Derivation of Human Salivary Epithelial Progenitors from Pluripotent Stem Cells via Activation of RA and Wnt Signaling.

Derivation of salivary gland epithelial progenitors (SGEPs) from human pluripotent stem cells (hPSCs) has great potential in developmental biology and regenerative medicine. At present, no efficient method is available to generate salivary gland cells from hPSCs. Here, we described for the first time a robust protocol for direct differentiation of hPSCs into SGEPs by mimicking retinoic acid and Wnt signaling. These hPSC-derived SGEPs expressed SOX9, KRT5, and KRT19, important progenitor markers of developing salivary glands. CD24 and α-SMA positive cells, capable of restoring the functions of injured salivary glands, were also present in SGEP cultures. Importantly, RNA-sequencing revealed that the SGEPs resembled the transcript profiles of human fetal submandibular glands. Therefore, we provided an efficient protocol to induce hPSCs differentiation into SGEPs. Our study provides a foundation for generating functional hPSCs derived salivary gland acinar cells and three-dimensional organoids, potentially serving as new models for basic study and future translational research.

Unicystic Mucoepidermoid Carcinoma: A Pitfall for Clinical and Pathologic Diagnosis.

Unicystic mucoepidermoid carcinoma (UC-MEC) is a rare MEC variant, and its diagnosis is frequently problematic. This study is aimed at summarizing its clinicopathologic characteristics, treatment, and prognosis and proposing key points to avoid missed diagnosis and misdiagnosis in clinical and pathological conditions. This retrospective study included 30 UC-MEC cases, and the clinical findings were collected from the clinical medical records. Radiographic features, histologic behaviors, MAML2 rearrangement by fluorescence in situ hybridization (FISH), and follow-up data were analyzed. Moreover, glandular odontogenic cyst (GOC) and cytadenoma (CA) were used as controls. In the UC-MEC group, 19 patients were female (63%), and 11 were male (37%). The mean patient age was 39.5 (range, 7-72 years). The affected locations included the jaw (8 maxillary, 3 mandibular) and salivary glands (7 parotid, 11 palates, and 1 floor of the mouth). The chief complaint was swelling; the lesions were all cystic, among which 66.7% were well circumscribed and 33.3% poorly defined. Microscopic examination showed two UC-MEC histologic subtypes. Type A presented as a single cyst with mural thickening (8/30, 27%) lined predominantly by epidermoid cells with interspersed intermediate and mucinous cells, and type B (22/30, 73%) showed infiltrative tumor islands in the cystic wall or the surrounding tissue. FISH analysis suggested that approximately 66.7% of UC-MEC harbored a MAML2 rearrangement. During the median follow-up period of 42 months (range, 6-120 months), all type A patients and 68% of type B patients who underwent complete surgical resection lived without relapse. Seven cases with type B cancer that underwent curettage initially had local recurrence. Clinicians and pathologists hardly recognize UC-MEC owing to its cystic architecture. Specific epidermoid, mucous, and intermediate tumor cells, and MAML2 fusion testing, are essential to avoid potential diagnostic pitfalls. Prompting and completing resection surgery with negative margins would have a favorable prognosis.

Clinicopathological and genetic study of a rare occurrence: Malignant transformation of fibrous dysplasia of the jaws.

BACKGROUND: Malignant transformation of fibrous dysplasia (FD) is very rare and little is known about this occurrence. METHODS: We present the detailed clinical course of three cases of osteosarcoma arising from FD of the jaws and explore the genetic aberrations by Sanger sequencing, whole-exome sequencing (WES) and immunohistochemistry (IHC). A literature review of important topics related to this occurrence was also performed. RESULTS: It was observed that patients with secondary sarcoma from FD showed a wide range of ages, with most during the third decade. Female and males were equally affected. Craniofacial bones and femurs were the most affected sites. High-risk factors for this occurrence included polyostotic FD, McCune-Albright syndrome and excess growth hormone. Notably, a potential relationship between thyroid hormones and sarcoma development was suggested in one patient, who began to show malignant features after hypothyroidism correction. Sanger sequencing revealed GNAS mutations of FD retained in all malignant tissues. Additionally, abnormal TP53 was demonstrated in all three cases by WES and IHC. WES also revealed two other driver mutations, ROS1 and CHD8, and large amounts of somatic copy number alterations (CNAs) where various oncogenes and tumour suppressors are located. CONCLUSION: This study demonstrated and reviewed the clinical features and risk factors for a rare occurrence, secondary sarcoma from FD, and provided important new knowledge about its genetics.

Generation of functional salivary gland tissue from human submandibular gland stem/progenitor cells.

BACKGROUND: Organ replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown. METHODS: Isolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin-eosin staining, Alcian blue-periodic acid-Schiff staining, and Masson's trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo. RESULTS: The isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland-specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (-) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion. CONCLUSION: FGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

Specific complexes derived from extracellular matrix facilitate generation of structural and drug-responsive human salivary gland microtissues through maintenance stem cell homeostasis.

Three-dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s-Ecx) and analyzed the components of the s-Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s-Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s-Ecx. s-Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s-Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.