Cell type-specific transforming growth factor-ß (TGF-ß) signaling in the regulation of salivary gland fibrosis and regeneration.

Salivary gland damage and hypofunction result from various disorders, including autoimmune Sjögren's disease (SjD) and IgG4-related disease (IgG4-RD), as well as a side effect of radiotherapy for treating head and neck cancers. There are no therapeutic strategies to prevent the loss of salivary gland function in these disorders nor facilitate functional salivary gland regeneration. However, ongoing aquaporin-1 gene therapy trials to restore saliva flow show promise. To identify and develop novel therapeutic targets, we must better understand the cell-specific signaling processes involved in salivary gland regeneration. Transforming growth factor-ß (TGF-ß) signaling is essential to tissue fibrosis, a major endpoint in salivary gland degeneration, which develops in the salivary glands of patients with SjD, IgG4-RD, and radiation-induced damage. Though the deposition and remodeling of extracellular matrix proteins are essential to repair salivary gland damage, pathological fibrosis results in tissue hardening and chronic salivary gland dysfunction orchestrated by multiple cell types, including fibroblasts, myofibroblasts, endothelial cells, stromal cells, and lymphocytes, macrophages, and other immune cell populations. This review is focused on the role of TGF-ß signaling in the development of salivary gland fibrosis and the potential for targeting TGF-ß as a novel therapeutic approach to regenerate functional salivary glands. The studies presented highlight the divergent roles of TGF-ß signaling in salivary gland development and dysfunction and illuminate specific cell populations in damaged or diseased salivary glands that mediate the effects of TGF-ß. Overall, these studies strongly support the premise that blocking TGF-ß signaling holds promise for the regeneration of functional salivary glands.

Parotid cancer: analysis of preoperative parameters for adaptation of the therapeutic strategy.

PURPOSE: To establish typical clinical and radiological profiles of primary low-grade parotid cancers in order to tailor therapeutic strategy. MATERIALS AND METHODS: Retrospective study of 57 patients operated on for primary parotid cancer between 2010 and 2021, with review of preoperative MRI and histopathology according to a standardized scoring grid. OBJECTIVE: To study prognostic factors and determine the preoperative clinical and radiological profile of low-grade cancers. RESULTS: Good prognostic factors for specific survival were: staging ≤ cT3 (p = 0.014), absence of adenopathy on cN0 MRI (p < 0.001), superficial lobe location (p = 0.033), pN0 (p < 0.001), absence of capsular rupture (p = 0.004), as well as the absence of peri-tumoral nodules (p = 0.033), intra-parotid adenopathies (p < 0.001), vascular emboli (p < 0.001), peri-neural sheathing (p = 0.016), nuclear atypia (p = 0.031), and necrosis (p = 0.002). It was not possible to define a reliable clinical and radiological profile for low-grade cancers (sensitivity 38%, specificity 79%). CONCLUSION: Our study demonstrated multiple factors of good prognosis, but it was not possible to define a clinical and radiological profile of patients likely to benefit from more limited surgery, nor to diagnose, a priori, low-grade cancers.

Bioengineered Salivary Gland Microtissues─A Review of 3D Cellular Models and their Applications.

Salivary glands (SGs) play a vital role in maintaining oral health through the production and release of saliva. Injury to SGs can lead to gland hypofunction and a decrease in saliva secretion manifesting as xerostomia. While symptomatic treatments for xerostomia exist, effective permanent solutions are still lacking, emphasizing the need for innovative approaches. Significant progress has been made in the field of three-dimensional (3D) SG bioengineering for applications in gland regeneration. This has been achieved through a major focus on cell culture techniques, including soluble cues and biomaterial components of the 3D niche. Cells derived from both adult and embryonic SGs have highlighted key in vitro characteristics of SG 3D models. While still in its first decade of exploration, SG spheroids and organoids have so far served as crucial tools to study SG pathophysiology. This review, based on a literature search over the past decade, covers the importance of SG cell types in the realm of their isolation, sourcing, and culture conditions that modulate the 3D microenvironment. We discuss different biomaterials employed for SG culture and the current advances made in bioengineering SG models using them. The success of these 3D cellular models are further evaluated in the context of their applications in organ transplantation and in vitro disease modeling.

A Prospective Evaluation of Tubarial Gland Doses With Acute Dysphagia and Treatment Tolerance in Head and Neck Cancer Patients.

Background This study prospectively analyzed the clinical significance of tubarial glands (TGs) doses in head and neck cancer (HNC) patients. Methods Patients diagnosed with HNC in Ankara Bilkent City Hospital, Turkey were analyzed. TGs volumes and doses were noted. The patients were evaluated in terms of acute dysphagia (AD) and radiation therapy (RT)-associated xerostomia. Results The median volume of the TGs was 3.5(2.1-5.9)cc. No increased standardized uptake values (SUV) were observed in the TGs. There was no significant relationship between TGs values and the third or sixth months of xerostomia after RT. There was a significant relationship between grade ≥2 AD and TGs-Dmean (p0.020); TGs-V25(%) (p0.007); TGs-V30(%) (p0.009); TGs-V40% (p0.011); TGs-V50% (p0.010), TGs-V60% (p0.045). In terms of the risk of grade ≥2 AD, the cut-off value of the TGs-Dmean was analyzed for 50 Gy, with 75% sensitivity and 73.3% specificity (p 0.020; AUC 0.746; 95% CI 0.561-0.929). Additionally for grade ≥2 AD, the cut-off value of the TGs-V25(%) was analyzed 78 with 81.3% sensitivity and 80.0% specificity (p 0.011; AUC 0.769; 95% CI 0.591-0.947). Conclusion A significant correlation was found between TGs doses and AD during RT. TGs-V25(%) value showed higher significance. In future studies, the clinical significance of TGs can be studied especially on this value. The relationship between TGs doses and xerostomia should be evaluated with a larger series.

Oral cavity salivary gland pleomorphic adenoma: a histomorphological case series.

BACKGROUND: Pleomorphic adenoma (PA), the most common benign salivary gland epithelial lesion, has a biphasic epithelial-mesenchymal pattern and great histopathological diversity. METHODS: This study's objective was to conduct a retrospective clinicopathological analysis, focusing on the histopathology characteristics of salivary gland PA. RESULTS: There were ten cases of pleomorphic adenoma. The mean age was 33.5 years and no gender predilection was observed. All the patients presented with an asymptomatic mass and the duration of presentation was 31.2 ± 19.4 months. The cellular subtype (50 %) of PA was the most common. Capsular infiltration and incomplete capsules occurred in 20 % of cases. All the cases had round (100 %) and myxoid stroma. The cellular subtype was more common in the major salivary glands; showed capsular abnormalities (incomplete capsule, absent capsule, and tumor infiltration); and had more plasmacytoid, angular, spindled non-luminal cells as well as inflammation and cystic degeneration. The classic subtype had more clear and oncocytic cells along with sebaceous and squamous differentiation. The stroma-rich subtype had the shortest duration of complaints (three months) and showed giant cell reaction. CONCLUSIONS: These findings confirm previous studies on the clinicopathological features of pleomorphic adenomas and highlight important morphologic characteristics like capsular invasion and squamous metaplasia, which can otherwise indicate malignancy.

PANoptosis induced by inflammatory cytokines promotes salivary glands radiation injury.

OBJECTIVE: Salivary glands are frequently damaged in patients undergoing radiotherapy for head and neck cancer. Whether PANoptosis, which is characterized by pyroptosis, apoptosis, and necroptosis, occurs during radiation injury to the salivary glands and its role remain unclear. MATERIALS AND METHODS: Radiation-induced injury models of mouse submandibular gland, as well as primary acinar cells and HSG cell lines were established to determine the presence of radiation-induced PANoptosis. Several programmed cell death inhibitors, PFTα, disulfiram, Nec-1 and zVAD, were used to compare the effects of different cell death pathway on radiation injury. The LEGENDplex™ Human Inflammation Panel was used to characterize the inflammatory landscape secreted by salivary gland cells after radiotherapy. RESULTS: Single 15Gy or 8Gy radiotherapy triggered PANoptosis in mouse submandibular gland or salivary gland cells. Compared to the suppression of pyroptosis, apoptosis, or necroptosis alone, the inhibition of PANoptosis is more effective in preventing radiation injury to the salivary glands (p < 0.0001). The levels of multiple inflammatory cytokines were significantly up-regulated in the supernatants of HSG cells within 48 h after IR. Neutralizing inflammatory cytokines are capable of inhibiting salivary glands PANoptosis. CONCLUSIONS: Inhibition of PANoptosis induced by inflammatory cytokines can effectively prevent radiation injury of salivary glands.

Dendritic cell subpopulations in carcinoma ex-pleomorphic adenoma: A multicenter study.

OBJECTIVE: Carcinoma ex-pleomorphic adenoma (CEXPA) represents a malignant transformation from a recurrent or primary pleomorphic adenoma (PA), and the immune response may be essential in this process. Therefore, in this study, we aimed to identify and quantify subpopulations of dendritic cells (DCs) in CEXPA, residual PA in CEXPA (rPA), and PA. MATERIALS AND METHODS: A multicenter study was performed collecting salivary gland tumor (SGT) samples from three Oral and Maxillofacial Pathology Centers. A tissue microarray containing 41 samples of CEXPA and 22 samples of PA was included in this study and submitted to immunohistochemical reactions against CD1a, CD83, CD207, and Ki67 antibodies. RESULTS: Both PA and rPA showed a higher quantification of CD207+ and CD83+ cells when compared to CEXPA (p < 0.001 and p < 0.01, respectively). There was also a difference when comparing the cell proliferation index between PA/rPA and CEXPA using the Ki-67 marker (p = 0.043). However, there was no difference in the DC population regarding clinical parameters such as sex, anatomical location, size, and metastases (p > 0.06). CONCLUSIONS: Immunohistochemical profile of DC subpopulations and cell proliferation biomarkers in SGTs can contribute as an important tool in the differentiation of benign and malignant tumors or detection of initial areas with malignant transformation.

Age-associated B cell infiltration in salivary glands represents a hallmark of Sjögren's-like disease in aging mice.

Sjögren's disease (SjD), characterized by circulating autoantibodies and exocrine gland inflammation, is typically diagnosed in women over 50 years of age. However, the contribution of age to SjD pathogenesis is unclear. C57BL/6 female mice at different ages were studied to investigate how aging influences the dynamics of salivary gland inflammation. Salivary glands were characterized for immune cell infiltration, inflammatory gene expression, and saliva production. At 8 months, gene expression of several chemokines involved in immune cell trafficking was significantly elevated. At this age, age-associated B cells (ABCs), a unique subset of B cells expressing the myeloid markers CD11b and/or CD11c, were preferentially enriched in the salivary glands compared to other organs like the spleen or liver. The salivary gland ABCs increased with age and positively correlated with increased CD4 T follicular helper cells. By 14 months, lymphocytic foci of well-organized T and B cells spontaneously developed in the salivary glands. In addition, the mice progressively developed high titers of serum autoantibodies. A subset of aged mice developed salivary gland dysfunction mimicking SjD patients. Our data demonstrates that aging is a significant confounding factor for SjD. Thus, aged female C57BL/6 mice are more appropriate and a valuable preclinical model for investigating SjD pathogenesis and novel therapeutic interventions.

Parotid hypersalivation after inferior salivatory nucleus glutamate/NMDA receptor excitation in the rat.

Although salivation is essential during eating behavior, little is known about the brainstem centers that directly control the salivary glands. With regard to the inferior salivatory nucleus (ISN), the site of origin of the parasympathetic preganglionic cell bodies that innervate the parotid glands, previous anatomical studies have located it within the rostrodorsal medullary reticular formation. However, to date there is no functional data that shows the secretory nature of the somas grouped in this region. To activate only the somas and rule out the activation of the efferent fibers from and the afferent fibers to the ISN, in exp. 1, NMDA neurotoxin was administered to the rostrodorsal medullary region and the secretion of saliva was recorded during the following hour. Results showed an increased secretion of parotid saliva but a total absence of submandibular-sublingual secretion. In exp. 2, results showed that the hypersecretion of parotid saliva after NMDA microinjection was completely blocked by the administration of atropine (a cholinergic blocker) but not after administration of dihydroergotamine plus propranolol (α and ß-adrenergic blockers, respectively). These findings suggest that the somata of the rostrodorsal medulla are secretory in nature, controlling parotid secretion via a cholinergic pathway. The data thus functionally supports the idea that these cells constitute the ISN.

Curcumin Effects on Age-Related Changes in Oral Immunity: An In Vivo Study.

The current study aimed to investigate the effects of ageing on oral immunity using ß-defensin (DEFB) 1/2 as a marker and evaluate the effects of curcumin (CUR) on these processes. The study sample included 30 male C57BL/6J mice divided into three groups based on the treatment method used. The young control (YC) and old control (OC) groups received 0.5% methylcellulose-400 (curcumin vehicle) orally for five days, whereas the CUR group of older mice received a curcumin solution suspended in 0.5% methylcellulose-400 (dose: 3.0 mg/kg body). DEFB1/2 and immune indicator levels were measured in the saliva and salivary glands posttreatment. The saliva volume and protein content were significantly reduced in the OC group compared to the YC group. CUR administration restored these parameters, decreased DEFB1 expression in the salivary gland and increased DEFB1/2 secretion and DEFB2 expression. These findings were supported by epigenetic gene regulation and partial cytokine activation from changes in WD40 repeat protein 5, Tumour Necrosis Factor alpha and interleukin-1beta. Curcumin can partially restore age-related changes in oral immune responses and promote oral health, thereby preventing frailty in the older population through a nutritional therapeutic pathway.

Senescence and fibrosis in salivary gland aging and disease.

Salivary gland hypofunction is highly prevalent in aged and diseased individuals leading to significant discomfort and morbidity. One factor that contributes to salivary gland hypofunction is cellular aging, or senescence. Senescent cells can impair gland function by secreting paracrine-acting growth factors and cytokines, known as senescence-associated secretory phenotype (SASP) factors. These SASP factors stimulate inflammation, propagate the senescent phenotype through the bystander effect, and stimulate fibrosis. As senotherapeutics that target senescent cells have shown effectiveness in limiting disease manifestations in other conditions, there is interest in the use of these drugs to treat salivary gland hypofunction. In this review, we highlight the contribution of senescence and fibrosis to salivary gland pathologies. We also discuss therapeutic approaches to eliminate or modulate the senescent SASP phenotype for treating age-related salivary gland diseases and extending health span.

The anterior lobe of the parotid gland：A CT sialographic study.

OBJECTIVES: To investigate the imaging and anatomic features of the anterior lobe (AL) of the superficial parotid gland. METHODS: Computed tomographic sialography examinations were undertaken for 142 parotid glands in 77 patients. Whole computer tomography (CT) data were analyzed using multi-planar reformation and maximum intensity projection to generate sialographic CT images. The tributary ducts of the superficial parotid gland were analyzed to classify the parotid morphology. Three-dimensional analyses were used to investigate the AL and its relationship with adjacent anatomic landmarks. RESULTS: Four major types (I-IV) and two minor types (V-VI) of the AL and the superficial parotid gland were observed. Type I AL (83/142) was contiguous and not separated from the retromandibular parotid gland. Type II AL (16/142) was detached from the retromandibular parotid gland with one to four tributary ducts. Type III AL (12/142) showed a small isolated lobe above the Stensen's duct around the anterior edge of the masseter. Type IV (28/142) showed the absence of the AL. Type V (3/142) shows the absence of the retromandibular parotid gland. Type VI (3/142) showed the presence of ectopic salivary gland beneath the Stensen's duct anterior to the retromandibular parotid gland. CONCLUSIONS: The AL gives rise to the morphological variations of the superficial parotid gland. AL also gives rise to the accessory parotid gland when it is detached from the retromandibular parotid gland.

Evaluation of the biological function of ribosomal protein S18 from cattle tick Rhipicephalus microplus.

Rhipicephalus (Boophilus) microplus, also known as the cattle tick, causes severe parasitism and transmits different pathogens to vertebrate hosts, leading to massive economic losses. In the present study, we performed a functional characterization of a ribosomal protein from R. microplus to investigate its importance in blood feeding, egg production and viability. Ribosomal protein S18 (RPS18) is part of the 40S subunit, associated with 18S rRNA, and has been previously pointed to have a secondary role in different organisms. Rhipicephalus microplus RPS18 (RmRPS18) gene expression levels were modulated in female salivary glands during blood feeding. Moreover, mRNA levels in this tissue were 10 times higher than those in the midgut of fully engorged female ticks. Additionally, recombinant RmRPS18 was recognized by IgG antibodies from sera of cattle naturally or experimentally infested with ticks. RNAi-mediated knockdown of the RmRPS18 gene was performed in fully engorged females, leading to a significant (29 %) decrease in egg production. Additionally, egg hatching was completely impaired, suggesting that no viable eggs were produced by the RmRPS18-silenced group. Furthermore, antimicrobial assays revealed inhibitory activities against gram-negative Escherichia coli and gram-positive Staphylococcus aureus bacteria, affecting bacterial growth. Data presented here show the important role of RmRPS18 in tick physiology and suggest that RmRPS18 can be a potential target for the development of novel strategies for tick control.

Oncocytic papillary cystadenoma of the larynx: a case report.

BACKGROUND: Cystadenoma of the salivary glands is a rare benign clinical condition affecting both major and minor salivary glands equally. It constitutes approximately 2% of total neoplasms and 4.2-4.7% of benign formations in minor salivary glands. Typically presenting as a slow-growing, painless neoplasm, it can be distinguished from Cystadenolymphoma (Whartin's Tumor) by the absence of lymphoid elements in histological examination. While mostly located in the oral cavity and oropharynx, it can also be found in sinonasal mucosa, and rare cases have been identified in the larynx. CASE PRESENTATION: A 75-year-old Caucasian woman presented to the ear, nose, and throat department with complaints of dysphonia and headaches persisting for several months. Dysphonia had developed months after an unspecified vocal cord surgery elsewhere. Flexible laryngoscopy identified a left-sided cystic swelling affecting the supraglottic space, leading to respiratory obstruction and dysphonia. Head and neck computed tomography confirmed a 1.9 × 1.7 cm bilobed cystic mass originating from the left Morgagni ventricle. Microlaryngoscopy with CO2 laser excision and biopsy revealed a histopathological diagnosis of oncocytic papillary cystadenoma. Post-surgery, the patient fully recovered from dysphonia, with no significant complications noted. Long-term clinical surveillance was advised to detect potential recurrences promptly. CONCLUSION: Ectopic minor salivary gland tumors, both benign and malignant, should be taken into consideration as potential differential diagnosis for any swelling arising within the upper digestive tract mucosa. Ears, nose, and throat clinical examination completed by videolaryngoscopy can easily point out the location of the mass. Imaging is mandatory for differential diagnosis and for surgical planning. Surgical excision can provide both diagnosis and definitive cure.

A histology guide for performing human cadaveric studies: SQIP 2024 what to look for with light microscopy.

Histological observation under light microscopy has long been used in human cadaveric studies. However, it can distort the interpretations of findings if not used appropriately; there is no guide for its proper use. The aim of this article is to revisit and discuss the correct use of histology in human cadaveric studies, following discussions with experts in multiple fields of medicine, and to create the first guide for such usage. We reached a consensus with the experts, agreeing that when this principle (structure, quantification, interaction, position: SQIP) is applied to histological observations, the findings will be interpreted correctly. Appropriate use of this recommendation can make human cadaveric studies more accurate and informative. This is the first histology guide for human cadaveric studies.

Transcriptomic response of citrus psyllid salivary glands to the infection of citrus Huanglongbing pathogen.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the key vector insect transmitting the Candidatus Liberibacter asiaticus (CLas) bacterium that causes the devastating citrus greening disease (Huanglongbing, HLB) worldwide. The D. citri salivary glands (SG) exhibit an important barrier against the transmission of HLB pathogen. However, knowledge on the molecular mechanism of SG defence against CLas infection is still limited. In the present study, we compared the SG transcriptomic response of CLas-free and CLas-infected D. citri using an illumine paired-end RNA sequencing. In total of 861 differentially expressed genes (DEGs) in the SG upon CLas infection, including 202 upregulated DEGs and 659 downregulated DEGs were identified. Functional annotation analysis showed that most of the DEGs were associated with cellular processes, metabolic processes, and the immune response. Gene ontology and Kyoto Encyclopaedia of Genes and Genomes enrichment analyses revealed that these DEGs were enriched in pathways involving carbohydrate metabolism, amino acid metabolism, the immune system, the digestive system, the lysosome, and endocytosis. A total of 16 DEGs were randomly selected to further validate the accuracy of RNA-Seq dataset by reverse-transcription quantitative polymerase chain reaction. This study provides substantial transcriptomic information regarding the SG of D. citri in response to CLas infection, which may shed light on the molecular interaction between D. citri and CLas, and provides new ideas for the prevention and control of citrus psyllid.

Will Nigella sativa oil protect parotid glands of rats against cranium gamma irradiation? Histological and immunohistochemical evaluation.

BACKGROUND: Radiation plays an essential role in treating malignancies. Radiation exposure of salivary glands often results in permanent loss of their functions; therefore, their protection against radiation is crucial. Nigella sativa oil (NSO) is a useful antioxidant against free radicals. The purpose of this study was to investigate the radio-protective effect of NSO on oxidative injury of parotid glands of gamma-irradiated rats. METHODS: Twenty-eight male albino rats were divided into four groups (n = 7): Group 1: Neither NSO nor radiation, Group 2: Rats received NSO 400 mg/kg, Group 3: Rats received 15 Gy cranium gamma irradiation & Group 4: Rats received gamma irradiation and NSO. Rats were sacrificed two weeks after the last NSO dose. Histological sections of parotid glands were stained with H&E, Masson's trichrome and anti-TGF-ß antibodies. Area percentage of Masson's trichrome and TGF-ß expression was morphometrically examined. RESULTS: Parotid glands of control and NSO groups revealed normal morphology. Gamma-irradiated glands showed loss of normal acinar architecture and slight acinar shrinkage. NSO treatment of gamma-irradiated glands preserved acinar outline and architecture. Masson's trichrome stained samples revealed trace amounts of collagen fibers in control and NSO groups, and excessive amounts of collagen fibers in gamma-irradiated group, in addition to few collagen fibers for gamma-irradiated glands treated with NSO. Additionally, control and NSO groups showed negative TGF-ß expression. Gamma-irradiated group showed high TGF-ß expression, while NSO treated gamma-irradiated group showed moderate TGF-ß expression. CONCLUSIONS: Gamma-irradiation adversely affected parotid glands, and in contrast, NSO seemed to positively counteract this adverse effect.

Pleomorphic Adenoma of the Nasal Vestibule: An Atypical Presentation.

Introduction: We introduce the third case reported in the literature of an atypical presentation of pleomorphic adenoma located in the nasal vestibule of a young patient who assisted at our clinic. Case Report: A young man with no important medical history consulted due to a painless mass-type slow-growth lesion associated with right nasal obstruction. He underwent surgical management and complete resection of the mass. The pathological study revealed a pleomorphic adenoma, confirmed by immunohistochemistry. Conclusion: This case confirms that pleomorphic adenomas can occur anywhere in the head and neck, even in areas without upper air-digestive tract mucosa.

Translational Insights in the Landscape of Salivary Gland Cancers: Ready for a New Era?

Salivary gland carcinomas (SGCs) are rare neoplasms, representing less than 10% of all head and neck tumors, but they are extremely heterogeneous from the histological point of view, their clinical behavior, and their genetics. The guidelines regarding their treatment include surgery in most cases, which can also play an important role in oligometastatic disease. Where surgery cannot be used, systemic therapy comes into play. Systemic therapy for many years has been represented by polychemotherapy, but recently, with the affirmation of translational research, it can also count on targeted therapy, at least in some subtypes of SGCs. Interestingly, in some SGC histotypes, predominant mutations have been identified, which in some cases behave as "driver mutations", namely mutations capable of governing the carcinogenesis process. Targeting these driver mutations may be an effective therapeutic strategy. Nonetheless, it is not always possible to have drugs suitable for targeting driver mutations-and targeting driver mutations is not always accompanied by a clinical benefit. In this review, we will analyze the main mutations predominant in the various histotypes of SGCs.

Exploring the Cocktail Factor Approach to Generate Salivary Gland Progenitors through Co-Culture Techniques.

BACKGROUND: The derivation of salivary gland (SG) progenitors from pluripotent stem cells (PSCs) presents significant potential for developmental biology and regenerative medicine. However, the existing protocols for inducing SG include limited factors, making it challenging to mimic the in vivo microenvironment of embryonic SGs. METHODS: We reported a cocktail factor approach to promote the differentiation of mouse embryonic stem cell (mESC)-derived oral epithelium (OE) into SG progenitors through a three-dimensional co-culture method. Upon confirming that the embryonic SG can promote the differentiation of mESC-derived OE, we performed RNA sequence analysis to identify factors involved in the differentiation of SG progenitors. RESULTS: Our findings highlight several efficient pathways related to SG development, with frequent appearances of four factors: IFN-γ, TGF-ß2, EGF, and IGF-1. The combined treatment using these cocktail factors increased the expression of key SG progenitor markers, including Sox9, Sox10, Krt5, and Krt14. However, absence of any one of these cocktail factors did not facilitate differentiation. Notably, aggregates treated with the cocktail factor formed SG epithelial-like structures and pre-bud-like structures on the surface. CONCLUSION: In conclusion, this study offers a novel approach to developing a differentiation protocol that closely mimics the in vivo microenvironment of embryonic SGs. This provides a foundation for generating PSC-derived organoids with near-physiological cell behaviors and structures.