MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

Putative role of endothelin receptor B in the development and maintenance of taste buds within the circumvallate papillae.

This study aimed to achieve a better understanding of taste receptor cell development relative to endothelin receptor B (ETB) in circumvallate papillae (CVP). ETB localization was assessed by immunohistochemistry during tongue development of the mouse. Co-localization of ETB with taste receptor type III cell marker, Synaptosomal-Associated Protein 25 kDa (SNAP25), was evident in both the developing and adult CVP. ETB was strongly localized in the stromal core region. As development progressed, ETB became localized in the CVP mesenchyme and partially in the epithelium. ETB and SNAP25 co-localization indicates that ETB may regulate innervation from the CVP mesenchyme to taste buds.

Surface Engineering of Natural Killer Cells with CD44-targeting Ligands for Augmented Cancer Immunotherapy.

Adoptive immunotherapy utilizing natural killer (NK) cells has demonstrated remarkable efficacy in treating hematologic malignancies. However, its clinical intervention for solid tumors is hindered by the limited expression of tumor-specific antigens. Herein, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thereby 2) sufficient presentation of the CD44 ligand (i.e., HA) onto NK cells for cancer targeting, without the need for genetic manipulation. Membrane-engineered NK cells can selectively recognize CD44-overexpressing cancer cells through HA-CD44 affinity and subsequently induce in situ activation of NK cells for cancer elimination. Therefore, the surface-engineered NK cells using HA-PEG-Lipid (HANK cells) establish an immune synapse with CD44-overexpressing MIA PaCa-2 pancreatic cancer cells, triggering the "recognition-activation" mechanism, and ultimately eliminating cancer cells. Moreover, in mouse xenograft tumor models, administrated HANK cells demonstrate significant infiltration into solid tumors, resulting in tumor apoptosis/necrosis and effective suppression of tumor progression and metastasis, as compared to NK cells and gemcitabine. Taken together, the HA-PEG-Lipid biomaterials expedite the treatment of solid tumors by facilitating a sequential recognition-activation mechanism of surface-engineered HANK cells, suggesting a promising approach for NK cell-mediated immunotherapy.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Crossing fibers may underlie the dynamic pulling forces of muscles that attach to cartilage at the tip of the nose.

The present study used microdissection, histology, and microcomputed tomography (micro-CT) with the aims of determining the prevalence and patterns of the depressor septi nasi (DSN) and orbicularis oris (OOr) muscles attached to the footplate of the medial crus (fMC) of the major alar cartilage, focusing on their crossing fibers. The DSN and OOr attached to the fMC of the major alar cartilage were investigated in 76 samples from 38 embalmed Korean adult cadavers (20 males, 18 females; mean age 70 years). The DSN, OOr, or both were attached to the fMC. When the DSN ran unilaterally or was absent, some OOr fibers ascended to attach to the fMC instead of the DSN in 20.6% of the samples. Crossing fibers of the DSN or OOr attached to the fMC were found in 82.4% of the samples. Bilateral and unilateral crossing fibers were found in 32.4% and 50.0%, respectively, and no crossing fibers were found in 17.6%. The DSN and OOr that attached to the fMC could be categorized into six types according to presence of the DSN and the crossing patterns of the DSN and OOr. Anatomical findings of the DSN and OOr that attached to the fMC were confirmed in histology and micro-CT images. These findings offer insights on anatomical mechanisms that may underlie the dynamic pulling forces generated by muscles that attach to the fMCs and on evolutionary variation observed in human facial expressions. They can also provide useful information for guiding rhinoplasty of the nasal tip.

Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction.

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo. The OSKM-induced forced dedifferentiation showed similar molecular features of intestinal regeneration, including a transition from homeostatic cell types to injury-responsive-like cell types. These injury-responsive-like cells, sharing gene signatures of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following damage. In contrast to normal intestinal regeneration involving Ptgs2 induction, the OSKM promotes autonomous production of prostaglandin E2 via epithelial Ptgs1 expression. These results indicate prostaglandin synthesis is a common mechanism for intestinal regeneration but involves a different enzyme when partial reprogramming is applied to the intestinal epithelium.

Effect of icariin surface treatment on the resorption of denuded roots after replantation in rat.

AIM: Limiting the incidence of resorption associated with delayed replantation of avulsed teeth is critical for long-term tooth survival. In this study, we assessed whether icariin, a natural product with anti-osteoclastic properties, could reduce root resorption in a rat model of tooth replantation. METHODOLOGY: Cytocompatibility of icariin (10, 20, 40 and 80 µM) was evaluated by CCK-8 proliferation assay in vitro, and an osteoclastogenesis assay was performed to evaluate the effect of icariin on the differentiation of rat bone marrow macrophages and human peripheral blood monocytes into tartrate-resistant acid phosphatase-stained (TRAP+ ) multinucleated giant cells (MNGCs). Differentiation of human periodontal ligament stem cells (hPDLSCs) treated with icariin (10 µM) was also evaluated at 5, 10 and 21 days of osteogenic induction. The first maxillary molars of five-week-old male Sprague-Dawley rats were extracted, denuded of PDL, then treated either with neutralized collagen solution (Carrier control) or icariin in collagen (3 µg/µL) before replantation into their sockets. The animals were euthanized 2 weeks post-surgery for micro-computed tomography (micro-CT) imaging and histological analyses. RESULTS: Icariin was cytocompatible and significantly reduced the differentiation of TRAP+ MNGCs in a dose-dependent manner compared to the control. Moreover, icariin enhanced alkaline phosphatase activity, expression of osteogenic marker genes and proteins, and calcium deposition in hPDLSCs. Micro-CT imaging of the replanted samples demonstrated a significantly higher volume of remaining roots in the icariin-treated group than in the control group. Histological analysis revealed a marked number of resorptive lacunae with TRAP activity in the control group, whereas icariin-treated samples showed signs of functional healing and reduced osteoclastic activity. CONCLUSIONS: Icariin was biocompatible and demonstrated potent anti-osteoclastic and pro-osteogenic properties that reduced resorption and promoted functional healing of denuded roots in a rat maxillary first molar model of replantation. These findings indicate that root surface treatment with icariin may be a clinically relevant and practical method for improving the retention and survival of teeth with compromised PDL after delayed replantation following traumatic avulsion.

Biological Function and Potential Applications of Garcinol in Human Dental Pulp Stem Cells.

INTRODUCTION: The regeneration of pulp tissue is crucial for true regenerative endodontic treatment, which requires a reduction in osteogenic differentiation. Garcinol, a histone acetyltransferase inhibitor, is a natural regulator that is known to suppress the osteogenic differentiation of dental pulp stem cells. In this study, the inhibitory effect of garcinol on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) was evaluated using three-dimensional culture under in vitro and in vivo conditions. METHODS: hDPSCs were obtained from caries-free third molars and cultured with 10 µM garcinol for 7 days in an ultra-low attachment plate. The cell stemness and expression of osteogenic differentiation-related genes were analyzed using reverse transcription-polymerase chain reaction and single-cell analysis. A transplantation experiment was performed in mice to investigate whether garcinol-treated hDPSCs showed restrained osteogenic differentiation. RESULTS: hDPSCs cultured in the U-shaped ultra-low attachment plate showed the highest expression of stemness-related genes. Garcinol-treated hDPSCs demonstrated downregulation of osteogenic differentiation, with lower expression of bone sialoprotein, which is related to bone formation, and higher expression of dentin sialophosphoprotein, which is related to dentin formation. However, the garcinol-treated hDPSCs did not show any alterations in their stemness. Consistent results were observed in the transplantation experiment in mice. CONCLUSIONS: Garcinol reduced the osteogenic differentiation of hDPSCs, which can contribute to true regenerative endodontic treatment.

Harnessing the dental cells derived from human induced pluripotent stem cells for hard tissue engineering.

INTRODUCTION: Most mineralized tissues in our body are present in bones and teeth. Human induced pluripotent stem cells (hiPSCs) are promising candidates for cell therapy to help regenerate bone defects and teeth loss. The extracellular matrix (ECM) is a non-cellular structure secreted by cells. Studies on the dynamic microenvironment of ECM are necessary for stem cell-based therapies. OBJECTIVES: We aim to optimize an effective protocol for hiPSC differentiation into dental cells without utilizing animal-derived factors or cell feeders that can be applied to humans and to mineralize differentiated dental cells into hard tissues. METHODS: For the differentiation of both dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) from hiPSCs, an embryoid body (EB) was formed from hiPSCs. hiPSC were differentiated into neural crest cells with an induction medium utilized in our previous study, and hiPSC-derived DECs were differentiated with a BMP-modulated customized medium. hiPSC-dental cells were then characterized, analyzed, and validated with transcriptomic analysis, western blotting, and RT-qPCR. To form mineralized tissues, hiPSC-derived DECs were recombined with hiPSC-derived DMCs encapsulated in various biomaterials, including gelatin methacryloyl (GelMA), collagen, and agar matrix. RESULTS: These hiPSC-derived dental cells are highly osteogenic and chondro-osteogenic in photocrosslinkable GelMA hydrogel and collagen type I microenvironments. Furthermore, hiPSC-derived dental cells in agar gel matrix induced the formation of a bioengineered tooth. CONCLUSION: Our study provides an approach for applying hiPSCs for hard tissue regeneration, including tooth and bone. This study has immense potential to provide a novel technology for bioengineering organs for various regenerative therapies.

Fabrication of functional ameloblasts from hiPSCs for dental application.

Tooth formation relies on two types of dental cell populations, namely, the dental epithelium and dental mesenchyme, and the interactions between these cell populations are important during tooth development. Although human-induced pluripotent stem cells (hiPSCs) can differentiate into dental epithelial and mesenchymal cells, organoid research on tooth development has not been established yet. This study focused on the hiPSC-derived human ameloblast organoid (hAO) using a three-dimensional (3D) culture system. hAOs had similar properties to ameloblasts, forming enamel in response to calcium and mineralization by interaction with the dental mesenchyme. hAOs simultaneously had osteogenic and odontogenic differentiation potential. Furthermore, hAOs demonstrated tooth regenerative potential upon interaction with the mouse dental mesenchyme. Our findings provide new insights into a suitable hiPSC-derived dental source and demonstrate that hAOs can be beneficial not only for tooth regeneration but also for the study of various dental diseases for which treatment has not been developed yet.

Ahnak is required to balance calcium ion homeostasis and smooth muscle development in the urinary system.

BACKGROUND: Various renal abnormalities, including hydronephrosis, polycystic kidney disease, and hydroureter, have been reported, and these abnormalities are present in DiGeorge syndrome, renal dysplasia, and acute kidney failure. Previous studies have shown that various genes are associated with renal abnormalities. However, the major target genes of nonobstructive hydronephrosis have not yet been elucidated. RESULTS: We examined neuroblast differentiation-associated protein Ahnak localization and analyzed morphogenesis in developing kidney and ureter. To investigated function of Ahnak, RNA-sequencing and calcium imaging were performed in wild type and Ahnak knockout (KO) mice. Ahnak localization was confirmed in the developing mouse kidneys and ureter. An imbalance of calcium homeostasis and hydronephrosis, which involves an expanded renal pelvis and hydroureter, was observed in Ahnak KO mice. Gene Ontology enrichment analysis on RNA-seq results indicated that 'Channel Activity', 'Passive Transmembrane Transporter Activity' and 'Cellular Calcium Ion Homeostasis' were downregulated in Ahnak KO kidney. 'Muscle Tissue Development', 'Muscle Contraction', and 'Cellular Calcium Ion Homeostasis' were downregulated in Ahnak KO ureter. Moreover, peristaltic movement of smooth muscle in the ureter was reduced in Ahnak KO mice. CONCLUSIONS: Abnormal calcium homeostasis causes renal disease and is regulated by calcium channels. In this study, we focused on Ahnak, which regulates calcium homeostasis in several organs. Our results indicate that Ahnak plays a pivotal role in kidney and ureter development, and in maintaining the function of the urinary system.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury.

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

MAST4 controls cell cycle in spermatogonial stem cells.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

Transcriptomic Comparison Analysis between Ameloblastoma and AM-1 Cell Line.

Cancer initiation and progression are profoundly along with the crosstalk between cancer cells and the surrounding stroma. Accumulating evidence has shown that the therapy targeting the extracellular matrix (ECM) would regress tumor growth and invasion in the most common carcinomas. However, it remains largely unexplored in several rare tumors like odontogenic tumors. Ameloblastoma (AM) is the representative odontogenic epithelial tumor in the jawbone, and it usually infiltrates into adjacent bone marrow and has unlimited growth capacity and a high potential for recurrence. This study aims to investigate the role of collagen-rich ECM during the invasion of AM. Transcriptomic analysis revealed that ECM- and epithelial-to-mesenchymal transition (EMT)-related genes were up-regulated in AM compared to ameloblastoma cell line, AM-1. Tumoroid forming analysis showed that Collagen-rich ECM is indispensable for AM progression, especially for aggressive growth patterns and collective invasion.

Unraveled roles of Cav1.2 in proliferation and stemness of ameloblastoma.

BACKGROUND: Transcriptome analysis has been known as a functional tool for cancer research recently. Mounting evidence indicated that calcium signaling plays several key roles in cancer progression. Despite numerous studies examining calcium signaling in cancer, calcium signaling studies in ameloblastoma are limited. RESULTS: In the present study, comparative transcriptome profiling of two representative odontogenic lesions, ameloblastoma and odontogenic keratocyst, revealed that Cav1.2 (CACNA1C, an L-type voltage-gated calcium channel) is strongly enriched in ameloblastoma. It was confirmed that the Ca2+ influx in ameloblastoma cells is mainly mediated by Cav1.2 through L-type voltage-gated calcium channel agonist and blocking reagent treatment. Overexpression and knockdown of Cav1.2 showed that Cav1.2 is directly involved in the regulation of the nuclear translocation of nuclear factor of activated T cell 1 (NFATc1), which causes cell proliferation. Furthermore, a tumoroid study indicated that Cav1.2-dependent Ca2+ entry is also associated with the maintenance of stemness of ameloblastoma cells via the enhancement of Wnt/ß-catenin signaling activity. CONCLUSION: In conclusion, Cav1.2 regulates the NFATc1 nuclear translocation to enhance ameloblastoma cell proliferation. Furthermore, Cav1.2 dependent Ca2+ influx contributes to the Wnt/ß-catenin activity for the ameloblastoma cell stemness and tumorigenicity. Our fundamental findings could have a major impact in the fields of oral maxillofacial surgery, and genetic manipulation or pharmacological approaches to Cav1.2 can be considered as new therapeutic options.

Mast4 determines the cell fate of MSCs for bone and cartilage development.

Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-ß and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-ß1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3ß and subsequent Smurf1 recruitment, promoted ß-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4-/- mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

Inhibition of L-type voltage-gated calcium channel-mediated Ca2+ influx suppresses the collective migration and invasion of ameloblastoma.

OBJECTIVES: Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non-detectable remnants during surgery, interrupting the complete elimination of cancer cells. METHODS: To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next-generation sequencing in detail. RESULTS: Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L-type voltage-gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L-type VGCC blocker revealed suppression of the Ca2+ -induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. CONCLUSION: Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM.

Ameloblastoma modifies tumor microenvironment for enhancing invasiveness by altering collagen alignment.

Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.

Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis.

Proteins expressed by the paired box gene 9 (PAX9) and Msh Homeobox 1 (MSX1) are intimately involved in tooth development (odontogenesis). The regulation of PAX9 and MSX1 protein turnover by deubiquitinating enzymes (DUBs) plausibly maintain the required levels of PAX9 and MSX1 during odontogenesis. Herein, we used a loss-of-function CRISPR-Cas9-mediated DUB KO library kit to screen for DUBs that regulate PAX9 and MSX1 protein levels. We identify and demonstrate that USP49 interacts with and deubiquitinates PAX9 and MSX1, thereby extending their protein half-lives. On the other hand, the loss of USP49 reduces the levels of PAX9 and MSX1 proteins, which causes transient retardation of odontogenic differentiation in human dental pulp stem cells and delays the differentiation of human pluripotent stem cells into the neural crest cell lineage. USP49 depletion produced several morphological defects during tooth development, such as reduced dentin growth with shrunken enamel space, and abnormal enamel formation including irregular mineralization. In sum, our results suggest that deubiquitination of PAX9 and MSX1 by USP49 stabilizes their protein levels to facilitate successful odontogenesis.

Anatomical connections among the depressor supercilii, levator labii superioris alaeque nasi, and inferior fibers of orbicularis oculi: Implications for variation in human facial expressions.

The aim of this study was to determine how the depressor supercilii (DS) connects to the levator labii superioris alaeque nasi (LLSAN) and inferior fibers of the orbicularis oculi (OOc INF) in the human midface. While grimacing, contraction of the DS with fibers connecting to the LLSAN and OOc INF can assist in pulling the medial eyebrow downward more than when these connecting fibers are not present. Contraction of these distinct connecting fibers between the DS and the LLSAN can also slightly elevate the nasal ala and upper lip. The DS was examined in 44 specimens of embalmed adult Korean cadavers. We found that the DS connected to the LLSAN or the OOc INF by muscle fibers or thin aponeuroses in 33 (75.0%) of the 44 specimens. The DS was connected to both the LLSAN and OOc INF by muscle fibers or aponeuroses and had no connection to either in 5 (11.4%) and 11 (25.0%) specimens, respectively. The DS was connected to the LLSAN by the muscle fibers and thin aponeuroses in 6 (13.6%) and 4 (9.1%) specimens, respectively. The DS was connected to the OOc INF by the muscle fibers and thin aponeuroses in 5 (11.4%) and 23 (52.3%) specimens, respectively. Our findings regarding the anatomical connections of the glabellar region DS to the midface LLSAN and OOc INF provide insights on the dynamic balance between the brow depressors such as the DS and brow-elevating muscle and contribute to understanding the anatomical origins of individual variation in facial expressions. These results can also improve the safety, predictability, and aesthetics of treatments for the glabellar region with botulinum toxin type A and can be helpful when performing electromyography.