis of autoimmune, immunodeficiency, and autoinflammatory diseases. Key Messages: The proteasome system is a pivotal molecular hub controlling immune visibility and immune escape. Understanding how distinct proteasome isoforms operate in different microenvironments will be essential for developing targeted immunomodulatory and proteasome-based therapeutic strategies.
Background: Bioelectrical signaling, a collection of electrical currents driven mainly by ionic fluxes and transmembrane potential gradients, is recognized as a central regulator of cell behavior, tissue patterning, and regeneration, extending far beyond classical roles in excitable tissues. While electrically conductive materials are widely used to deliver exogenous stimulation, the specific contribution of intrinsic biomaterial charge (fixed charges, counterions, piezoelectric polarization, electric field) to bioelectrical signaling remains comparatively underexplored. Summary: This review focuses on how charged biomaterials influence bioelectrical signaling and downstream human cell responses. We first outline the fundamentals of bioelectricity, including electrochemical gradients, Nernst potentials, and resting membrane potential as key regulators of ion flux, metabolism, and cell fate. We then discuss major classes of charged biomaterials (charged polymers, ionically conductive hydrogels, piezoelectric systems, and charged nanoparticles) and how their charge density, polarity, and dynamic behavior shape protein adsorption, integrin-mediated signaling, or membrane potential. Mechanistic links between material charge and some signaling pathways are highlighted in the context of cell adhesion, viability, migration, proliferation, and differentiation. Finally, we examine tissue-specific applications in cardiac tissue engineering, bone regeneration, neural interfaces, and skin wound healing and immunomodulation. Key Messages: Charged biomaterials have the potential to modulate bioelectrical signaling not only through electronic conductivity but also via electrostatic fields, Donnan effects, and piezoelectric or ionic charge transport. The charge density of the biomaterials often plays a more dominant role than charge polarity in regulating protein adsorption and cell proliferation, whereas polarity can bias lineage-specific differentiation and immunomodulation. In many regenerative systems, the effects of charge are confounded with mechanical and biochemical cues; mechanistic studies that isolate charge-driven bioelectric phenomena remain limited. Although it is known that surface charge affects the adaptive and innate immune systems, such as through macrophage polarization, the broader consequences of charge-mediated immune modulation remain inadequately explored. Therefore, a more comprehensive discussion and studies on the cell-biomaterial charge interactions are warranted to advance the field and guide future biomaterial design.
INTRODUCTION:Calcineurin-NFAT is an important pathway that regulates skeletal muscle regeneration. Homer 2 that modulates signal transduction in the central nervous system directly binds to NFATc1. However, its role is not fully understood in skeletal muscle regeneration. We aimed to investigate the change of Homer 2 protein levels and expression patterns during muscle regeneration. METHODS:Male ICR mice (12 weeks) were used in the experiment (n = 6/group). Their left tibialis anterior (TA) muscle was damaged via intramuscular injection of 0.5% bupivacaine hydrochloride (100 μL). The TA muscles of both legs were dissected at 2, 4, and 6 days post-injection and were subjected to immunofluorescence staining with Homer 2, NFATc1, and muscle regeneration markers (Pax7 and myogenin). We calculated their expression frequency by quantifying the immunoreactivity per 500 nuclei. RESULTS:We observed Homer 2 immunoreactivity in TA muscles at 2, 4, and 6 days post-injection. Homer 2 and Pax7, a satellite cell marker, were co-localized in mononuclear cells also in regenerating TA muscles. Many Homer 2-positive mononuclear cells expressed myogenin. The frequency of Homer 2- and NFATc1-positive cells significantly increased at 4 and 6 days rather than 2 days post-injection. Interestingly, co-immunoprecipitation experiments of Homer 2 and NFATc1 showed markedly increased levels at 4 days. CONCLUSION:We demonstrated that expression of Homer 2 protein increases in TA muscle regeneration. Homer 2 may act in the muscle regeneration process via the calcineurin-NFAT pathway.
BACKGROUND:Peripheral nerve injuries (PNIs) disrupt sensory and motor circuits, affecting millions worldwide and often resulting in lasting functional deficits. Autologous nerve grafting remains the surgical benchmark for segmental defects, yet limited graft length, donor site morbidity, and inconsistent outcomes - particularly in long (>30 mm) or proximal lesions - underscore the need for engineered alternatives. Although parallel strategies seek to enhance autograft outcomes (e.g., polyethylene glycol fusion, growth factor sleeves, post-repair electrical stimulation), this review focuses specifically on engineered replacements. Over the past decade, convergent advances in cell biology, biomaterials science, and biofabrication have produced tissue-engineered conduits intended to serve as effective alternatives to autografts. Unlike previous reviews that address these domains separately, this work treats cells, biomaterials, and advanced engineering as a unified design problem, benchmarking outcomes with standardized quantitative metrics across both short-gap rodent and long-gap large-animal models and articulating specific design-readiness targets as a translational framework. SUMMARY:This review synthesizes the past decade (2015-2025) of progress, identified through a structured narrative search of PubMed, Google Scholar, Web of Science, and Scopus (160 articles included), in three domains: (1) cell-based therapies, including Schwann cells, mesenchymal stem cells, induced pluripotent stem cell (iPSC)-derived glia, neural stem/progenitor cells, and olfactory ensheathing cells (OECs), emphasizing their roles in neurotrophic support, immunomodulation, and remyelination; (2) biomaterial scaffolds, spanning natural polymers, synthetic polymers, and hybrid composites, with design parameters tailored to mimic native nerve microarchitecture and degradation timelines; and (3) integrated strategies that couple living cells with architecturally and biochemically instructive scaffolds, including pre-seeded conduits, injectable hydrogel systems, conductive/electroactive designs, and three-dimensional bioprinted constructs. Functional outcomes are evaluated across short-gap (≤15 mm) rodent and long-gap (≥30 mm) large-animal models using motor metrics (Sciatic Functional Index [SFI], compound muscle action potential, nerve conduction velocity) and, where reported, sensory assessments (von Frey thresholds, two-point discrimination), enabling cross-study benchmarking against quantitative scaffold design-readiness criteria. KEY MESSAGES:Well-designed, cell-seeded conduits consistently achieve ≥85% of motor functional recovery compared to autografts in rodent models of ≤15 mm nerve gaps, with certain platforms nearing equivalence in 20-30 mm defects in large animals; sensory recovery, though less frequently reported, shows parallel trends in studies employing standardized sensory testing. Persistent barriers - including rapid vascularization of long grafts, acidic degradation by-products from synthetic polyesters, mitigation of fibrotic by-products, donor-to-donor cell variability, and scalable good manufacturing practice-compliant manufacturing of combination products - remain central to translation. Clinical translation is promising for cell-seeded biodegradable conduits in digital nerve repairs (≤30 mm gaps), with future prospects for obstetric brachial plexus injuries and longer term horizons for iPSC-based, patient-specific grafts. A next-generation framework integrating optimized biomaterials, function-tuned cells, and advanced fabrication - guided by the quantitative design criteria codified here - will enable predictable, high-quality repair of complex PNIs.
INTRODUCTION:Temperature plays a crucial role in embryonic development, particularly in oviparous species that experience natural fluctuations during incubation. Unlike viviparous embryos developing under stable maternal conditions, chick embryos are exposed to external temperature variability that can influence cellular regulatory processes. Notch signaling, a conserved pathway essential for maintaining the neural progenitor cell (NPC) pool, is regulated by endosomal recycling; however, the mechanism by which temperature modulates this process across species remains poorly understood. METHODS:To model environmental challenges experienced by chick embryos, we examined the effects of a physiologically relevant low temperature (30°C) on Dll1-mediated Notch signaling in chick and mouse NPCs. RESULTS:Under hypothermic conditions, Dll1 reporter signals increased in chick NPCs but decreased in mouse NPCs, correlating with corresponding changes in Notch activity. Chick NPCs maintained progenitor gene expression at low temperature, whereas mouse NPCs showed reduced expression. Mechanistic analyses revealed that distinct recycling pathways are differentially required for Notch signaling across temperatures and species. Chick NPCs maintained Notch signaling through both fast recycling (FR) and slow recycling (SR) under normothermia and additional degradation under hypothermia, whereas mouse NPCs relied on SR and degradation under normothermia and showed impaired recycling under hypothermia. CONCLUSION:Together, these findings reveal species-specific adaptations in the temperature-dependent endocytic regulation of Notch signaling, highlighting how environmental temperature influences the maintenance of neural progenitors across species.
BACKGROUND:Early human embryonic development sets the trajectory for health across the life course. Any aberrations in this process are associated with a heightened burden of adverse pregnancy outcomes. Accordingly, a comprehensive understanding of early human embryogenesis, together with the development of faithful research models, is pivotal for elucidating pregnancy physiology and pathophysiology. However, as embryonic development occurs within the uterus, direct observation has been severely limited by ethical and technical constraints. SUMMARY:To overcome these challenges, in vitro culture (IVC) systems have emerged as powerful platforms for studying early embryonic development under controlled conditions. These systems support human embryo development to the primitive streak anlage stage (near the 14-day ethical boundary), whereas nonhuman primate models sustain growth to neurulation and early organogenesis (up to E25), thereby bridging the gap between implantation and complex organ formation. In parallel, studies of rare early-stage primate embryos obtained from clinical procedures have yielded complementary insights into these developmental processes. In this review, we summarize recent progress in early primate embryo development research, emphasize the critical role of IVC systems in elucidating developmental processes, and discuss the integration of these experimental models with spatiotranscriptomic atlases to establish a more comprehensive framework for early human embryonic development. KEY MESSAGES:Primate IVC systems offer accessible platforms for high-resolution dynamic observation and perturbation, while in vivo embryos provide physiologically faithful references. Coupling these complementary approaches reconstructs the trajectory of early human development, establishing a robust framework to decipher the causes of birth defects and facilitate mechanism-guided drug screening.
Introduction: Human endometrium is one of peculiar tissues capable of scarless regeneration after injury during every menstrual cycle, birth, or surgery. However, it is disputable whether this feature should be attributed to specific regulatory factors of wound environment and menstrual fluid (MF) or to tissue-specific properties of endometrial mesenchymal stromal cells (eMSCs), which are pivotal participants of wound healing. We aimed to elucidate the role of eMSC tissue specificity in wound healing. Methods: We evaluated changes of eMSC transcriptomic profile in response to MF and their potency to granulation tissue formation in vitro in comparison with those of stromal cells from scar-forming organs - dermal and adipose MSC (dMSC and adMSC). Results: We have found that MF contains numerous inflammatory factors and induces a profound inflammatory response in both eMSC and dMSC, but a tissue-specific component was identified in their transcriptome profiles. Furthermore, transcriptomic tissue specificity was stable and present prior to MF treatment as well as after it, so we validated our findings against in vivo single-cell RNA-sequencing data from Human Protein Atlas. Tissue specificity traits were related to embryonic development and morphogenesis, suggesting a putative contribution of "developmental imprinting" in its establishment. Using in vitro models of fibroplasia, angiogenesis, and extracellular matrix deposition, we showed that eMSC lack the ability to induce these processes in contrast to dMSC and adMSC. Finally, we refined transcription factors (TFs) from stable tissue-specific genes that may explain the unique properties of eMSC and endometrium itself and can serve as potential targets to induce regeneration in scar-forming organs. Conclusion: eMSCs possess tissue-specific properties including stable expression of TFs that may explain the scarless regeneration of endometrium.
Introduction: Otic organoids differentiated from human pluripotent stem cells are three-dimensional in vitro cultures that broadly mimic the complexity and functionality of the human inner ear. They provide a valuable model for developmental and disease-related studies. However, current protocols differ substantially in efficiency and reproducibility. In this study, we investigated whether different stem cell maintenance media influence the differentiation of keratinocyte-derived induced pluripotent stem cells (kiPSCs) into the otic lineage. Methods: kiPSCs were cultured in either a self-made FTDA medium or the commercially available PeproGrow™ human embryonic stem cell medium and subsequently subjected to an established otic differentiation protocol. Early developmental stages, including the pre-placodal region, the otic placode, and pro-neural sensory regions, were analyzed using immunofluorescence and gene expression profiling. Results: While no significant differences were observed in iPSC maintenance or pluripotency between the two media, distinct differences emerged during otic differentiation. Media composition influenced the expression of placodal, otic, and pro-sensory markers at multiple stages, indicating differential responsiveness to otic induction cues. Conclusion: Our findings demonstrate that stem cell maintenance media composition is a critical determinant of subsequent otic lineage differentiation. These results provide guidance for optimizing stem cell culture conditions and improving the reproducibility of otic organoid differentiation protocols.
Introduction: Mesenchymal stromal cells (MSCs) play a crucial role in tissue repair and exhibit anti-inflammatory properties, making them promising for regenerative medicine. Dental tissue-derived MSCs, such as stromal cells from human exfoliated deciduous teeth (SHEDs) and dental pulp stromal cells (DPSCs), express neural markers and hold the potential for treating neurodegenerative diseases. Nonetheless, their relative ability to differentiate into neurons is still unclear. Given their shared embryonic origin, we hypothesised that SHEDs and DPSCs possess similar potential for neuronal differentiation along with intrinsic expression of neuronal markers. The objective of this study was to compare their differentiation abilities under standardised conditions, evaluate neuronal markers pro- and post-neuronal induction, and compare the neuronal differentiation potential of SHEDs and DPSCs. METHODS:SHEDs (n = 3) and DPSCs (n = 3) were collected with ethical approval, cultured, and characterised according to established MSC criteria. Clonogenicity, proliferation, senescence, and trilineage differentiation were assessed. Neuronal differentiation was induced for 21 days and evaluated using flow cytometry (SRY-Box Transcription Factor 1 [SOX1], SRY-Box Transcription Factor 2 [SOX2], glial fibrillary acidic protein [GFAP], doublecortin, nestin, CD56, CD146), immunofluorescence for βIII-tubulin, reverse transcription polymerase chain reaction for tubulin 3 (TUB3), and microtubule-associated protein 2 (MAP2). RESULTS:Both SHEDs and DPSCs exhibited MSC characteristics. SHEDs showed higher clonogenicity. Early neuronal markers (e.g., SOX1, nestin, GFAP, βIII-tubulin) were detected pre- and post-induction in both cell types without significant intergroup differences. No significant expression of TUB3 and MAP2 was observed. CONCLUSION:SHEDs and DPSCs show comparable neuronal marker expression profiles, suggesting similar early neuronal differentiation potential. These findings support using undifferentiated SHEDs and DPSCs in neuroregenerative strategies, offering cost-effective and safer alternatives to pre-differentiated cells. .
Introduction: While caudal foregut development in human fetuses has been outlined in previous research, the formation of its border region remains unclear. This study aimed to visualize the precise timeline of caudal foregut boundary formation. Methods: Three-dimensional images of the foregut from T1-weighted scans of 24 fetuses (crown–rump length [CRL]: 34–103 mm) were analyzed to measure the wall thickness and lumen diameter at nine specific sites. The internal structure in the border region was verified using histological sections and diffusion tensor imaging (DTI) tractography. Results: The lower esophageal and pyloric canal walls were thicker in samples with a CRL ≥50 mm. The esophageal wall at the esophageal hiatus, where the lower esophageal sphincter is located, was particularly thick in samples with a CRL ≥88 mm. Increased wall thickness at the esophageal hiatus and pyloric canal resulted in a narrower lumen. The pyloric canal lumen narrowed from its distal to proximal sections. The lumen diameter-to-wall thickness ratio at the esophageal hiatus and proximal pyloric was negatively correlated with CRL. The thickened esophageal wall at the esophageal hiatus had a thick submucosa, and all layers in the pyloric canal thickened with growth. DTI tractography revealed that the lower esophageal wall mainly comprised longitudinal fibers, whereas the pyloric canal wall consisted solely of circular fibers, with fractional anisotropy increasing with growth. Conclusion: This study provides a comprehensive timeline of normal caudal foregut boundary formation during the early human fetal period, thereby improving the understanding of congenital foregut obstruction pathogenesis.
Introduction: Surgical reconstruction is recommended for cleft palate patients. However, improper muscle reconstruction can cause scarring and velopharyngeal insufficiency. Understanding the developmental patterns of soft palatal musculature is crucial for improving treatments. Although mice are commonly used to investigate soft palate development, the uvula deficiency in mice limits their applicability. This study aims to compare the developmental characteristics of soft palate muscles in miniature pigs and mice at various stages to better understand the patterns in large mammals. Methods: Specimens of soft palate were collected from human embryos, miniature pigs and mice at different stages. Furthermore, comprehensive gross observations, Hematoxylin and Eosin staining, and immunohistochemical analyses for myosin heavy chain and Hypermethylated cancer 1 (Hic1) were conducted on multiple dissected sections. Results: Mature soft palatal musculature exhibited anatomical and histological similarities across the three species. Notably, miniature pigs exhibited the uvula structure and uvula muscle development comparable to humans. Embryonic day 40 (E40, equivalent to human embryonic week 11, E11w) represented the early developmental stage of the uvula muscle, characterized by scattered muscle cells not yet coalescing into multinucleated fibres. By E45 (aligned with human E12w), the muscle bundles reached maturity, exhibiting two oriented fibre bundles flanking the midline. Levator veli palatini muscle and palatopharyngeus of miniature pigs exhibited a distinct course pattern due to the presence of the uvula muscle. Hic1+ perimysial cells were observed at the extending edge of the developing palatopharyngeus of miniature pigs, indicating a potential guiding role in the migration of myogenic cells. Conclusion: Characterized by the uvula muscle, the spatiotemporal dynamic process of soft palatal musculature of miniature pigs was revealed. Miniature pigs exhibit a similar structure of the uvula muscle to that of humans and therefore serve as a promising model for researching soft palatal musculature development of large mammals in the future.
INTRODUCTION:Cytosolic carboxypeptidase 1 (CCP1) is a deglutamylase that antagonizes polyglutamylation. Mutations in human CCP1 gene cause a severe disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), which is characterized by marked growth retardation. However, the role and mechanisms of CCP1 in skeletal development remain unclear. METHODS:In this study, we used CCP1 knockout (CCP1-KO) mice to assess bone mass changes by micro-CT, HE, alkaline phosphatase (ALP) staining, tartrate-resistant acid phosphatase staining and immunofluorescence staining. Changes in osteogenic differentiation, proliferation, and migration capacity of bone marrow mesenchymal stem cells (BMSCs) were assessed by ALP, alizarin red (ARS) staining, quantitative real-time PCR, EdU staining, and cell scratching assay. Then, tubulin glutamylation and primary cilia of BMSCs after deletion of CCP1 was analyzed by Western blot and immunofluorescence staining. Finally, CB839, an inhibitor of glutamine metabolism, was used to detect changes in the osteogenic differentiation ability and primary cilia of BMSCs after reducing the elevated glutamylation level. RESULTS:CCP1-KO mice exhibited phenotypes relevant to humans, including reduced body size, decreased bone mass, and reduced bone density during growth and development. CCP1 deficiency impairs the proliferation, migration, and osteogenic differentiation of BMSCs. Meanwhile, the number of pre-osteoblasts derived from BMSCs is decreased, leading to impaired osteogenesis. At the cellular level, CCP1 loss results in aberrant tubulin glutamylation, increased microtubule glutamylation, and shortened primary cilia in BMSCs. Finally, reduction of abnormally elevated tubulin glutamylation was efficacious for promoting osteogenic differentiation of BMSCs and restoring primary cilia length of BMSCs. CONCLUSION:We propose that CCP1 plays a critical role in regulating BMSCs differentiation and promotes osteogenesis by modulating the post-translational modifications of tubulin, with a view to provide new targets for the prevention and treatment of hard tissue diseases.
Introduction: Endo-beta-N-acetylglucosaminidase (ENGASE) is one of the key enzymes involved in the structural and functional regulations of glycoproteins. Although its enzymatic activities and applications have been well studied in vitro, its biological function in vivo yet remains to be illustrated. In this study, the biological function of ENGASE in Caenorhabditis elegans was explored in detail. METHODS:An Engase gene knockout in C. elegans (CeEng-1 or CeEngase) was constructed and subjected to a panel of phenotypical and glycomics analysis. In addition, in vitro and in vivo ENGASE inhibition assays were performed. RESULTS:Engase knockout worm's adaptivity to environmental stresses (heat and osmotic) was significantly improved, and its longevity was also increased mildly. A clustered change in basement membrane proteins (e.g., LAM-1, LAM-2, and EPI-1) was illustrated by N-glycopeptide analysis, suggesting that ENGASE is involved in a basement membrane-based stress regulation. Then, the heat stress phenotype was further supported by in vivo CeEngase knockdown assay and in vitro and in vivo small compound inhibitory assay of CeENGASE, indicating that ENGASE is a potential drug target for stress management. CONCLUSION:Engase is actively involved in a basement membrane-mediated stress adaptation and could serve as a potential target for healthcare products. .
Fibroblasts are central to variety of homeostatic events such as wound healing and tissue regeneration. However, their pathologic activation is thought to play roles in a variety of diseases not only limited to fibrosis, foreign body reaction, scleroderma but also cancer metastasis. Biophysical properties of the extracellular matrix (ECM) deposited by an activated fibroblast determine whether there is a pro-regenerative or scarring response. Compared to aged fibroblasts, embryonic fibroblasts were shown to deposit a pro-regenerative ECM characterized by early hyaluronic acid (HA) deposition and increased levels of pro-regenerative collagens such as type III collagen. Since HA is also a regulator of collagen organization, we propose that early accumulation of HA by fibroblasts can facilitate a pro-regenerative matrix formation. Given that the molecular weights of HA present in pro-regenerative matrix are higher than synthetic HA, we strategize attracting HA synthesized by fibroblasts. In this study, we used a synthetic peptide sequence known to have affinity to HA as a strategy to instruct fibroblasts to retain HA on the surface. We hypothesized that hyaluronic acid binding peptide (HABP) may instruct fibroblast endogenous HA deposition onto functionalized surfaces. We functionalized silica glass surfaces with HABP using aminoorganosilane mediated chemisorption and screened primary human dermal fibroblasts (hDF) for cell morphology, cytoskeletal arrangement, and alpha-smooth muscle actin (α-SMA) expression. Our results show HABP treated surfaces retain higher levels of HA on silica glass compared to control surfaces on fibroblast derived matrices. Analysis of α-SMA shows increased α-SMA expression on hDFs and increased stress fiber formation. HABP treated surfaces were found to have reduced α-SMA expression. The physical features of collagen fibers deposited by fibroblasts were also organized differently in the presence of HABP. We propose that HABPs are a potentially viable strategy to instruct pro-regenerative fibroblasts and can be used therapeutically to treat fibrotic diseases.
Background: Hydrogels are widely used as extracellular matrix (ECM)-mimetic biomaterials, but most lack the nanofibrous hierarchy of the native extracellular matrix, which is essential for regulating human stem cells (hSCs) behavior. Nanofibrous-composite hydrogels address this limitation by incorporating fibrillar cues, either intrinsically formed, dispersed within the matrix, or applied at the surface, to better replicate the structural and mechanotopographical features of the stem cell niche. Summary: This review systematically compares three nanofiber-hydrogel architectures: self-assembling nanofiber matrices, hydrogels with encapsulated electrospun fibers, and hydrogels surface-decorated with fibrous coatings. We examine how differences in fiber chemistry, stiffness, degradability, and spatial organization regulate key hSCs' behaviors, including adhesion, viability, morphology, proliferation, migration, differentiation, and secretion. Polymeric, natural, hybrid, magnetic, and bioactive nanoparticle reinforced fibers are each discussed to highlight how each configuration generates distinct biophysical and biochemical cues. By linking fabrication strategies to resulting cellular outcomes, this review outlines architecture-specific advantages and limitations that inform the rational design of next-generation ECM-mimetic scaffolds. Key Messages: Nanofibrous hydrogels bridge the gap between conventional hydrogel mechanics and the nanoscale organization of the native ECM, enabling more physiologically relevant control of hSCs’ behavior. Each architecture provides distinct structural and mechanobiological cues suited to different therapeutic or manufacturing goals. Hybrid and multifunctional fiber systems, such as magnetic systems, ion-releasing platforms, and nanoparticle-enhanced fibers, deliver synergistic biochemical and mechanical signals that enhance differentiation and paracrine activity. Understanding how fiber properties and organization influence cell responses provides a roadmap for designing ECM-mimetic biomaterials optimized for scalable hSCs expansion and regenerative applications.
Cytosolic carboxypeptidase 1 (CCP1) is a deglutamylase that antagonizes polyglutamylation. Mutations in human CCP1 gene cause a severe disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), which is characterized by marked growth retardation. However, the role and mechanisms of CCP1 in skeletal development remain unclear. In this study, we used CCP1 knockout (CCP1-KO) mice to assess bone mass changes by micro-CT, HE, alkaline phosphatase (ALP) staining, tartrateresistant acid phosphatase (TRAP) staining and immunofluorescence staining. Changes in osteogenic differentiation, proliferation and migration capacity of bone marrow mesenchymal stem cells (BMSCs) were assessed by ALP, alizarin red (ARS) staining, quantitative real-time PCR (qRT-PCR), EdU staining and cell scratching assay. Then, tubulin glutamylation and primary cilia of BMSCs after deletion of CCP1 was analyzed by western blot (WB) and immunofluorescence staining. Finally, CB839, an inhibitor of glutamine metabolism, was used to detect changes in the osteogenic differentiation ability and primary cilia of BMSCs after reducing the elevated glutamylation level. CCP1-KO mice exhibited phenotypes relevant to humans, including reduced body size, decreased bone mass, and reduced bone density during growth and development. CCP1 deficiency impairs the proliferation, migration and osteogenic differentiation of BMSCs. Meanwhile, the number of pre-osteoblasts derived from BMSCs is decreased, leading to impaired osteogenesis. At the cellular level, CCP1 loss results in aberrant tubulin glutamylation, increased microtubule glutamylation, and shortened primary cilia in BMSCs. Finally, reduction of abnormally elevated tubulin glutamylation was efficacious for promoting osteogenic differentiation of BMSCs and restoring primary cilia length of BMSCs. We propose that CCP1 plays a critical role in regulating BMSCs differentiation and promotes osteogenesis by modulating the post-translational modifications (PTM) of tubulin, with a view to provide new targets for the prevention and treatment of hard tissue diseases.
