Ductal Cell Marker Research Articles

Identification and culture of functional salivary gland ductal epithelial cells.

Sialadenitis is a prevalent salivary gland disease resulting in decreased salivary flow rate. To date, little is known about the exact changes and mechanism of ductal cells in sialadenitis. This study aims to establish an efficient method to identify and isolate ductal cells, thereby facilitating further research on this specific cell type. Immunofluorescence for cytokeratin 13 and cytokeratin 19 was conducted in salivary glands to confirm their specificity as ductal cell markers. The dissected ducts were assessed through PCR and Western blot of cytokeratin 19 and digested by dispase and collagenase. The functionality of the isolated ductal cells was determined by measuring intracellular calcium. Cytokeratin 19 and cytokeratin 13 were expressed in all segments of human ducts. Cytokeratin 19 was limited to ducts excluding granular convoluted tubules in rat and mouse. The purities of the obtained ductal cells were approximately 98% in humans and 93% in rats. Furthermore, intracellular free calcium increased with time and concentration of carbachol treatment. Cytokeratin 19 serves as a dependable marker for identifying ductal cells in salivary glands, except for granular convoluted tubules. Moreover, we have successfully developed an efficient method for isolating ductal cells from salivary glands.

Abstract 1076: Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies

Abstract Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including Adenoid cystic carcinoma (ACC), Acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In this study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, total 13,643 salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2, and myoepithelial cells/stromal cells, with differentially expressed genes of each group. The localization of each cell group was anatomically validated by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Gene ontology analysis for this group included “epithelial cell differentiation” confirming the intercalated duct. Furthermore, to elucidate the potential etiologic relationship between normal salivary glands cell type and salivary gland carcinoma histology, we compared the expression of each cluster marker to SGC public RNA-seq data. In comparison between AciCC and ACC, we used normalized RNA-seq data of 54 ACC and 21 AciCC. ACC had significantly less expression of acinar cells markers (STATH, CST2, and LPO) compared to other markers expression(***P = 4 × 10−51), unlike AciCC (P = 0.54), suggesting a possible contribution of acinar cells to the carcinogenesis of AciCC. In another cohort, we compared 16 SDC, and 38 MECA with publicly available RNA-seq data. Remarkably, HES1 and ELF3, each of two different ductal cell markers, were significantly upregulated in SDC consistent with origin of SDC from ductal cells (P=0.001, P=0.0007, respectively). Consistent with bulk RNA-seq results, expression of HES1 in IHC using clinical human SDC and MECA samples was higher in SDC than MECA, suggesting that the expression of the derived cells is involved in salivary gland carcinogenesis. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship. On the other hand, there was no significant difference in myoepithelial marker ACTA2 expression between SDC and MECA, suggesting that the markers of origin are not always highly expressed, depending on cancer subtypes. Although publicly available SGC RNA-seq data used in this study are limited, the results remarkably reflect the diverse modes of carcinogenesis of SGC. These findings might provide a better understanding of the molecular basis of SGC and treatment strategies for SGC. Citation Format: Takuya Nakagawa, Chanond Nasamran, Jingjing Hu, Prakriti Sen, Theresa Guo, Kathleen Fisch, Joseph Califano. Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1076.

CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

Defining the relationship of salivary gland malignancies to novel cell subpopulations in human salivary glands using single nucleus RNA-sequencing.

Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.

The Expression of Two Distinct Sets of Glycolytic Enzymes Reveals Differential Effects of Glycolytic Reprogramming on Pancreatic Ductal Tumorigenesis in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is associated with enhanced aerobic glycolysis through elevated glucose uptake and the upregulated expression of genes encoding rate-limiting glycolytic enzymes. However, the direct impact of altered glycolytic pathways on pancreatic tumor progression has not been thoroughly investigated. Here, we utilized two strains of BAC transgenic mice with pancreatic expression of two distinct sets of glycolytic genes each arranged in a polycistronic fashion (PFKFB3-HK2-GLUT1 and LDHA-PDK1, respectively) to investigate the role of altered glycolysis on the development of pancreatic ductal tumor development in the Pdx1-Cre; LSL-KrasG12D mice. The overexpression of the two sets of glycolytic genes exhibited no significant effects on tumor development in the 4-5-month-old mice (the PanIN2 lesions stage). In the 9-10-month-old mice, the overexpression of PFKFB3-HK2-GLUT1 significantly accelerated PanIN3 progression, exhibiting elevated levels of ductal cell marker CK19 and tumor fibrosis. Surprisingly, the overexpression of LDHA-PDK1 significantly attenuated the progression of PanIN3 in the 9-10-month-old mice with significantly downregulated levels of CK19 and fibrosis. Therefore, distinct set of glycolytic enzymes that are involved in different glycolytic routes exhibited contrasting effects on pancreatic ductal tumor development depending on the tumor stages, providing novel insights into the complexity of the glycolytic pathway in the perspective of PDAC development and therapy.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

The effect and mechanism of gene Fam20a on the development and function of salivary glands in mice

ObjectiveThe influence of the knockout of gene Fam20a on mice salivary glands was studied in this research, to provide a potential gene therapeutic target for salivary gland dysfunction. DesignThe control group with genotype Fam20af/f and conditional knockout (cKO) group with Fam20af/f;K14-Cre were constructed with Cre-Loxp. The influence of Fam20a on the salivary glands was studied in terms of morphology, functionality and molecular mechanism. ResultsIn terms of morphology, the cross-sectional area ratio of ductal to the total was reduced in the cKO mice, while that of extracellular matrix to the total was increased. At the sub-microscopic level, the knockout of Fam20a led to abnormal sub-microscopic structure of the duct cells. Functionally, saliva flow rate was significantly reduced in cKO mice. The result was consistent with the change of acinar cell marker Aquaporin 5 which was abnormally diffusely expressed in the cytoplasm of acinar cells. Meanwhile, the expression of ductal cell markers Cytokeratin 7 and nerve growth factor β were significantly decreased, suggesting the abnormal development and function of the duct cells. The research on the mechanism reveals that the loss of Fam20a led to the decreased expression and varied localization of bone morphogenetic protein 4 (BMP4), and a significant decrease of the proportion of phosphorylated extracellular signal-regulated protein1/2 (ERK1/2) to total ERK1/2. These changes suggested that the loss of Fam20a attenuated the activity of the BMP/ERK signaling pathway. ConclusionsFam20a affects the morphology and function of salivary glands, probably by attenuating the activity of the BMP/ERK signaling pathway.

Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function.

Progress in the development of salivary gland regenerative strategies is limited by poor maintenance of the secretory function of salivary gland cells (SGCs) in vitro. To reduce the precipitous loss of secretory function, a modified approach to isolate intact acinar cell clusters and intercalated ducts (AIDUCs), rather than commonly used single cell suspension, is investigated. This isolation approach yields AIDUCs that maintain many of the cell-cell and cell-matrix interactions of intact glands. Encapsulation of AIDUCs in matrix metalloproteinase (MMP)-degradable PEG hydrogels promotes self-assembly into salivary gland mimetics (SGm) with acinar-like structure. Expression of Mist1, a transcription factor associated with secretory function, is detectable throughout the in vitro culture period up to 14 days. Immunohistochemistry also confirms expression of acinar cell markers (NKCC1, PIP and AQP5), duct cell markers (K7 and K5), and myoepithelial cell markers (SMA). Robust carbachol and ATP-stimulated calcium flux is observed within the SGm for up to 14 days after encapsulation, indicating that secretory function is maintained. Though some acinar-to-ductal metaplasia is observed within SGm, it is reduced compared to previous reports. In conclusion, cell-cell interactions maintained within AIDUCs together with the hydrogel microenvironment may be a promising platform for salivary gland regenerative strategies.

CK19 Predicts Recurrence and Prognosis of HBV Positive HCC

BackgroundCytokeratin is associated with the recurrence and metastasis of some cancers and tends to increase the malignancy of the disease. It is getting more and more attention in cancer research. Abnormal expression of cytokeratin 19 (CK19) has been reported as an important prognostic factor in cancers. CK19 is a marker of bile duct cells, liver progenitor cells (HPCs), and early hepatoblasts, and its expression is associated with poor prognosis in patients diagnosed with hepatocellular carcinoma (HCC). The purpose of this study was to evaluate the predictive value of CK19 for tumor recurrence after radical resection in patients with hepatitis B virus (HBV) positive HCC. MethodsThis study was a retrospective study conducted in two institutions. A total of 674 patients with HBV positive HCC who underwent radical HCC resection from January 2010 to May 2020 were included in this study. Chi-square test or Fisher’s exact test was used to compare the classification variables and continuous variables were compared by t-test or Wilcoxon rank sum test. Cox regression model was used for univariate and multi-variable survival analyses. Based on the results of the multi-variable analyses of Cox regression, the nomogram of 2-year recurrence-free survival (RFS) was plotted. The model was validated internally in the Hangzhou cohort (training set) and then externally in the Lanzhou cohort (test set) and the effectiveness of the model was tested. ResultsFor all 674 patients, 223 cases (33.1%) were positive and 451 cases (66.9%) were negative for CK19. The 2-year RFS rate was higher in patients with CK19 negative than in patients with CK19 positive. In the training set, correlation analysis showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001) and microvascular invasion (P = 0.020). In the test set, CK19 expression was correlated with postoperative platelet (P = 0.038), satellite nodules (P = 0.003), microvascular invasion (P = 0.011), and maximum tumor size (P = 0.039). Univariate Cox regression correlation analyses showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001), and microvascular invasion (P = 0.020). Training and test sets showed that postoperative platelet (> 300/L), CK19, satellite nodules in the training set, microvascular invasion, maximum tumor size, and tumor boundary were adverse factors for predicting RFS. Multi-variable analyses showed that in the training set, postoperative platelet > 300/L (hazard ratios (HR) = 2.753, 95% confidence interval (95%CI):1.234–6.142, P = 0.013), CK19 (HR = 1.410, 95%CI:1.006–1.976, P = 0.046), satellite nodule (HR = 1.476, 95%CI:1.026–2.120, P = 0.036), microvascular invasion (HR = 2.927, 95%CI:2.006–4.146, P < 0.001), incomplete tumor capsule (HR = 1.539, 95%CI:1.012–2.341, P = 0.044) were independent prognostic indicator of poor RFS. In the test set, postoperative platelet > 300/L (HR = 2.816, 95%CI:1.043–7.603, P = 0.041), CK19 (HR = 1.586, 95%CI:1.016–2.475, P = 0.042), satellite nodule (HR = 1.706, 95%CI:1.067–2.728, P = 0.026), microvascular invasion (HR = 1.611, 95%CI:1.034–2.510, P = 0.035), and tumor without capsule (HR = 1.870, 95%CI:1.120–3.120, P = 0.017) were independent prognostic indicators of poor RFS. The C-index for the nomogram was 0.698 (95%CI: 0.654–0.742) and the C-index for the test set was 0.670 (95%CI: 0.616–0.724). Both internal and external verification showed good results in identification and calibration. ConclusionCK19 plays a key role in tumor malignancy through overexpression and the expression of CK19 is an independent adverse factor affecting recurrence; therefore, CK19 can be used as a potential biomarker to predict adverse prognosis after surgery and adjuvant therapy in HCC patients.

PD49-03 GENETIC AND EPIGENETIC PROFILING INDICATES THE PROXIMAL TUBULE ORIGIN OF RENAL CANCERS IN END-STAGE RENAL DISEASE

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (PD49)1 Sep 2021PD49-03 GENETIC AND EPIGENETIC PROFILING INDICATES THE PROXIMAL TUBULE ORIGIN OF RENAL CANCERS IN END-STAGE RENAL DISEASE Hiroki Ishihara, Satoshi Yamashita, Yu-Yu Liu, Naoko Hattori, Omar El-Omar, Takashi Ikeda, Hironori Fukuda, Kazuhiko Yoshida, Toshio Takagi, Sekiko Taneda, Tsunenori Kondo, Yoji Nagashima, Kazunari Tanabe, and Toshikazu Ushijima Hiroki IshiharaHiroki Ishihara More articles by this author , Satoshi YamashitaSatoshi Yamashita More articles by this author , Yu-Yu LiuYu-Yu Liu More articles by this author , Naoko HattoriNaoko Hattori More articles by this author , Omar El-OmarOmar El-Omar More articles by this author , Takashi IkedaTakashi Ikeda More articles by this author , Hironori FukudaHironori Fukuda More articles by this author , Kazuhiko YoshidaKazuhiko Yoshida More articles by this author , Toshio TakagiToshio Takagi More articles by this author , Sekiko TanedaSekiko Taneda More articles by this author , Tsunenori KondoTsunenori Kondo More articles by this author , Yoji NagashimaYoji Nagashima More articles by this author , Kazunari TanabeKazunari Tanabe More articles by this author , and Toshikazu UshijimaToshikazu Ushijima More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002071.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: End-stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of two major histopathological types: clear-cell RCCs (ESRD-ccRCCs) and acquired cystic disease (ACD)-associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. METHODS: We investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD-ccRCCs and 7 ACD-associated RCCs to identify their molecular alterations and cellular origins. RESULTS: Targeted sequencing of 409 cancer-related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD-ccRCCs harbored frequent VHL mutations while ACD-associated RCCs did not. CNA analysis showed that ESRD-ccRCCs had a frequent loss of chromosome 3p while ACD-associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD-ccRCCs had methylation profiles similar to those of sporadic ccRCCs while ACD-associated RCCs had profiles similar to those of papillary RCCs (Figure 1). Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD-ccRCCs and ACD-associated RCCs had high expressions of proximal tubule cell marker genes while chromophobe RCCs had high expressions of distal tubule cell/collecting duct cell marker genes (Figures 2 and 3). CONCLUSIONS: ESRD-ccRCCs and ACD-associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these two histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron. Source of Funding: This research was supported by AMED under Grant Number JP19cm0106451 to Toshikazu Ushijima, JSPS KAKENHI under Grant Number JP18H02704 to Toshikazu Ushijima, and JSPS KAKENHI under Grant Number 17K11162 to Yoji Nagashima © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e844-e845 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiroki Ishihara More articles by this author Satoshi Yamashita More articles by this author Yu-Yu Liu More articles by this author Naoko Hattori More articles by this author Omar El-Omar More articles by this author Takashi Ikeda More articles by this author Hironori Fukuda More articles by this author Kazuhiko Yoshida More articles by this author Toshio Takagi More articles by this author Sekiko Taneda More articles by this author Tsunenori Kondo More articles by this author Yoji Nagashima More articles by this author Kazunari Tanabe More articles by this author Toshikazu Ushijima More articles by this author Expand All Advertisement Loading ...

115-LB: Liver-Derived FGF21 Mediates the Promoting Effect of Glucagon Receptor Antagonism on Beta-Cell Regeneration in Type 2 Diabetic Mice

Objective: Based on the clear role for glucagon in the pathogenesis of diabetes, glucagon-blocking therapies have been pursued as a therapeutic strategy for this disease. Here, we used REMD 2.59, a human antagonistic glucagon receptor (GCGR) monoclonal antibody (mAb), to clarify the effects of GCGR mAb in promoting pancreatic β cell regeneration in type 2 diabetes (T2D), and its potential mechanism. Methods: GCGR mAb was administrated weekly for 4 weeks in db/db mice and HFD/STZ-induced T2D mice, including different lineage-tracing mice. The plasma insulin and glucagon were determined by using ELISA. The evaluation of islet morphology was performed with immunofluorescent analysis. GCGR mAb was also treated in the systemic Fgf21 gene knockout (Fgf21-/-) and hepatocyte-specific Fgf21 gene knockout (Fgf21Hep-/-) T2D mice. Results: Treatment with GCGR mAb lowered blood glucose, improved glucose tolerance, and elevated plasma glucagon and insulin levels in both db/db mice and HFD/STZ-induced T2D mice. The β cell dedifferentiation as indicated by β cell lineage-tracing was blocked by GCGR mAb. The β cell proliferation as indicated by insulin+ and BrdU+ cells was boosted by GCGR mAb. The α-to-β cell conversion, as evaluated by glucagon+ insulin+ cells and confirmed by α cell lineage-tracing, was facilitated by GCGR mAb. After GCGR mAb treatment, some insulin+ cells were located in the duct region or even colocalized with duct cell markers, suggestive of duct-derived β cell neogenesis that was also verified by Ngn3+ cell lineage-tracing. Notably, GCGR mAb could still improve glucose tolerance and increase β cell mass, but these effects were attenuated in Fgf21-/- and Fgf21Hep-/- T2D mice compared with WT T2D mice. Conclusions: GCGR mAb promotes β cell regeneration via inhibiting β cell dedifferentiation and promoting β cell self-replication, α-to-β cell conversion and duct-derived β cell neogenesis in T2D mice, which is partly mediated by liver-derived FGF21. Disclosure X. Cui: None. T. Wei: None. J. Feng: None. J. Yang: None. H. Yan: Board Member; Self; REMD Biotherapeutics, Other Relationship; Spouse/Partner; REMD Biotherapeutics. R. Wei: None. T. Hong: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp &amp; Dohme Corp., Novo Nordisk, Sanofi. Funding National Natural Science Foundation of China (81830022)

The effect of fibroblast growth factor 7 on human dental pulp stem cells for differentiation to AQP5-positive and αSMA-positive cells in vitro and in vivo.

ObjectivesTransplantation of stem cells into wounds has become popular in regeneration therapies. As stem cells for transplantation, human dental pulp stem cells (hDPSCs) are known to be pluripotent cells that are relatively easy to collect from the pulp of deciduous or wisdom teeth. The purpose of this study was to investigate whether hDPSCs treated with fibroblast growth factor 7 (FGF7) would contribute to the regeneration of wounded rat submandibular glands (SMGs).Materials and methodsIn in vitro studies, hDPSCs were treated with or without FGF7 and mRNA expression levels were examined at days 3, 7 and 14 using qRT‐PCR. The target genes analyzed were BMI1 as an undifferentiated marker, AQP5 as an acinar cell marker, CK19 as a ductal epithelial cell marker, αSMA as a myoepithelial cell marker and VIMENTIN as a fibroblast marker. In in vivo studies, hDPSCs treated with or without FGF7 for 14 days were mixed with type I collagen gels and were transplanted into wounded rat SMGs. Hematoxylin–Eosin and immunohistochemical staining were performed at days 3 and 7, and the numbers of positive cells were counted. The primary antibodies used were against BMI1, AQP5, αSMA, PanCK and VIMENTIN.ResultsIn the in vitro studies, mRNA levels of BMI1 were decreased and αSMA were increased at days 3, 7 and 14, while AQP5 was increased at day 14 in the FGF7 group. In the in vivo studies, the proliferation of hDPSCs and cell islands was observed at day 7 in the FGF7 group. Few BMI1‐positive cells were observed, while numbers of AQP5‐positive and αSMA‐positive cells were increased at days 3 and 7 in the FGF7 group. Moreover, cell islands were AQP5‐positive.ConclusionThese results suggest that FGF7‐treated hDPSCs differentiate into AQP5‐positive and αSMA‐positive cells. Moreover, AQP5‐positive cell aggregations were formed.

Axin2+ Peribiliary Glands in the Periampullary Region Generate Biliary Epithelial Stem Cells That Give Rise to Ampullary Carcinoma

Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited because of a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer. We examined the expression pattern of the Wnt target gene Axin2 in extrahepatic bile ducts. We then applied lineage tracing to investigate whether Axin2-expressing cells from PBGs contribute to biliary regeneration and carcinogenesis using Axin2-CreERT mice. Wnt signaling activation, marked by Axin2, was limited to PBGs located in the periampullary region. Lineage tracing showed that Axin2-expressing periampullary PBG cells are capable of self-renewal and supplying new biliary epithelial cells (BECs) to the luminal surface. Additionally, the expression pattern of Axin2 and the mature ductal cell marker CK19 were mutually exclusive in periampullary region, and fate tracing of CK19+ luminal surface BECs showed gradual replacement by CK19- cells, further supporting the continuous replenishment of new BECs from PBGs to the luminal surface. We also found that Wnt signal enhancer R-spondin3 secreted from Myh11-expressing stromal cells, corresponding to human sphincter of Oddi, maintained the periampullary Wnt signal-activating niche. Notably, introduction of PTEN deletion into Axin2+ PBG cells, but not CK19+ luminal surface BECs, induced ampullary carcinoma whose development was suppressed by Wnt inhibitor. A specific cell population receiving Wnt-activating signal in periampullary PBGs functions as biliary epithelial stem/progenitor cells and also the cellular origin of ampullary carcinoma.

Secretome Analysis of Inductive Signals for BM-MSC Transdifferentiation into Salivary Gland Progenitors.

Severe dry mouth in patients with Sjögren’s Syndrome, or radiation therapy for patients with head and neck cancer, significantly compromises their oral health and quality of life. The current clinical management of xerostomia is limited to palliative care as there are no clinically-proven treatments available. Previously, our studies demonstrated that mouse bone marrow-derived mesenchymal stem cells (mMSCs) can differentiate into salivary progenitors when co-cultured with primary salivary epithelial cells. Transcription factors that were upregulated in co-cultured mMSCs were identified concomitantly with morphological changes and the expression of acinar cell markers, such as α-amylase (AMY1), muscarinic-type-3-receptor(M3R), aquaporin-5(AQP5), and a ductal cell marker known as cytokeratin 19(CK19). In the present study, we further explored inductive molecules in the conditioned media that led to mMSC reprogramming by high-throughput liquid chromatography with tandem mass spectrometry and systems biology. Our approach identified ten differentially expressed proteins based on their putative roles in salivary gland embryogenesis and development. Additionally, systems biology analysis revealed six candidate proteins, namely insulin-like growth factor binding protein-7 (IGFBP7), cysteine-rich, angiogenetic inducer, 61(CYR61), agrin(AGRN), laminin, beta 2 (LAMB2), follistatin-like 1(FSTL1), and fibronectin 1(FN1), for their potential contribution to mMSC transdifferentiation during co-culture. To our knowledge, our study is the first in the field to identify soluble inductive molecules that drive mMSC into salivary progenitors, which crosses lineage boundaries.

Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease.

End‐stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear‐cell RCCs (ESRD‐ccRCCs) and acquired cystic disease (ACD)‐associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD‐ccRCCs and 7 ACD‐associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer‐related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD‐ccRCCs harbored frequent VHL mutations, while ACD‐associated RCCs did not. CNA analysis showed that ESRD‐ccRCCs had a frequent loss of chromosome 3p while ACD‐associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD‐ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD‐associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD‐ccRCCs and ACD‐associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD‐ccRCCs and ACD‐associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.

Dapagliflozin promotes beta cell regeneration by inducing pancreatic endocrine cell phenotype conversion in type 2 diabetic mice

BackgroundClinical trials and animal studies have shown that sodium-glucose co-transporter type 2 (SGLT2) inhibitors improve pancreatic beta cell function. Our study aimed to investigate the effect of dapagliflozin on islet morphology and cell phenotype, and explore the origin and possible reason of the regenerated beta cells. MethodsTwo diabetic mouse models, db/db mice and pancreatic alpha cell lineage-tracing (glucagon-β-gal) mice whose diabetes was induced by high fat diet combined with streptozotocin, were used. Mice were treated by daily intragastric administration of dapagliflozin (1 mg/kg) or vehicle for 6 weeks. The plasma insulin, glucagon and glucagon-like peptide-1 (GLP-1) were determined by using ELISA. The evaluation of islet morphology and cell phenotype was performed with immunofluorescence. Primary rodent islets and αTC1.9, a mouse alpha cell line, were incubated with dapagliflozin (0.25–25 μmol/L) or vehicle in the presence or absence of GLP-1 receptor antagonist for 24 h in regular or high glucose medium. The expression of specific markers and hormone levels were determined. ResultsTreatment with dapagliflozin significantly decreased blood glucose in the two diabetic models and upregulated plasma insulin and GLP-1 levels in db/db mice. The dapagliflozin treatment increased islet and beta cell numbers in the two diabetic mice. The beta cell proliferation as indicated by C-peptide and BrdU double-positive cells was boosted by dapagliflozin. The alpha to beta cell conversion, as evaluated by glucagon and insulin double-positive cells and confirmed by using alpha cell lineage-tracing, was facilitated by dapagliflozin. After the dapagliflozin treatment, some insulin-positive cells were located in the duct compartment or even co-localized with duct cell markers, suggestive of duct-derived beta cell neogenesis. In cultured primary rodent islets and αTC1.9 cells, dapagliflozin upregulated the expression of pancreatic endocrine progenitor and beta cell specific markers (including Pdx1) under high glucose condition. Moreover, dapagliflozin upregulated the expression of Pcsk1 (which encodes prohormone convertase 1/3, an important enzyme for processing proglucagon to GLP-1), and increased GLP-1 content and secretion in αTC1.9 cells. Importantly, the dapagliflozin-induced upregulation of Pdx1 expression was attenuated by GLP-1 receptor antagonist. ConclusionsExcept for glucose-lowering effect, dapagliflozin has extra protective effects on beta cells in type 2 diabetes. Dapagliflozin enhances beta cell self-replication, induces alpha to beta cell conversion, and promotes duct-derived beta cell neogenesis. The promoting effects of dapagliflozin on beta cell regeneration may be partially mediated via GLP-1 secreted from alpha cells.

VDR in salivary gland homeostasis and cancer

The vitamin D receptor (VDR) and its ligand 1,25(OH)2D3 (1,25D) impact differentiation and exert anti-tumor effects in many tissues, but its role in salivary gland has yet to be defined. Using immunohistochemistry (IHC), we have detected strong VDR expression in murine and human salivary gland ducts. Compared to normal gland, VDR protein expression was retained in differentiated human pleomorphic adenoma (PA) but was undetectable in undifferentiated PA and in carcinomas, suggesting deregulation of VDR during salivary cancer progression. To gain insight into the potential role of VDR in salivary cancer, we assessed the effects of vitamin D in vivo and in vitro. Despite the presence of VDR in salivary gland, chronic dietary vitamin D restriction did not alter morphology of the salivary epithelium in C57/Bl6 mice. The localization of VDR in ductal epithelium prompted us to examine the effects of 1,25D in an established cell line (mSGc) derived from normal murine submandibular gland (SMG). This previously characterized cell line consists of multiple stem, progenitor and differentiated cell types as determined by mutually exclusive cellular expression of basal, ductal and myoepithelial markers. We demonstrated VDR expression and regulation of VDR target genes Vdr and Postn by 1,25D in mSGc, indicating functional ligand-mediated transcriptional activity. The effect of VDR signaling on epithelial differentiation markers was assessed by qPCR and IHC in mSGc cells treated with 1,25D. We found that 1,25D reduced mRNA expression of the basal cell progenitor marker keratin 5 (K5) and increased expression of the differentiated ductal cell marker keratin 7 (K7). Further, we found that 1,25D significantly decreased the number of proliferating cells, including proliferating K5+ cells. Characterization of cell cycle by Muse cytometry indicated 1,25D treatment decreased cells in S, G2, and M phase. The inhibition of K5+ cell proliferation by 1,25D is of particular interest because K5+ basal cells contribute to a wide variety of salivary tumor types. Our studies suggest that 1,25D alters cancer-relevant progenitor and differentiation markers in the salivary gland.

Coxsackievirus-B4 Infection of Human Primary Pancreatic Ductal Cell Cultures Results in Impairment of Differentiation into Insulin-Producing Cells.

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

MIST1, an Inductive Signal for Salivary Amylase in Mesenchymal Stem Cells.

Sjögren’s syndrome (SjS) is an autoimmune disease that destroys the salivary glands and results in severe dry mouth. Mesenchymal stem cell (MSC) transplantation has been recently proposed as a promising therapy for restoring cells in multiple degenerative diseases. We have recently utilized advanced proteomics biochemical assays to identify the key molecules involved in the mesenchymal-epithelial transition (MET) of co-cultured mouse bone-marrow-derived MSCs mMSCs with primary salivary gland cells. Among the multiple transcription factors (TFs) that were differentially expressed, two major TFs were selected: muscle, intestine, and stomach expression-1 (MIST1) and transcription factor E2a (TCF3). These factors were assessed in the current study for their ability to drive the expression of acinar cell marker, alpha-salivary amylase 1 (AMY1), and ductal cell marker, cytokeratin19 (CK19), in vitro. Overexpression of MIST1-induced AMY1 expression while it had little effect on CK19 expression. In contrast, TCF3 induced neither of those cellular markers. Furthermore, we have identified that mMSCs express muscarinic-type 3 receptor (M3R) mainly in the cytoplasm and aquaporin 5 (AQP5) in the nucleus. While MIST1 did not alter M3R levels in mMSCs, a TCF3 overexpression downregulated M3R expressions in mMSCs. The mechanisms for such differential regulation of glandular markers by these TFs warrant further investigation.

Effects of Neuropeptide Substance P on Proliferation and β-Cell Differentiation of Adult Pancreatic Ductal Cells.

Purpose: The pancreas is innervated by sensory nerves, parasympathetic and sympathetic nerves. The classical neurotransmitters, acetylcholine and noradrenaline, and some kind of neuropeptides are contained in the terminals of these nerves. Neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) co-released from the primary sensory fibers have been identified as the key neurotransmitters in pancreas. Pancreatic ductal epithelium cells are one of the important sources of the pancreatic islet β-cell neogenesis. We hypothesized that SP and CGRP might play a role on proliferation of ductal cells and differentiation of ductal cells toward the β-cell neogenesis.Methods: Primary ductal cells of rat pancreas at the third passage (P3) were used. The identification of P3 cells were confirmed with flow cytometry analysis and immunostaining by CK19 (the ductal cell marker). Proliferation of ductal cells was verified by CCK-8 assay and Ki67 immunostaining. Differentiation of ductal cells was determined with immunostaining and flow cytometry. Possible mechanism was explored by testing the key proteins of Wnt signaling using Western blot analysis.Results: Our data showed that SP but not CGRP promoted proliferation of ductal cells. Moreover, NK-1 receptor antagonist L-703,606 blocked the SP-induced stimulation of proliferation. The results of Western blot analysis showed that L-703,606 attenuated the effects of substance P on NK1R, GSK-3β, and β-catenin expression. However, SP did not directly induce the differentiation of ductal cells into β-cells, and did not promote the progression of ductal cells to differentiate into more insulin-produced cells in induction medium.Conclusions: These findings suggested that SP but not CGRP promoted proliferation of adult pancreatic ductal cells. SP promoted proliferation of ductal cells but not differentiation into β-cells. NK1R and Wnt signaling pathway might be involved in the mechanism of promoting the proliferation of ductal cells by SP. Findings in this study indicated the lack of SP might be a possible indicator for the initial of diabetes. SP could also be used as a drug candidate for the treatment of diabetes.