TLR8 Agonist VTX-2337 and Pegylated Liposomal Doxorubicin Hydrochloride or Paclitaxel in Treating Patients With Recurrent or Persistent Ovarian Epithelial, Fallopian Tube, or Peritoneal Cavity Cancer

ClinicalTrials.gov

2014-12-23

Malignant Ovarian Mixed Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Cystadenocarcinoma; Ovarian Serous Cystadenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Ovarian Carcinoma

Notch-Induced Expression of FZD7 Requires Noncanonical NOTCH3 Signaling in Human Breast Epithelial Cells.

PubMed

Bhat, Vasudeva; Sun, Yu Jia; Weger, Steve; Raouf, Afshin

2016-04-01

The evolutionarily conserved Notch and Wnt signaling pathways have demonstrated roles in normal mammary gland development and in breast carcinogenesis. We previously reported that in human mammary gland, signaling through NOTCH3 alone regulates the commitment of the undifferentiated bipotential progenitors to the luminal cell fate, indicating that NOTCH3 may regulate the expression of unique genes apart from the other Notch receptors. In this study, we used gain of function and loss of function experiments and found that a Wnt signaling receptor, Frizzled7 (FZD7), is a unique and nonredundant target of NOTCH3 in human breast epithelial cells. Interestingly, neither the constitutively active forms of NOTCH1-2, 4 nor loss of expression of these receptors were able to alter expression of FZD7 in human breast epithelial cells. We further show that FZD7-expressing cells are found more frequently in the luminal progenitor-enriched subpopulation of cells obtained from breast reduction samples compared with the undifferentiated bipotent progenitors. Also, we show that NOTCH3-induced expression of FZD7 occurs in the absence of CSL (CBF1-Suppressor of Hairless-Lag-1). Our data suggest that noncanonical Notch signaling through NOTCH3 could modulate Wnt signaling via FZD7 and in this way, might be involved in luminal cell differentiation.

Notch Signaling Modulates MUC16 Biosynthesis in an In Vitro Model of Human Corneal and Conjunctival Epithelial Cell Differentiation

PubMed Central

Xiong, Linjie; Woodward, Ashley M.

2011-01-01

Purpose. Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation. Methods. HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Results. Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions. These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface. PMID:21508102

Vaccine Therapy in Treating Patients With Stage IIIC-IV Ovarian Epithelial, Fallopian Tube, or Primary Peritoneal Cavity Cancer Following Surgery and Chemotherapy

ClinicalTrials.gov

2017-10-12

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Tumor; Fallopian Tube Mucinous Neoplasm; Fallopian Tube Serous Neoplasm; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Cystadenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Frequent loss of claudin-4 expression in dedifferentiated and undifferentiated endometrial carcinomas.

PubMed

Tessier-Cloutier, Basile; Soslow, Robert A; Stewart, Colin J R; Köbel, Martin; Lee, Cheng-Han

2018-04-19

Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non-fermentable (SWI/SNF) complex proteins. Claudin-4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF-deficient undifferentiated carcinomas but not in SWI/SNF-deficient sarcomas. The aim of this study was to examine claudin-4 expression in UECs/DDECs and other high-grade uterine carcinomas. We examined claudin-4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF-deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin-4 expression were considered to be negative. Nearly all SWI/SNF-deficient, and most SWI/SNF-proficient, UECs/DDECs showed a complete absence of claudin-4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin-4 expression. Only one SWI/SNF-deficient DDEC showed focal expression of claudin-4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin-4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. Claudin-4 expression can be absent or very focal in a subset of high-grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF-deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin-4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down-regulation of claudin-4, a tight junction protein, in SWI/SNF-deficient UECs/DDECs further supports the undifferentiated nature of these tumours. © 2018 John Wiley & Sons Ltd.

Ultrastructural study of the semicircular canal cells of the frog Rana esculenta.

PubMed

Oudar, O; Ferrary, E; Feldmann, G

1988-03-01

The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in an attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes.

Pluripotency of adult stem cells derived from human and rat pancreas

NASA Astrophysics Data System (ADS)

Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells are undifferentiated epithelial cells which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we ad...

Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia.

PubMed

Mouallif, Mustapha; Albert, Adelin; Zeddou, Mustapha; Ennaji, My Mustapha; Delvenne, Philippe; Guenin, Samuel

2014-08-01

Undifferentiated cell Transcription Factor 1 (UTF1) is a chromatin-bound protein involved in stem cell differentiation. It was initially reported to be restricted to stem cells or germinal tissues. However, recent work suggests that UTF1 is also expressed in somatic cells and that its expression may increase during carcinogenesis. To further clarify the expression profile of UTF1, we evaluated UTF1 expression levels immunohistochemically in eight normal human epithelia (from breast, prostate, endometrium, bladder, colon, oesophagus, lung and kidney) and their corresponding tumours as well as in several epithelial cell lines. We showed UTF1 staining in normal and tumour epithelial tissues, but with varying intensities according to the tissue location. In vitro analyses also revealed that UTF1 is expressed in somatic epithelial cell lines even in the absence of Oct4A and Sox2, its two main known regulators. The comparison of UTF1 levels in normal and tumoral tissues revealed significant overexpression in endometrial and prostatic adenocarcinomas, whereas lower intensity of the staining was observed in renal and colic tumours, suggesting a potential tissue-specific function of UTF1. Altogether, these results highlight a potential dual role for UTF1, acting either as an oncogene or as a tumour suppressor depending on the tissue. These findings also question its role as a specific marker for stem cells. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Paclitaxel, Polyglutamate Paclitaxel, or Observation in Treating Patients With Stage III or Stage IV Ovarian Epithelial, Peritoneal Cancer, or Fallopian Tube Cancer

ClinicalTrials.gov

2017-05-03

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation.

PubMed

Kulshreshtha, Ankur; Ahmad, Tanveer; Agrawal, Anurag; Ghosh, Balaram

2013-04-01

Exosomes are nanovesicles involved in intercellular communication. Their roles in various diseases are often contextual, depending on the cell type producing them. Although few studies hint toward the proinflammatory role of bronchoalveolar lavage fluid-derived exosomes in asthmatic progression, the cell types in lungs associated with exosome-mediated crosstalk and their resultant effects remain unexplored. It is well established that exosome-mediated cellular communication can influence disease phenotypes. This study explores exosome-mediated cellular crosstalk between structural and immune cells in asthma pathogenesis. Exosomes were isolated and detected from bronchoalveolar lavage fluid of control and asthmatic mice and were quantified by using a bead-based assay. Involvement of epithelial cells and macrophages were established by using immunohistochemical techniques in lung tissue sections. The role of IL-13 in exosome production was ascertained by using various in vitro and in vivo techniques. Exosome secretion was blocked in in vitro and in vivo settings by using a chemical inhibitor, and the effects on various asthmatic features were studied. Using combinatorial in vitro and in vivo approaches, we found that exosome secretion and production of exosome-associated proteins are higher in lungs of asthmatic mice compared with that seen in sham mice. Asthma is marked by enhanced secretion of exosomes by epithelial cells, but not macrophages, under the influence of IL-13. These epithelial cell exosomes induce proliferation and chemotaxis of undifferentiated macrophages. On the other hand, GW4869, which inhibited exosome production, resulted in a reduced population of proliferating monocytes and alleviation of various asthmatic features. Under the influence of IL-13, epithelial cell-derived exosomes can induce enhanced proliferation and chemotaxis of undifferentiated macrophages in the lungs during asthmatic inflammatory conditions. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Remodeling of the abdominal epithelial monolayer during the larva-pupa-adult transformation of Manduca.

PubMed

Nardi, James B; Bee, Charles Mark; Wallace, Catherine Lee

2018-06-01

During metamorphosis of insect epithelial monolayers, cells die, divide, and rearrange. In Drosophila undifferentiated diploid cells destined to form the adult cuticle of each abdominal segment segregate early in development from the surrounding polyploid larval epithelial cells of that segment as eight groups of diploid histoblast cells. The larval polyploid cells are programmed to die and be replaced by divisions and rearrangements of histoblast cells. By contrast, abdominal epithelial cells of Manduca larvae form a monolayer of cells representing different ploidy levels with no definitive segregation of diploid cells destined to form adult structures. These epithelial cells of mixed ploidy levels produce a thick smooth larval cuticle with sparsely distributed sensory bristles. Adult descendants of this larval monolayer produce a thinner cuticle with densely packed scale cells. The transition between these differentiated states of Manduca involves divisions of cells, changes in ploidy levels, and sorting of certain polyploid cells into circular rosette patches to minimize contacts of these polyploid cells with surrounding cells of equal or smaller size. Cells within the rosettes and some surrounding cells are destined to die and be replaced by remaining epithelial cells of uniform size and ploidy at pupa-adult apolysis. Copyright © 2018 Elsevier Inc. All rights reserved.

Nasal Epithelial Cells as Surrogates for Bronchial Epithelial Cells in Airway Inflammation Studies

PubMed Central

McDougall, Catherine M.; Blaylock, Morgan G.; Douglas, J. Graham; Brooker, Richard J.; Helms, Peter J.; Walsh, Garry M.

2008-01-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, αvβ3, and αvβ5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1β and TNF-α were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation. PMID:18483420

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.

PubMed

McDougall, Catherine M; Blaylock, Morgan G; Douglas, J Graham; Brooker, Richard J; Helms, Peter J; Walsh, Garry M

2008-11-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.

Undifferentiated carcinoma of the esophagus: a clinicopathological study of 16 cases☆

PubMed Central

Singhi, Aatur D.; Seethala, Raja R.; Nason, Katie; Foxwell, Tyler J.; Roche, Robyn L.; McGrath, Kevin M.; Levy, Ryan M.; Luketich, James D.; Davison, Jon M.

2015-01-01

Summary Undifferentiated carcinoma of the esophagus is a rare histologic variant of esophageal carcinoma. Using criteria based on studies of undifferentiated carcinomas arising at other sites, we have collected 16 cases of resected esophageal undifferentiated carcinomas. Patients ranged in age from 39 to 84 years (mean, 65.5 years) and were predominantly male (94%). The tumors were characterized by an expansile growth pattern of neoplastic cells organized in solid sheets and without significant glandular, squamous, or neuroendocrine differentiation. The neoplastic cells had a syncytial-like appearance, little intervening stroma, and patchy tumor necrosis. In a subset of cases, the tumor cells adopted a sarcomatoid (n = 2), rhabdoid (n = 1), or minor component (<5%) of glandular morphology (n = 3). In 1 case, reactive osteoclast-like giant cells were found interspersed among the neoplastic cells. Lymphovascular invasion, perineural invasion, and lymph node metastases were identified in 88%, 56%, and 81% of cases, respectively. In 12 (75%) specimens, the background esophageal mucosa was notable for Barrett esophagus. Consistent with the epithelial nature of these neoplasms, cytokeratin positivity was identified in all cases. In addition, SALL4 expression was present in 8 (67%) of 12 cases. Follow-up information was available for 15 (94%) of 16 patients, all of whom were deceased. Survival after surgery ranged from 1 to 50 months (mean, 11.9 months). Before death, 67% patients had documented locoregional recurrence and/or distant organ metastases. In summary, esophageal undifferentiated carcinomas are aggressive neoplasms and associated with a high incidence of recurrence and/or metastases and a dismal prognosis. PMID:25582499

3D Bioprinted Artificial Trachea with Epithelial Cells and Chondrogenic-Differentiated Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Bae, Sang-Woo; Lee, Kang-Woog; Park, Jae-Hyun; Lee, JunHee; Jung, Cho-Rok; Yu, JunJie; Kim, Hwi-Yool; Kim, Dae-Hyun

2018-05-31

Tracheal resection has limited applicability. Although various tracheal replacement strategies were performed using artificial prosthesis, synthetic stents and tissue transplantation, the best method in tracheal reconstruction remains to be identified. Recent advances in tissue engineering enabled 3D bioprinting using various biocompatible materials including living cells, thereby making the product clinically applicable. Moreover, clinical interest in mesenchymal stem cell has dramatically increased. Here, rabbit bone marrow-derived mesenchymal stem cells (bMSC) and rabbit respiratory epithelial cells were cultured. The chondrogenic differentiation level of bMSC cultured in regular media (MSC) and that in chondrogenic media (d-MSC) were compared. Dual cell-containing artificial trachea were manufactured using a 3D bioprinting method with epithelial cells and undifferentiated bMSC (MSC group, n = 6) or with epithelial cells and chondrogenic-differentiated bMSC (d-MSC group, n = 6). d-MSC showed a relatively higher level of glycosaminoglycan (GAG) accumulation and chondrogenic marker gene expression than MSC in vitro. Neo-epithelialization and neo-vascularization were observed in all groups in vivo but neo-cartilage formation was only noted in d-MSC. The epithelial cells in the 3D bioprinted artificial trachea were effective in respiratory epithelium regeneration. Chondrogenic-differentiated bMSC had more neo-cartilage formation potential in a short period. Nevertheless, the cartilage formation was observed only in a localized area.

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

PubMed Central

Yip, Yim Ling; Pang, Pei Shin; Deng, Wen; Tsang, Chi Man; Zeng, Musheng; Hau, Pok Man; Man, Cornelia; Jin, Yuesheng; Yuen, Anthony Po Wing; Tsao, Sai Wah

2013-01-01

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma. PMID:24167620

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

PubMed

Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors

PubMed Central

Mori, Munemasa; Mahoney, John E.; Stupnikov, Maria R.; Paez-Cortez, Jesus R.; Szymaniak, Aleksander D.; Varelas, Xaralabos; Herrick, Dan B.; Schwob, James; Zhang, Hong; Cardoso, Wellington V.

2015-01-01

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition. PMID:25564622

Ion Transport by Pulmonary Epithelia

PubMed Central

Hollenhorst, Monika I.; Richter, Katrin; Fronius, Martin

2011-01-01

The lung surface of air-breathing vertebrates is formed by a continuous epithelium that is covered by a fluid layer. In the airways, this epithelium is largely pseudostratified consisting of diverse cell types such as ciliated cells, goblet cells, and undifferentiated basal cells, whereas the alveolar epithelium consists of alveolar type I and alveolar type II cells. Regulation and maintenance of the volume and viscosity of the fluid layer covering the epithelium is one of the most important functions of the epithelial barrier that forms the outer surface area of the lungs. Therefore, the epithelial cells are equipped with a wide variety of ion transport proteins, among which Na+, Cl−, and K+ channels have been identified to play a role in the regulation of the fluid layer. Malfunctions of pulmonary epithelial ion transport processes and, thus, impairment of the liquid balance in our lungs is associated with severe diseases, such as cystic fibrosis and pulmonary oedema. Due to the important role of pulmonary epithelial ion transport processes for proper lung function, the present paper summarizes the recent findings about composition, function, and ion transport properties of the airway epithelium as well as of the alveolar epithelium. PMID:22131798

Expression and Function of αvβ3 and αvβ5 Integrins in the Developing Pancreas

PubMed Central

Cirulli, Vincenzo; Beattie, Gillian M.; Klier, George; Ellisman, Mark; Ricordi, Camillo; Quaranta, Vito; Frasier, Francine; Ishii, Jennifer K.; Hayek, Alberto; Salomon, Daniel R.

2000-01-01

Cell–cell and cell–matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that αvβ3 and αvβ5, two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of αvβ3 and αvβ5 integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins αvβ3 and αvβ5 and their ligands to morphogenetic events in the human endocrine pancreas. PMID:10995448

Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

PubMed

Ude, Victor C; Brown, David M; Viale, Luca; Kanase, Nilesh; Stone, Vicki; Johnston, Helinor J

2017-08-23

Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm 2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm 2 for CuO NMs, and 4.25 μg/cm 2 for copper sulphate (CuSO 4 ), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO 4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO 4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO 4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P app ); a measure of barrier permeability to CuO NMs. For all experiments, CuSO 4 was used as an ionic control. CuO NMs and CuSO 4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO 4 translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO 4 decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted. CuO NMs and CuSO 4 stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the P app and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO 4 . Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

PubMed

Wice, B M; Gordon, J I

1992-01-01

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)

Synchronous Pulmonary Malignancies: Atypical Presentation of Mantle Cell Lymphoma Masking a Lung Malignancy

PubMed Central

Masha, Luke; Zinchuk, Andrey; Boosalis, Valia

2015-01-01

We present a case of a pleural space malignancy masked by an atypical presentation of mantle cell lymphoma. Our patient presented with a large pleural effusion and right sided pleural studding, initially attributed to a new diagnosis of mantle cell lymphoma. Rare atypical epithelial cells were also seen amongst the clonal population of lymphocytes. The patient lacked systemic manifestations of mantle cell lymphoma and did not improve with chemotherapy. A pleural biopsy ultimately revealed the presence of an undifferentiated carcinoma, favoring a lung primary. A discussion of synchronous pleural space malignancies involving lymphomas is given. PMID:26500732

Synchronous Pulmonary Malignancies: Atypical Presentation of Mantle Cell Lymphoma Masking a Lung Malignancy.

PubMed

Masha, Luke; Zinchuk, Andrey; Boosalis, Valia

2015-09-07

We present a case of a pleural space malignancy masked by an atypical presentation of mantle cell lymphoma. Our patient presented with a large pleural effusion and right sided pleural studding, initially attributed to a new diagnosis of mantle cell lymphoma. Rare atypical epithelial cells were also seen amongst the clonal population of lymphocytes. The patient lacked systemic manifestations of mantle cell lymphoma and did not improve with chemotherapy. A pleural biopsy ultimately revealed the presence of an undifferentiated carcinoma, favoring a lung primary. A discussion of synchronous pleural space malignancies involving lymphomas is given.

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

PubMed

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

PubMed

Hagen, S J; Trier, J S

1988-07-01

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

Bevacizumab and Intravenous or Intraperitoneal Chemotherapy in Treating Patients With Stage II-III Ovarian Epithelial Cancer, Fallopian Tube Cancer, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-05-25

Malignant Ovarian Mixed Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Cystadenocarcinoma; Ovarian Serous Cystadenocarcinoma; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

PubMed

Klymenko, T; Gu, Q; Herbert, I; Stevenson, A; Iliev, V; Watkins, G; Pollock, C; Bhatia, R; Cuschieri, K; Herzyk, P; Gatherer, D; Graham, S V

2017-10-11

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log 2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress. IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle. Copyright © 2017 American Society for Microbiology.

A Novel Role for Integrin-linked Kinase in Epithelial Sheet Morphogenesis

PubMed Central

Vespa, Alisa; D'Souza, Sudhir J.A.; Dagnino, Lina

2005-01-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration. PMID:15975904

ZEB1 overexpression associated with E-cadherin and microRNA-200 downregulation is characteristic of undifferentiated endometrial carcinoma.

PubMed

Romero-Pérez, Laura; López-García, M Ángeles; Díaz-Martín, Juan; Biscuola, Michele; Castilla, M Ángeles; Tafe, Laura J; Garg, Karuna; Oliva, Esther; Matias-Guiu, Xavier; Soslow, Robert A; Palacios, José

2013-11-01

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression (∼33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis of E-cadherin and ZEB1 can aid in the differential diagnosis of the more agressive undifferentiated endometrial carcinomas from grade 3 endometrioid carcinomas.

Ceftaroline modulates the innate immune and host defense responses of immunocompetent cells exposed to cigarette smoke.

PubMed

Bruno, A; Cipollina, C; Di Vincenzo, S; Siena, L; Dino, P; Di Gaudio, F; Gjomarkaj, M; Pace, E

2017-09-05

Cigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system's response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. The present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. The constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. Ceftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Endometrial and ovarian carcinomas with undifferentiated components: clinically aggressive and frequently underrecognized neoplasms.

PubMed

Tafe, Laura J; Garg, Karuna; Chew, Ivy; Tornos, Carmen; Soslow, Robert A

2010-06-01

Carcinomas of the endometrium and ovary with undifferentiated components are uncommon neoplasms that are likely underdiagnosed. They are important to recognize as they have been shown to be clinically aggressive. We identified 32 carcinomas with undifferentiated components as defined by Silva and co-workers, 26 endometrial and 6 of ovarian origin. The patient age ranged from 21 to 76 years (median 55); 40% of patients were

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

PubMed Central

Sumiyoshi, Mika; Ricciuto, Jessica; Tisdale, Ann; Gipson, Ilene K.; Mantelli, Flavio; Argüeso, Pablo

2008-01-01

Purpose Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal–limbal epithelial (HCLE) cells. Methods Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-α-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell–cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Results Benzyl-α-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-α-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. Conclusions These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface–associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye). PMID:18172093

Paclitaxel and Carboplatin With or Without Bevacizumab in Treating Patients With Stage II, Stage III, or Stage IV Ovarian Epithelial Cancer, Primary Peritoneal Cancer, or Fallopian Tube Cancer

ClinicalTrials.gov

2017-12-28

Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Mixed Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinofibroma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

PubMed Central

Liévin-Le Moal, Vanessa

2013-01-01

SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

Epithelial alterations adjacent to invasive squamous carcinoma of the vulva.

PubMed

Gómez Rueda, N; García, A; Vighi, S; Belardi, M G; Cardinal, L; di Paola, G

1994-07-01

The slides of 64 vulvectomy specimens from vulvar squamous carcinoma were reviewed in order to study the histopathologic changes adjacent to the neoplasia. Normal epithelium was found in 7 cases (11%) and epithelial alterations adjacent to carcinoma in 59 (89%). The epithelial alterations found were: nonneoplastic epithelial disorder (NNDV) in 38 cases (59%) and vulvar intraepithelial neoplasia (VIN) in 19 (30%). The distribution of NNDV was: 20 cases of epithelial hyperplasia (EH) (31%), 6 of lichen sclerosus (9%) and 12 of the mixed type (19%). Sixteen cases of VIN 3 (25%) were undifferentiated, and three cases were differentiated VIN. Eighteen of 19 VIN cases were associated with NNDV, and 8 cases of undifferentiated VIN were associated with human papillomavirus infection. There was no apparent relationship between the associated lesions and tumor size, depth of invasion, lymph node metastases and clinical stage. Nevertheless, we found a significantly higher frequency of associated lesions in poorly differentiated tumors (P > .01). The most important finding was a high association between EH (50%) and VIN (30%) with carcinoma. VIN cases were almost always (95%) associated with EH.

Spectral Monitoring of Surfactant Clearance during Alveolar Epithelial Type II Cell Differentiation

PubMed Central

Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

2008-01-01

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment. PMID:18820234

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

PubMed

Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

2013-01-01

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

Penile Analogue of Stratified Mucin-Producing Intraepithelial Lesion of the Cervix: The First Described Case. A Diagnostic Pitfall.

PubMed

Michal, Michael; Michal, Michal; Miesbauerova, Marketa; Hercogova, Jana; Skopalikova, Barbora; Kazakov, Dmitry V

2016-05-01

The authors report a case where undifferentiated (classic) penile intraepithelial neoplasia was associated with the presence of goblet cells throughout the full epithelial thickness and which later progressed into an invasive carcinoma. The lesion evolved in three consecutive biopsies from only surface epithelium occupying numerous goblet cells in the first to variably sized solid nodules in the dermis composed of atypical squamous and/or basaloid cells intermixed with numerous goblet cells in the third biopsy. Both cellular components expressed CK7 and p16 protein. Human Papillomavirus (HPV) genotyping revealed high risk HPV type 16. To the best of our knowledge, this is the first description of such a lesion occurring on the penis, which can be considered the penile analogue of cervical stratified mucin-producing intraepithelial lesion (SMILE). The correct diagnosis was rendered retrospectively, after recognition of the existence of a vulvar lesion resembling cervical SMILE. The initial biopsy was misinterpreted as extramammary Paget disease, which also constitutes the main pitfall in the differential diagnosis. Another important differential diagnosis is penile/vulvar mucinous metaplasia. The finding of atypical squamous epithelial cells positive for p16 associated with mucinous cells present throughout the full epithelial thickness is a clue to the diagnosis of penile SMILE.

Epstein-Barr virus infection and nasopharyngeal carcinoma.

PubMed

Tsao, Sai Wah; Tsang, Chi Man; Lo, Kwok Wai

2017-10-19

Epstein-Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Author(s).

[Expression of embryonic markers in pterygium derived mesenchymal cells].

PubMed

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Dedifferentiated Liposarcoma With Epithelioid/Epithelial Features.

PubMed

Makise, Naohiro; Yoshida, Akihiko; Komiyama, Motokiyo; Nakatani, Fumihiko; Yonemori, Kan; Kawai, Akira; Fukayama, Masashi; Hiraoka, Nobuyoshi

2017-11-01

Dedifferentiated liposarcoma (DDLPS) demonstrates a variety of growth patterns, and their histologic resemblance to other spindle cell mesenchymal tumors has been widely recognized. However, epithelioid morphology in DDLPS has only rarely been documented. Here, we report 6 cases of DDLPS with striking epithelioid/epithelial features. The patients were 5 men and 1 woman with a median age of 61 years. All tumors were located in the internal trunk. During follow-up of 1 to 41 months, local recurrence, distant metastases, and tumor-related death occurred in 4, 2, and 4 patients, respectively. Beside well-differentiated liposarcoma component and conventional high-grade spindle cell morphology, all tumors focally exhibited growth comprising small or large epithelioid cells in diffuse or sheet-like proliferation. Rhabdoid cells were present in 2 cases. All 5 tumors tested harbored MDM2 amplification. Cytokeratin and/or epithelial membrane antigen were at least focally positive in all 5 tumors tested. One case contained a small focus of novel heterologous epithelial differentiation with acinar structures, wherein cytokeratin, MOC31, and claudin-4 were diffusely expressed and H3K27me3 expression was lost. DDLPS with epithelioid/epithelial features may lead to misdiagnosis of carcinoma or mesothelioma, and their diagnosis should be based on correlation with clinicopathologic and molecular findings. The epithelioid morphology in DDLPS may suggest an aggressive behavior based on this small series. In addition, we document 2 cases of MDM2-amplified undifferentiated neoplasm with epithelioid features in the internal trunk that lacked association with well-differentiated liposarcoma histology and showed rapid clinical course. Whether these latter tumors belong to DDLPS with epithelioid features requires further study.

Primary Culture of Undifferentiated Pleomorphic Sarcoma: Molecular Characterization and Response to Anticancer Agents

PubMed Central

Recine, Federica; Mercatali, Laura; Miserocchi, Giacomo; Spadazzi, Chiara; Liverani, Chiara; Bongiovanni, Alberto; Pieri, Federica; Casadei, Roberto; Riva, Nada; Fausti, Valentina; Amadori, Dino; Ibrahim, Toni

2017-01-01

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS. PMID:29292724

Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs?

PubMed

Nigam, Sanjay K

2013-12-01

Branching morphogenesis is critical to the development of organs such as kidney, lung, mammary gland, prostate, pancreas, and salivary gland. Essentially, an epithelial bud becomes an iterative tip-stalk generator (ITSG) able to form a tree of branching ducts and/or tubules. In different organs, branching morphogenesis is governed by similar sets of genes. Epithelial branching has been recapitulated in vitro (or ex vivo) using three-dimensional cell culture and partial organ culture systems, and several such systems relevant to kidney tissue engineering are discussed here. By adapting systems like these it may be possible to harness the power inherent in the ITSG program to propagate and engineer epithelial tissues and organs. It is also possible to conceive of a universal ITSG capable of propagation that may, by recombination with organ-specific mesenchymal cells, be used for engineering many organ-like tissues similar to the organ from which the mesenchyme cells were derived, or toward which they are differentiated (from stem cells). The three-dimensional (3D) branched epithelial structure could act as a dynamic branching cellular scaffold to establish the architecture for the rest of the tissue. Another strategy-that of recombining propagated organ-specific ITSGs in 3D culture with undifferentiated mesenchymal stem cells-is also worth exploring. If feasible, such engineered tissues may be useful for the ex vivo study of drug toxicity, developmental biology, and physiology in the laboratory. Over the long term, they have potential clinical applications in the general fields of transplantation, regenerative medicine, and bioartificial medical devices to aid in the treatment of chronic kidney disease, diabetes, and other diseases.

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

2018-01-01

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

High expression of SALL4 and fascin, and loss of E-cadherin expression in undifferentiated/dedifferentiated carcinomas of the endometrium

PubMed Central

Onder, Semen; Taskin, Orhun Cig; Sen, Fatma; Topuz, Samet; Kucucuk, Seden; Sozen, Hamdullah; Ilhan, Ridvan; Tuzlali, Sitki; Yavuz, Ekrem

2017-01-01

Abstract Undifferentiated/dedifferentiated endometrial carcinomas (UCE/DCEs) of the endometrium are rare tumors with poor prognosis. There are few clinicopathologic studies with detailed immunohistochemical analysis regarding UCE/DCEs. We evaluated the diagnostic value of a selected tumor stem-cell marker and epithelial-mesenchymal transition (EMT) markers, in addition to previously studied markers in identifying UCE/DCEs from other types of high-grade endometrial carcinomas. Eleven cases of UCE/DCEs with complete clinical follow-up that were diagnosed between 2006 and 2015 were included in the study. For immunohistochemical comparison, 11 clinically matched cases for each type of other high-grade endometrial carcinomas (high-grade endometrioid (F3-EC), serous [SC], and clear cell carcinoma [CCC]) were used as a control group. An immunohistochemical analysis including fascin, SALL4, E-cadherin, and β-catenin, in addition to epithelial and neuroendocrine markers was performed in each case. The majority of UCE/DCEs displayed diffuse expression of fascin (81.9%) and loss of E-cadherin expression (54.5%). SALL4 expression was detected in 36.3% of the UCE/DCE cases. SALL4 expression was significantly more frequent in UCE/DCEs than all other high-grade carcinomas (P < 0.001). Loss of E-cadherin and fascin expression was significantly more frequent in UCE/DCEs than high-grade endometrioid and clear cell adenocarcinomas (P = 0.012, 0.014 and P = 0.01, 0.003, respectively). We suggest that loss of E-cadherin expression together with fascin and SALL4 immunopositivity in addition to morphologic features have an impact in differential diagnosis of UCE/DCEs from other high-grade endometrial carcinomas. PMID:28272224

Cytological lesions in the midgut of Tribolium confusum larvae exposed to gamma radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jafri, R.H.; Ismail, M.

1977-01-01

The major cytological lesions in Tribolium confusum after irradiation were displayed by the midgut epithelium. At 24 hr following exposure to 5.3 kR, the regenerative cells called nidi appeared numerous. They gradually disappeared with increases in dosage and time in accordance with Arndt-Schulze's Law. The columnar epithelial cells and their nuclei appeared swollen and vacuolated on the fifth and twelfth day following exposure to 5.3 kR. They appeared disorganized and shed into the lumen of the midgut on the twelfth and fifth day following 50- and 70-kR irradiation, respectively. The basement membrane and the muscularis appeared loose on the fifthmore » and twelfth day dollowing 70-kR irradiation. It was observed that once the catabolic activity, i.e., histolysis, was initiated in the midgut, it continued to accelerate with increasing dose and time. Thus, the last effects at low doses, 5.3 and 10 kR, appeared as immediate effects at high doses, 50 and 70 kR. The differentiated cells, i.e., columnar epithelial cells, appeared radioresistant as compared to undifferentiated cells, i.e., regenerative cells, which appeared radiosensitive in accordance with the principle of Bergonie and Tribondeau.« less

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

PubMed Central

Kietzmann, Leonie; Guhr, Sebastian S.O.; Meyer, Tobias N.; Ni, Lan; Sachs, Marlies; Panzer, Ulf; Stahl, Rolf A.K.; Saleem, Moin A.; Kerjaschki, Dontscho; Gebeshuber, Christoph A.

2015-01-01

Parietal epithelial cells have been identified as potential progenitor cells in glomerular regeneration, but the molecular mechanisms underlying this process are not fully defined. Here, we established an immortalized polyclonal human parietal epithelial cell (hPEC) line from naive human Bowman’s capsule cells isolated by mechanical microdissection. These hPECs expressed high levels of PEC-specific proteins and microRNA-193a (miR-193a), a suppressor of podocyte differentiation through downregulation of Wilms’ tumor 1 in mice. We then investigated the function of miR-193a in the establishment of podocyte and PEC identity and determined whether inhibition of miR-193a influences the behavior of PECs in glomerular disease. After stable knockdown of miR-193a, hPECs adopted a podocyte-like morphology and marker expression, with decreased expression levels of PEC markers. In mice, inhibition of miR-193a by complementary locked nucleic acids resulted in an upregulation of the podocyte proteins synaptopodin and Wilms’ tumor 1. Conversely, overexpression of miR-193a in vivo resulted in the upregulation of PEC markers and the loss of podocyte markers in isolated glomeruli. Inhibition of miR-193a in a mouse model of nephrotoxic nephritis resulted in reduced crescent formation and decreased proteinuria. Together, these results show the establishment of a human PEC line and suggest that miR-193a functions as a master switch, such that glomerular epithelial cells with high levels of miR-193a adopt a PEC phenotype and cells with low levels of miR-193a adopt a podocyte phenotype. miR-193a–mediated maintenance of PECs in an undifferentiated reactive state might be a prerequisite for PEC proliferation and migration in crescent formation. PMID:25270065

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

NUTM1 Gene Fusions Characterize a Subset of Undifferentiated Soft Tissue and Visceral Tumors.

PubMed

Dickson, Brendan C; Sung, Yun-Shao; Rosenblum, Marc K; Reuter, Victor E; Harb, Mohammed; Wunder, Jay S; Swanson, David; Antonescu, Cristina R

2018-05-01

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

CLOCK regulates mammary epithelial cell growth and differentiation

PubMed Central

Crodian, Jennifer; Suárez-Trujillo, Aridany; Erickson, Emily; Weldon, Bethany; Crow, Kristi; Cummings, Shelby; Chen, Yulu; Shamay, Avi; Mabjeesh, Sameer J.; Plaut, Karen

2016-01-01

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland. PMID:27707717

In vitro selection and characterization of new probiotic candidates from table olive microbiota.

PubMed

Botta, Cristian; Langerholc, Tomaz; Cencič, Avrelija; Cocolin, Luca

2014-01-01

To date, only a few studies have investigated the complex microbiota of table olives in order to identify new probiotic microorganisms, even though this food matrix has been shown to be a suitable source of beneficial lactic acid bacteria (LAB). Two hundred and thirty eight LAB, belonging to Lactobacillus plantarum, Lactobacillus pentosus and Leuconostoc mesenteroides species, and isolated from Nocellara Etnea table olives, have been screened in this survey through an in vitro approach. A simulation of transit tolerance in the upper human gastrointestinal tract, together with autoaggregation and hydrophobicity, have been decisive in reducing the number of LAB to 17 promising probiotics. None of the selected strains showed intrinsic resistances towards a broad spectrum of antibiotics and were therefore accurately characterized on an undifferentiated and 3D functional model of the human intestinal tract made up of H4-1 epithelial cells. As far as the potential colonization of the intestinal tract is concerned, a high adhesion ratio was observed for Lb. plantarum O2T60C (over 9%) when tested in the 3D functional model, which closely mimics real intestinal conditions. The stimulation properties towards the epithelial barrier integrity and the in vitro inhibition of L. monocytogenes adhesion and invasion have also been assessed. Lb. plantarum S1T10A and S11T3E enhanced trans-epithelial electrical resistance (TEER) and therefore the integrity of the polarized epithelium in the 3D model. Moreover, S11T3E showed the ability to inhibit L. monocytogenes invasion in the undifferentiated epithelial model. The reduction in L. monocytogenes infection, together with the potential enhancement of barrier integrity and an adhesion ratio that was above the average in the 3D functional model (6.9%) would seem to suggest the Lb. plantarum S11T3E strain as the most interesting candidate for possible in vivo animal and human trials.

The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues.

PubMed

Kosheleva, N V; Saburina, I N; Zurina, I M; Gorkun, A A; Borzenok, S A; Nikishin, D A; Kolokoltsova, T D; Ustinova, E E; Repin, V S

2016-01-01

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.

PubMed

Goldspink, Deborah A; Gadsby, Jonathan R; Bellett, Gemma; Keynton, Jennifer; Tyrrell, Benjamin J; Lund, Elizabeth K; Powell, Penny P; Thomas, Paul; Mogensen, Mette M

2013-09-01

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.

Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells.

PubMed

Luxen, Sylvia; Noack, Deborah; Frausto, Monika; Davanture, Suzel; Torbett, Bruce E; Knaus, Ulla G

2009-04-15

Duox NADPH oxidases generate hydrogen peroxide at the air-liquid interface of the respiratory tract and at apical membranes of thyroid follicular cells. Inactivating mutations of Duox2 have been linked to congenital hypothyroidism, and epigenetic silencing of Duox is frequently observed in lung cancer. To study Duox regulation by maturation factors in detail, its association with these factors, differential use of subunits and localization was analyzed in a lung cancer cell line and undifferentiated or polarized lung epithelial cells. We show here that Duox proteins form functional heterodimers with their respective DuoxA subunits, in close analogy to the phagocyte NADPH oxidase. Characterization of novel DuoxA1 isoforms and mispaired Duox-DuoxA complexes revealed that heterodimerization is a prerequisite for reactive oxygen species production. Functional Duox1 and Duox2 localize to the leading edge of migrating cells, augmenting motility and wound healing. DuoxA subunits are responsible for targeting functional oxidases to distinct cellular compartments in lung epithelial cells, including Duox2 expression in ciliated cells in an ex vivo differentiated lung epithelium. As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development.

Prenatal metanephrogenesis of the camel: morphological evidence of epithelial-mesenchymal interaction.

PubMed

Osman, A-H K; Farouk, S M; Eidaroos, H; Abbott, L C; Ahmed, A A

2014-04-01

The present investigation examined histogenesis of epithelial, stromal and angiogenic elements of the prenatal camel permanent or metanephric kidney. The primitive metanephros was first observed at the 13-mm crown vertebral rump length (CVRL) stage as an ovoid structure composed of a centrally located epithelial ureteric bud and peripheral circumscribed masses of undifferentiated mesenchymal cells. The first morphological evidence of glomerulogenesis was observed at the 28-mm CVRL stage. Developing renal corpuscles became obvious at the 35-mm CVRL stage. At the 60-mm CVRL stage, the epithelial renal pelvis gave rise to tubular branches that extended towards the cortical zone. These branches represented the presumptive collecting ducts. Differentiation of renal tubules into the proximal and distal convoluted tubules was observed at the 95-mm CVRL stage. At the 130-mm CVRL stage, the renal medulla was clearly delineated into medullary pyramids, which in association with the corresponding cortical caps formed the morphological basis of the renal lobar formation. A gradual nephrogenic decline was noticed from the 940-mm CVRL on; however, the process of nephrogenesis persisted throughout all the studied foetal stages. © 2013 Blackwell Verlag GmbH.

Partitioning-Defective 1a/b Depletion Impairs Glomerular and Proximal Tubule Development.

PubMed

Akchurin, Oleh; Du, Zhongfang; Ramkellawan, Nadira; Dalal, Vidhi; Han, Seung Hyeok; Pullman, James; Müsch, Anne; Susztak, Katalin; Reidy, Kimberly J

2016-12-01

The kidney is a highly polarized epithelial organ that develops from undifferentiated mesenchyme, although the mechanisms that regulate the development of renal epithelial polarity are incompletely understood. Partitioning-defective 1 (Par1) proteins have been implicated in cell polarity and epithelial morphogenesis; however, the role of these proteins in the developing kidney has not been established. Therefore, we studied the contribution of Par1a/b to renal epithelial development. We examined the renal phenotype of newborn compound mutant mice carrying only one allele of Par1a or Par1b. Loss of three out of four Par1a/b alleles resulted in severe renal hypoplasia, associated with impaired ureteric bud branching. Compared with kidneys of newborn control littermates, kidneys of newborn mutant mice exhibited dilated proximal tubules and immature glomeruli, and the renal proximal tubular epithelia lacked proper localization of adhesion complexes. Furthermore, Par1a/b mutants expressed low levels of renal Notch ligand Jag1, activated Notch2, and Notch effecter Hes1. Together, these data demonstrate that Par1a/b has a key role in glomerular and proximal tubule development, likely via modulation of Notch signaling. Copyright © 2016 by the American Society of Nephrology.

A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baylin, S.B.; Gazdar, A.F.; Minna, J.D.

1982-08-01

Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or humanmore » fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.« less

ETV4 is a useful marker for the diagnosis of CIC-rearranged undifferentiated round-cell sarcomas: a study of 127 cases including mimicking lesions.

PubMed

Le Guellec, Sophie; Velasco, Valérie; Pérot, Gaëlle; Watson, Sarah; Tirode, Franck; Coindre, Jean-Michel

2016-12-01

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes*

PubMed Central

Park, Dayoung; Brune, Kristin A.; Mitra, Anupam; Marusina, Alina I.; Maverakis, Emanual; Lebrilla, Carlito B.

2015-01-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. PMID:26355101

Morphological Analysis of Human Induced Pluripotent Stem Cells During Induced Differentiation and Reverse Programming

PubMed Central

Magniez, Aurélie; Oudrhiri, Noufissa; Féraud, Olivier; Bacci, Josette; Gobbo, Emilie; Proust, Stéphanie; Turhan, Ali G.

2014-01-01

Abstract The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. PMID:25371857

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures.

PubMed

Meller, D; Pires, R T F; Tseng, S C G

2002-04-01

Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics.

PubMed

Kulkarni, Bina B; Tighe, Patrick J; Mohammed, Imran; Yeung, Aaron M; Powe, Desmond G; Hopkinson, Andrew; Shanmuganathan, Vijay A; Dua, Harminder S

2010-09-29

The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye. Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC). LEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence. Our transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.

Cancer of the Esophagus and Esophagogastric Junction: An Eighth Edition Staging Primer

PubMed Central

Rice, Thomas W.; Ishwaran, Hemant; Ferguson, Mark K.; Blackstone, Eugene H.; Goldstraw, Peter

2017-01-01

This primer for eighth edition staging of esophageal and esophagogastric epithelial cancers presents separate classifications for the clinical (cTNM), pathologic (pTNM), and postneoadjuvant pathologic (ypTNM) stage groups, which are no longer shared. For pTNM, pT1 has been subcategorized as pT1a and pT1b for the subgrouping pStage I adenocarcinoma and squamous cell carcinoma. A new, simplified esophagus-specific regional lymph node map has been introduced. Undifferentiated histologic grade (G4) has been eliminated; additional analysis is required to expose histopathologic cell type. Location has been removed as a category for pT2N0M0 squamous cell cancer. The definition of the esophagogastric junction has been revised. ypTNM stage groups are identical for both histopathologic cell types, unlike those for cTNM and pTNM. PMID:27810391

In the absence of (early) invasive carcinoma, vulvar intraepithelial neoplasia associated with lichen sclerosus is mainly of undifferentiated type: new insights in histology and aetiology.

PubMed

van Seters, M; ten Kate, F J W; van Beurden, M; Verheijen, R H M; Meijer, C J L M; Burger, M P M; Helmerhorst, T J M

2007-05-01

Differentiated vulvar intraepithelial neoplasia (VIN) is presumed to be the precursor of invasive squamous cell carcinoma (SCC) of the vulva. It is commonly assumed that differentiated VIN is related to lichen sclerosus (LS). However, evidence for this is limited to a small number of studies describing epithelial alterations adjacent to vulvar SCC. To study the histology and human papillomavirus (HPV) status in patients with a history of both LS and VIN without coexistent SCC. Original biopsy specimens and surgical specimens of patients retrieved from the pathology files were revised for the presence of LS, VIN and (early) invasive SCC, specifically focused on the two different types of VIN: differentiated and undifferentiated. Thereafter, VIN lesions were tested for the presence of HPV DNA. Twenty-seven patients fulfilled the criteria for LS and VIN without SCC. In all 27 patients, LS was found to be related to undifferentiated VIN. Grading yielded the following results: VIN 1 (n=10), VIN 2 (n=11) and VIN 3 (n=6). Additionally, VIN lesions from 26 patients could be tested for the presence of HPV DNA. HPV DNA, predominantly type 16, was present in 8 (31%) of them. Seven of these eight patients had VIN 2 or 3. During follow-up, three patients progressed to (early) invasive carcinoma. In two of these patients, differentiated VIN was observed overlying early invasive SCC. VIN related to LS without coexisting SCC is likely to be undifferentiated, in contrast to what was previously thought. HPV DNA was demonstrated in 31% of the lesions, and was strongly related to high-grade VIN.

In the absence of (early) invasive carcinoma, vulvar intraepithelial neoplasia associated with lichen sclerosus is mainly of undifferentiated type: new insights in histology and aetiology

PubMed Central

van Seters, M; Kate, F J W ten; van Beurden, M; Verheijen, R H M; Meijer, C J L M; Burger, M P M; Helmerhorst, T J M

2007-01-01

Background Differentiated vulvar intraepithelial neoplasia (VIN) is presumed to be the precursor of invasive squamous cell carcinoma (SCC) of the vulva. It is commonly assumed that differentiated VIN is related to lichen sclerosus (LS). However, evidence for this is limited to a small number of studies describing epithelial alterations adjacent to vulvar SCC. Aim To study the histology and human papillomavirus (HPV) status in patients with a history of both LS and VIN without coexistent SCC. Methods Original biopsy specimens and surgical specimens of patients retrieved from the pathology files were revised for the presence of LS, VIN and (early) invasive SCC, specifically focused on the two different types of VIN: differentiated and undifferentiated. Thereafter, VIN lesions were tested for the presence of HPV DNA. Results Twenty‐seven patients fulfilled the criteria for LS and VIN without SCC. In all 27 patients, LS was found to be related to undifferentiated VIN. Grading yielded the following results: VIN 1 (n = 10), VIN 2 (n = 11) and VIN 3 (n = 6). Additionally, VIN lesions from 26 patients could be tested for the presence of HPV DNA. HPV DNA, predominantly type 16, was present in 8 (31%) of them. Seven of these eight patients had VIN 2 or 3. During follow‐up, three patients progressed to (early) invasive carcinoma. In two of these patients, differentiated VIN was observed overlying early invasive SCC. Conclusions VIN related to LS without coexisting SCC is likely to be undifferentiated, in contrast to what was previously thought. HPV DNA was demonstrated in 31% of the lesions, and was strongly related to high‐grade VIN. PMID:16714399

Lymphoepithelial Carcinoma of the Nasolacrimal Duct: Clinical, Radiologic, and Immunopathologic Features.

PubMed

Jakobiec, Frederick A; Stagner, Anna M; Rubin, Peter A D

Undifferentiated lymphoepithelial carcinoma (exhibiting both begin lymphoid and malignant epithelial components) most commonly arises in the head and neck, especially in the nasopharynx. It may also be encountered in various ocular adnexal sites, including the nasolacrimal duct. A 63-year-old woman developed a swelling in the region of the right lacrimal sac accompanied by epiphora. CT scanning revealed an enlargement of the nasolacrimal duct from the lacrimal sac to the inferior nasal meatus. A biopsy during dacryocystorhinostomy for symptomatic epiphora revealed hypercellular sheets of small lymphocytes which were interpreted as evidence for a chronic dacryocystitis. Two years later the subtotally excised lesion had substantially grown in size. Repeat CT scans demonstrated an inferonasal anterior orbital mass with further enlargement of the nasolacrimal duct with a solid mass in its lumen, and bone erosion. The biopsy combined a rich background of lymphocytes within which were clusters of undifferentiated carcinoma cells that were cytokeratin and p63 positive. Critical review of the earlier biopsy led to the detection of the same cells, but in smaller numbers, that had been overlooked. An awareness of the possibility of lymphoepithelial carcinoma of the lacrimal sac/duct should improve diagnostic accuracy with the aid of immunohistochemistry. Radiation therapy is often successful in managing this highly sensitive malignant tumor.

Establishment and characterization of a human uterine endometrial undifferentiated carcinoma cell line, TMG-L.

PubMed

Hasegawa, Kiyoshi; Suzuki, Machiko; Ishikawa, Kunimi; Yasue, Akira; Kato, Rina; Nakamura, Azumi; Kuroki, Jun; Udagawa, Yasuhiro

2003-03-01

A new cell line of human uterine endometrial undifferentiated carcinoma, designated as TMG-L, was established from the metastatic lymph node of 56-year-old patient TMG-L cells have been cultured with Ham's F-12 medium supplemented with 10% FCS and grew as a loosely adherent monolayer with polygonal or spindle-shaped cells exhibiting poor cell-cell contact and piled up against each other, showing a tendency to grow as floating cells. The doubling time of this cell line was about 48 hours, and chromosomal analysis revealed aneuploidy at passage 25. The cells formed tumors in SCID mouse, the histology of which was similar to that of undifferentiated carcinoma component of primary tumor. TMG-L cells showed the loss of expression and membranous localization of either E-cadherin or alpha-catenin, implied corresponding loss of their adhesive function. And this dysfunction implicated the biological aggressive behavior of uterine endometrial undifferentiated carcinoma. This cell line appears to provide a useful system for studying uterine undifferentiated carcinoma in vivo and in vitro.

Carboplatin, Paclitaxel, Bevacizumab, and Veliparib in Treating Patients With Newly Diagnosed Stage II-IV Ovarian Epithelial, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-03-22

Fallopian Tube Carcinosarcoma; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Neoplasm; Fallopian Tube Transitional Cell Carcinoma; Ovarian Brenner Tumor; Ovarian Carcinosarcoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Seromucinous Tumor; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIA Fallopian Tube Cancer AJCC v6 and v7; Stage IIA Ovarian Cancer AJCC V6 and v7; Stage IIB Fallopian Tube Cancer AJCC v6 and v7; Stage IIB Ovarian Cancer AJCC v6 and v7; Stage IIC Fallopian Tube Cancer AJCC v6 and v7; Stage IIC Ovarian Cancer AJCC v6 and v7; Stage IIIA Fallopian Tube Cancer AJCC v7; Stage IIIA Ovarian Cancer AJCC v6 and v7; Stage IIIA Primary Peritoneal Cancer AJCC v7; Stage IIIB Fallopian Tube Cancer AJCC v7; Stage IIIB Ovarian Cancer AJCC v6 and v7; Stage IIIB Primary Peritoneal Cancer AJCC v7; Stage IIIC Fallopian Tube Cancer AJCC v7; Stage IIIC Ovarian Cancer AJCC v6 and v7; Stage IIIC Primary Peritoneal Cancer AJCC v7; Stage IV Fallopian Tube Cancer AJCC v6 and v7; Stage IV Ovarian Cancer AJCC v6 and v7; Stage IV Primary Peritoneal Cancer AJCC v7; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells.

PubMed

Muranaka, Shikibu; Fujita, Hirofumi; Fujiwara, Takuzo; Ogino, Tetsuya; Sato, Eisuke F; Akiyama, Jitsuo; Imada, Isuke; Inoue, Masayasu; Utsumi, Kozo

2005-01-01

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.

PubMed

Park, Dayoung; Brune, Kristin A; Mitra, Anupam; Marusina, Alina I; Maverakis, Emanual; Lebrilla, Carlito B

2015-11-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

PubMed

Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

2017-05-01

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

PubMed

Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

High prevalence of atypical hyperplasia in the endometrium of patients with epithelial ovarian cancer.

PubMed

Mingels, Marjanka J J M; Masadah, Rina; Geels, Yvette P; Otte-Höller, Irene; de Kievit, Ineke M; van der Laak, Jeroen A W M; van Ham, Maaike A P C; Bulten, Johan; Massuger, Leon F A G

2014-08-01

The aim of the present study is to determine the prevalence of endometrial premalignancies in women diagnosed with epithelial ovarian cancer (EOC). Endometrial and ovarian specimens of 186 patients with EOC were retrospectively selected using the nationwide pathology network and registry, and sections were comprehensively reviewed: 136 (73%) serous, 19 (10%) endometrioid, 15 (8%) mucinous, seven (4%) clear cell, and nine (5%) undifferentiated. Immunohistochemical phenotypes were compared for patients with serous EOC with concurrent endometrial pathology. In 31%, endometrial (pre)malignancy was found: carcinoma in 3%, endometrial intraepithelial carcinoma (EIC) in 4%, and atypical hyperplasia in 24%. Atypical hyperplasia was found in 47% of endometrioid EOCs but in 7% to 33% of other subtypes. Body mass index was higher concurrent to atypical hyperplasia (P=.001). Serous EOC and EIC immunophenotypes were comparable, whereas atypical hyperplasia was expressed differently. Apart from synchronous endometrial carcinoma, endometrial premalignancies should be taken into account when determining optimal treatment for women diagnosed with EOC. Copyright© by the American Society for Clinical Pathology.

p63 protein is essential for the embryonic development of vibrissae and teeth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rufini, Alessandro; Weil, Miguel; McKeon, Frank

2006-02-17

Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 inmore » the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression.« less

Fine-needle aspiration cytology of hemangiopericytoma: report of two cases.

PubMed

Sawh, R N; Lele, S M; Borkowski, J; Ventura, K C; Zaharopoulos, P; Logroño, R

2000-09-01

The fine-needle aspiration biopsy (FNAB) findings in two cases of hemangiopericytoma (HP), arising in the parotid gland and on the inner chest wall, respectively, are reported. Smear preparations in each case showed cytologic features of an undifferentiated spindle-cell neoplasm, whereas a core needle biopsy specimen of the chest wall mass showed a spindle-cell tumor with a "staghorn-like" arrangement of endothelium-lined vascular channels. Immunostains performed on this core biopsy, and on the surgical resection specimens in both cases, showed positive staining of tumor cells for vimentin and CD34, with negative staining for a variety of smooth muscle, epithelial, neural, and neuroendocrine markers. Electron microscopy performed in one case further supported the diagnosis of HP. With adequate sampling and appropriate use of ancillary studies, a diagnosis of HP can be reliably suggested on the basis of FNAB and core biopsy of a soft-tissue mass. Copyright 2000 Wiley-Liss, Inc.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Superficial EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion: a new variant of Ewing-like tumors with locoregional lymph node metastasis.

PubMed

Machado, Isidro; Cruz, Julia; Lavernia, Javier; Rubio, Luis; Campos, Jorge; Barrios, María; Grison, Camille; Chene, Virginie; Pierron, Gaelle; Delattre, Olivier; Llombart-Bosch, Antonio

2013-12-01

The present study describes a new case of EWSR1-negative undifferentiated sarcoma with CIC/DUX4 gene fusion. This case is similar to tumors described as primitive undifferentiated round cell sarcomas that occur mainly in the trunk and display an aggressive behavior. To our knowledge, this is the first report of such a tumor presenting locoregional lymph node metastasis. In view of previous studies that prove the existence of a particular variant of undifferentiated sarcoma with Ewing-like morphology and CIC/DUX-4 gene fusion, a search for this gene fusion in all undifferentiated round cell sarcomas should be considered if a conclusive diagnosis cannot be reached following other conventional studies. Although additional cases with more extensive follow-up studies are needed, we believe that EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion should be added to the list of new sarcoma variants with the possibility of lymph node metastasis.

Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.

PubMed

Tee, Jing Yang; Vaghjiani, Vijesh; Liu, Yu Han; Murthi, Padma; Chan, James; Manuelpillai, Ursula

2013-01-01

Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-β1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.

Drosophila Glypicans Regulate Follicle Stem Cell Maintenance and Niche Competition.

PubMed

Su, Tsu-Yi; Nakato, Eriko; Choi, Pui Yee; Nakato, Hiroshi

2018-04-09

Adult stem cells reside in specialized microenvironments, called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. Here, we show that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and FSC competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hyper-competitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNAi knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition. Copyright © 2018, Genetics.

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

PubMed Central

Tano, Keiko; Yasuda, Satoshi; Kuroda, Takuya; Saito, Hirohisa; Umezawa, Akihiro; Sato, Yoji

2014-01-01

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process. PMID:25347300

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

PubMed

El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

PubMed

Yoshioka, Hirotaka; Minamizaki, Tomoko; Yoshiko, Yuji

2015-11-01

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.

NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60.

PubMed

Dong, Jing-Mei; Zhao, Sheng-Guo; Huang, Guo-Yin; Liu, Qing

2004-06-01

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.

Involvement of CRF2 signaling in enterocyte differentiation

PubMed Central

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-01-01

AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases. PMID:28811708

Involvement of CRF2 signaling in enterocyte differentiation.

PubMed

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

PubMed

Kim, Mee-Hae; Matsubara, Yoshifumi; Fujinaga, Yukako; Kino-Oka, Masahiro

2018-02-01

Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thalidomide induces apoptosis in undifferentiated human induced pluripotent stem cells.

PubMed

Tachikawa, Saoko; Nishimura, Toshinobu; Nakauchi, Hiromitsu; Ohnuma, Kiyoshi

2017-10-01

Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium

PubMed Central

Faris, Andrea N.; Ganesan, Shyamala; Chattoraj, Asamanja; Chattoraj, Sangbrita S.; Comstock, Adam T.; Unger, Benjamin L.; Hershenson, Marc B.

2016-01-01

Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia. PMID:27119973

Vorinostat and Azacitidine in Treating Patients With Locally Recurrent or Metastatic Nasopharyngeal Cancer or Nasal Natural Killer T-Cell Lymphoma

ClinicalTrials.gov

2018-04-20

Adult Nasal Type Extranodal NK/T-Cell Lymphoma; Recurrent Nasopharyngeal Keratinizing Squamous Cell Carcinoma; Recurrent Nasopharyngeal Undifferentiated Carcinoma; Stage IV Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage IV Nasopharyngeal Undifferentiated Carcinoma AJCC v7

Altered expression of epithelial mesenchymal transition and pluripotent associated markers by sex steroid hormones in human embryonic stem cells.

PubMed

Jeon, So-Ye; Hwang, Kyung-A; Kim, Cho-Won; Jeung, Eui-Bae; Choi, Kyung-Chul

2017-07-01

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.

Phosphoproteomics profiling suggests a role for nuclear βΙPKC in transcription processes of undifferentiated murine embryonic stem cells.

PubMed

Costa-Junior, Helio Miranda; Garavello, Nicole Milaré; Duarte, Mariana Lemos; Berti, Denise Aparecida; Glaser, Talita; de Andrade, Alexander; Labate, Carlos A; Ferreira, André Teixeira da Silva; Perales, Jonas Enrique Aguilar; Xavier-Neto, José; Krieger, José Eduardo; Schechtman, Deborah

2010-12-03

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, βIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of βΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one βIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect βΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting βΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express βΙPKC and βΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that βIPKC takes part in the processes that maintain ESCs in their undifferentiated state.

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

PubMed

Jia, Dongya; Jolly, Mohit Kumar; Kulkarni, Prakash; Levine, Herbert

2017-06-22

Waddington's epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of "landscape" in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an "attractor" that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of "cancer attractors"-hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes "recanalization", i.e., the exit from "cancer attractors" and re-entry into "normal attractors", is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.

Nivolumab and Ipilimumab in Treating Patients With Rare Tumors

ClinicalTrials.gov

2018-06-27

Acinar Cell Carcinoma; Adenoid Cystic Carcinoma; Adrenal Cortex Carcinoma; Adrenal Gland Pheochromocytoma; Anal Canal Neuroendocrine Carcinoma; Anal Canal Undifferentiated Carcinoma; Appendix Mucinous Adenocarcinoma; Bartholin Gland Transitional Cell Carcinoma; Bladder Adenocarcinoma; Cervical Adenocarcinoma; Cholangiocarcinoma; Chordoma; Colorectal Squamous Cell Carcinoma; Desmoid-Type Fibromatosis; Endometrial Transitional Cell Carcinoma; Endometrioid Adenocarcinoma; Esophageal Neuroendocrine Carcinoma; Esophageal Undifferentiated Carcinoma; Extrahepatic Bile Duct Carcinoma; Fallopian Tube Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Fibromyxoid Tumor; Gastric Neuroendocrine Carcinoma; Gastric Squamous Cell Carcinoma; Gastrointestinal Stromal Tumor; Giant Cell Carcinoma; Intestinal Neuroendocrine Carcinoma; Intrahepatic Cholangiocarcinoma; Lung Carcinoid Tumor; Lung Sarcomatoid Carcinoma; Major Salivary Gland Carcinoma; Malignant Odontogenic Neoplasm; Malignant Peripheral Nerve Sheath Tumor; Malignant Testicular Sex Cord-Stromal Tumor; Metaplastic Breast Carcinoma; Metastatic Malignant Neoplasm of Unknown Primary Origin; Minimally Invasive Lung Adenocarcinoma; Mixed Mesodermal (Mullerian) Tumor; Mucinous Adenocarcinoma; Mucinous Cystadenocarcinoma; Nasal Cavity Adenocarcinoma; Nasal Cavity Carcinoma; Nasopharyngeal Carcinoma; Nasopharyngeal Papillary Adenocarcinoma; Nasopharyngeal Undifferentiated Carcinoma; Oral Cavity Carcinoma; Oropharyngeal Undifferentiated Carcinoma; Ovarian Adenocarcinoma; Ovarian Germ Cell Tumor; Ovarian Mucinous Adenocarcinoma; Ovarian Squamous Cell Carcinoma; Ovarian Transitional Cell Carcinoma; Pancreatic Acinar Cell Carcinoma; Pancreatic Neuroendocrine Carcinoma; Paraganglioma; Paranasal Sinus Adenocarcinoma; Paranasal Sinus Carcinoma; Parathyroid Gland Carcinoma; Pituitary Gland Carcinoma; Placental Choriocarcinoma; Placental-Site Gestational Trophoblastic Tumor; Primary Peritoneal High Grade Serous Adenocarcinoma; Pseudomyxoma Peritonei; Rare Disorder; Scrotal Squamous Cell Carcinoma; Seminal Vesicle Adenocarcinoma; Seminoma; Serous Cystadenocarcinoma; Small Intestinal Adenocarcinoma; Small Intestinal Squamous Cell Carcinoma; Spindle Cell Neoplasm; Squamous Cell Carcinoma of the Penis; Teratoma With Malignant Transformation; Testicular Non-Seminomatous Germ Cell Tumor; Thyroid Gland Carcinoma; Tracheal Carcinoma; Transitional Cell Carcinoma; Undifferentiated Gastric Carcinoma; Ureter Adenocarcinoma; Ureter Squamous Cell Carcinoma; Urethral Adenocarcinoma; Urethral Squamous Cell Carcinoma; Vaginal Adenocarcinoma; Vaginal Squamous Cell Carcinoma, Not Otherwise Specified; Vulvar Carcinoma

Blastema induction in aneurogenic state and Prrx-1 regulation by MMPs and FGFs in Ambystoma mexicanum limb regeneration.

PubMed

Satoh, Akira; makanae, Aki; Hirata, Ayako; Satou, Yutaka

2011-07-15

Urodele amphibians can regenerate amputated limbs. It has been considered that differentiated dermal tissues generate multipotent and undifferentiated cells called blastema cells during limb regeneration. In early phases of limb regeneration, blastema cells are induced by nerves and the apical epithelial cap (AEC). We had previously investigated the role of neurotrophic factors in blastema or blastema-like formation consisting of Prrx-1 positive cells. A new system suitable for investigating early phases of limb regeneration, called the accessory limb model (ALM), was recently developed. In this study, we performed a comparative transcriptome analysis between a blastema and wound using ALM. Matrix metalloproteinase (MMP) and fibroblast growth factor (FGF) signaling components were observed to be predominantly expressed in ALM blastema cells. Furthermore, we found that MMP activity induced a blastema marker gene, Prrx-1, in vitro, and FGF signaling pathways worked in coordination to maintain Prrx-1 expression and ALM blastema formation. Furthermore, we demonstrated that these two activities were sufficient to induce an ALM blastema in the absence of a nerve in vivo. Copyright © 2011 Elsevier Inc. All rights reserved.

Transgenic Expression of Interferon-γ in Mouse Stomach Leads to Inflammation, Metaplasia, and Dysplasia

PubMed Central

Syu, Li-Jyun; El-Zaatari, Mohamad; Eaton, Kathryn A.; Liu, Zhiping; Tetarbe, Manas; Keeley, Theresa M.; Pero, Joanna; Ferris, Jennifer; Wilbert, Dawn; Kaatz, Ashley; Zheng, Xinlei; Qiao, Xiotan; Grachtchouk, Marina; Gumucio, Deborah L.; Merchant, Juanita L.; Samuelson, Linda C.; Dlugosz, Andrzej A.

2013-01-01

Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H+/K+ ATPase β promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear β-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion. PMID:23036899

Cloning from stem cells: different lineages, different species, same story.

PubMed

Oback, Björn

2009-01-01

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Apoptosis and cell proliferation in the development of gastric carcinomas: associations with c-myc and p53 protein expression.

PubMed

Ishii, Hideaki H; Gobé, Glenda C; Pan, Wenshen; Yoneyama, Juichi; Ebihara, Yoshiro

2002-09-01

Patients with gastric carcinomas have a poor prognosis and low survival rates. The aim of the present paper was to characterize cellular and molecular properties to provide insight into aspects of tumor progression in early compared with advanced gastric cancers. One hundred and nine graded gastric carcinomas (early or advanced stage, undifferentiated or differentiated type) with paired non-cancer tissue were studied to define the correlation between apoptosis (morphology, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling), cell proliferation (Ki-67 expression, morphology) and expression and localization of two proteins frequently having altered expression in cancers, namely p53 and c-myc. Overall, apoptosis was lower in early stage, differentiated and undifferentiated gastric carcinomas compared with advanced-stage cancers. Cell proliferation was comparatively high in all stages. There was a high level of p53 positivity in all stages. Only the early- and advanced-stage undifferentiated cancers that were p53 positive had a significantly higher level of apoptosis (P < 0.05). Cell proliferation was significantly greater (P < 0.05) only in the early undifferentiated cancers that had either c-myc or p53-positivity. The results indicate that low apoptosis and high cell proliferation combine to drive gastric cancer development. The molecular controls for high cell proliferation of the early stage undifferentiated gastric cancers involve overexpression of both p53 and c-myc. Overexpression of p53 may also control cancer development in that its expression is associated with higher levels of apoptosis in early and late-stage undifferentiated, cancers. Copyright 2002 Blackwell Publishing Asia Pty Ltd

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

mTORC1 is essential for leukemia propagation but not stem cell self-renewal

PubMed Central

Hoshii, Takayuki; Tadokoro, Yuko; Naka, Kazuhito; Ooshio, Takako; Muraguchi, Teruyuki; Sugiyama, Naoyuki; Soga, Tomoyoshi; Araki, Kimi; Yamamura, Ken-ichi; Hirao, Atsushi

2012-01-01

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence. PMID:22622041

Stem cells in clinical trials for treatment of retinal degeneration.

PubMed

Klassen, Henry

2016-01-01

After decades of basic science research involving the testing of regenerative strategies in animal models of retinal degenerative diseases, a number of clinical trials are now underway, with additional trials set to begin shortly. These efforts will evaluate the safety and preliminary efficacy of cell-based products in the eyes of patients with a number of retinal conditions, notably including age-related macular degeneration, retinitis pigmentosa and Stargardt's disease. This review considers the scientific work and early trials with fetal cells and tissues that set the stage for the current clinical investigatory work, as well the trials themselves, specifically those either now completed, underway or close to initiation. The cells of interest include retinal pigment epithelial cells derived from embryonic stem or induced pluripotent stem cells, undifferentiated neural or retinal progenitors or cells from the vascular/bone marrow compartment or umbilical cord tissue. Degenerative diseases of the retina represent a popular target for emerging cell-based therapeutics and initial data from early stage clinical trials suggest that short-term safety objectives can be met in at least some cases. The question of efficacy will require additional time and testing to be adequately resolved.

MV-NIS or Investigator's Choice Chemotherapy in Treating Patients With Ovarian, Fallopian, or Peritoneal Cancer

ClinicalTrials.gov

2018-04-27

Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Clear Cell Tumor; Malignant Ovarian Endometrioid Tumor; Malignant Ovarian Serous Tumor; Ovarian Seromucinous Carcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Undifferentiated Pleomorphic Sarcoma after Pirfenidone Use: A Case Report

PubMed Central

Moore, Christine A; Kapila, Aaysha

2018-01-01

Introduction Pirfenidone was approved in 2014 for the treatment of idiopathic pulmonary fibrosis. Pirfenidone inhibits several factors such as tissue growth factor-β and platelet-derived growth factor, leading to decreased epithelial and fibroblast proliferation and collagen synthesis. The drug improves progression-free survival and is well tolerated, with minimal side effects. However, data on its long-term effects are lacking. Case Presentation We present a rare case in which an undifferentiated pleomorphic sarcoma developed in a 59-year-old man with idiopathic pulmonary fibrosis who was treated with pirfenidone for more than a year. Discussion Undifferentiated pleomorphic sarcoma, also known as malignant fibrous histiocytoma, is a soft-tissue sarcoma arising from fibroblasts. The disease presents in the extremities and the trunk of elderly patients, and rarely in the retroperitoneum. Surgical excision is the mainstay of treatment; however, recurrence is common in the form of lung and lymph node metastases. In this report we review this rare malignancy and highlight the need for postmarketing longitudinal studies to determine additional adverse effects in patients with idiopathic pulmonary fibrosis who are on pirfenidone therapy. PMID:29702057

Fine structure of the midgut epithelium in the millipede Telodeinopus aoutii (Myriapoda, Diplopoda) with special emphasis on epithelial regeneration.

PubMed

Rost-Roszkowska, M M; Kszuk-Jendrysik, M; Marchewka, A; Poprawa, I

2018-01-01

The midgut of millipedes is composed of a simple epithelium that rests on a basal lamina, which is surrounded by visceral muscles and hepatic cells. As the material for our studies, we chose Telodeinopus aoutii (Demange, 1971) (Kenyan millipede) (Diplopoda, Spirostreptida), which lives in the rain forests of Central Africa. This commonly reared species is easy to obtain from local breeders and easy to culture in the laboratory. During our studies, we used transmission and scanning electron microscopes and light and fluorescent microscopes. The midgut epithelium of the species examined here shares similarities to the structure of the millipedes analyzed to date. The midgut epithelium is composed of three types of cells-digestive, secretory, and regenerative cells. Evidence of three types of secretion have been observed in the midgut epithelium: merocrine, apocrine, and microapocrine secretion. The regenerative cells of the midgut epithelium in millipedes fulfill the role of midgut stem cells because of their main functions: self-renewal (the ability to divide mitotically and to maintain in an undifferentiated state) and potency (ability to differentiate into digestive cells). We also confirmed that spot desmosomes are common intercellular junctions between the regenerative and digestive cells in millipedes.

Bevacizumab, Cisplatin, Radiation Therapy, and Fluorouracil in Treating Patients With Stage IIB, Stage III, Stage IVA, or Stage IVB Nasopharyngeal Cancer

ClinicalTrials.gov

2018-01-04

Stage II Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage III Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage III Nasopharyngeal Undifferentiated Carcinoma AJCC v7; Stage IV Nasopharyngeal Keratinizing Squamous Cell Carcinoma AJCC v7; Stage IV Nasopharyngeal Undifferentiated Carcinoma AJCC v7

Rhabdoid and Undifferentiated Phenotype in Renal Cell Carcinoma: Analysis of 32 Cases Indicating a Distinctive Common Pathway of Dedifferentiation Frequently Associated With SWI/SNF Complex Deficiency.

PubMed

Agaimy, Abbas; Cheng, Liang; Egevad, Lars; Feyerabend, Bernd; Hes, Ondřej; Keck, Bastian; Pizzolitto, Stefano; Sioletic, Stefano; Wullich, Bernd; Hartmann, Arndt

2017-02-01

Undifferentiated (anaplastic) and rhabdoid cell features are increasingly recognized as adverse prognostic findings in renal cell carcinoma (RCC), but their molecular pathogenesis has not been studied sufficiently. Recent studies identified alterations in the Switch Sucrose nonfermentable (SWI/SNF) chromatin remodeling complex as molecular mechanisms underlying dedifferentiation and rhabdoid features in carcinomas of different organs. We herein have analyzed 32 undifferentiated RCCs having in common an undifferentiated (anaplastic) phenotype, prominent rhabdoid features, or both, irrespective of the presence or absence of conventional RCC component. Cases were stained with 6 SWI/SNF pathway members (SMARCB1, SMARCA2, SMARCA4, ARID1A, SMARCC1, and SMARCC2) in addition to conventional RCC markers. Patients were 20 males and 12 females aged 32 to 85 years (mean, 59). A total of 22/27 patients with known stage presented with ≥pT3. A differentiated component varying from microscopic to major component was detected in 20/32 cases (16 clear cell and 2 cases each chromophobe and papillary RCC). The undifferentiated component varied from rhabdoid dyscohesive cells to large epithelioid to small monotonous anaplastic cells. Variable loss of at least 1 SWI/SNF complex subunit was noted in the undifferentiated/rhabdoid component of 21/32 cases (65%) compared with intact or reduced expression in the differentiated component. A total of 15/17 patients (88%) with follow-up died of metastatic disease (mostly within 1 y). Only 2 patients were disease free at last follow-up (1 and 6 y). No difference in survival, age distribution, or sex was observed between the SWI/SNF-deficient and the SWI/SNF-intact group. This is the first study exploring the role of SWI/SNF deficiency as a potential mechanism underlying undifferentiated and rhabdoid phenotype in RCC. Our results highlight the association between the aggressive rhabdoid phenotype and the SWI/SNF complex deficiency, consistent with studies on similar neoplasms in other organs. Thorough sampling of such tumors that are usually huge and locally advanced is necessary for recognizing the clone of origin and hence for proper subtyping and also for differentiating them from undifferentiated urothelial carcinoma.

Cellular Plasticity-Targeted Therapy in Head and Neck Cancers.

PubMed

Shang, W; Zhang, Q; Huang, Y; Shanti, R; Alawi, F; Le, A; Jiang, C

2018-06-01

Head and neck cancer is one of the most frequent human malignancies worldwide, with a high rate of recurrence and metastasis. Head and neck squamous cell carcinoma (HNSCC) is cellularly and molecularly heterogeneous, with subsets of undifferentiated cancer cells exhibiting stem cell-like properties, called cancer stem cells (CSCs). Epithelial-mesenchymal transition, gene mutation, and epigenetic modification are associated with the formation of cellular plasticity of tumor cells in HNSCC, contributing to the acquisition of invasive, recurrent, and metastatic properties and therapeutic resistance. Tumor microenvironment (TME) plays a supportive role in the initiation, progression, and metastasis of head and neck cancer. Stromal fibroblasts, vasculature, immune cells, cytokines, and hypoxia constitute the main components of TME in HNSCC, which contributes not only to the acquisition of CSC properties but also to the recurrence and therapeutic resistance of the malignancies. In this review, we discuss the potential mechanisms underlying the development of cellular plasticity, especially the emergence of CSCs, in HNSCC. We also highlight recent studies implicating the complex interplays among TME components, plastic CSCs, tumorigenesis, recurrence, and therapeutic resistance of HNSCC. Finally, we summarize the treatment modalities of HNSCC and reinforce the novel concept of therapeutic targeting CSCs in HNSCC.

Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells.

PubMed

Singer, Bernhard B; Scheffrahn, Inka; Kammerer, Robert; Suttorp, Norbert; Ergun, Suleyman; Slevogt, Hortense

2010-01-18

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

Undifferentiated Neuroblastoma Cells Are More Sensitive to Photogenerated Oxidative Stress Than Differentiated Cells.

PubMed

Lee, Chu-I; Perng, Jing-Huei; Chen, Huang-Yo; Hong, Yi-Ren; Wang, Jyh-Jye

2015-09-01

Neuroblastoma is one of the most aggressive cancers and has a complex form of differentiation. We hypothesized that advanced cellular differentiation may alter the susceptibility of neuroblastoma to photodynamic treatment (PDT) and confer selective survival advantage. We demonstrated that hematoporphyrin uptake by undifferentiated SH-SY5Y cells was lower than that of differentiated counterparts, yet the former were more susceptible to PDT-induced oxidative stress killing. Photogenerated reactive oxygen species (ROS) in undifferentiated cells efficiently stimulated cell cycle arrest at G2/M phase, mitochondrial apoptotic pathway activation, the sustained phosphorylation of Akt/GSK-3β and ERK. Differentiated cells with more resistance to PDT exhibited a ROS-independent and a prolonged activation of ERK. Both SH-SY5Y cells exposed to PDT exhibited ROS-independent p38 and JNK activation. These results may have important implications for neuroblastoma patients undergoing photodynamic therapy. © 2015 Wiley Periodicals, Inc.

Nestin is expressed in HMB-45 negative melanoma cells in dermal parts of nodular melanoma.

PubMed

Kanoh, Maho; Amoh, Yasuyuki; Tanabe, Kenichi; Maejima, Hideki; Takasu, Hiroshi; Katsuoka, Kensei

2010-06-01

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells can differentiate into neurons, glia, keratocytes, smooth muscle cells and melanocytes in vitro. These pluripotent nestin-expressing stem cells are keratin 15 (K15)-negative, suggesting that they are in a relatively undifferentiated state. Recent studies suggest that the epithelial stem cells are important in tumorigenesis, and nestin expression is thought to be important in tumorigenesis. In the present study, we examined the expression of the hair follicle and neural stem cell marker nestin, as well as S-100 and HMB-45, in melanoma. Nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in all five cases of amelanotic nodular melanomas. Moreover, nestin immunoreactivity was observed in the dermal parts in seven of 10 cases of melanotic nodular melanomas. Especially, nestin immunoreactivity was observed in the HMB-45-negative melanoma cells in the dermal parts of all 10 cases of HMB-45-negative amelanotic and melanotic nodular melanomas. On the other hand, nestin expression was negative in 10 of 12 cases of superficial spreading melanoma. These results suggest that nestin is an important marker of HMB-45-negative melanoma cells in the dermal parts of patients with nodular melanoma.

Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

PubMed

Kim, Hwa-Young; Baek, Song; Han, Na Rae; Lee, Eunsong; Park, Choon-Keun; Lee, Seung Tae

2018-05-29

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α 3 , α 5 , α 6 , α 9 , α V , and β 1 subunits, but not the α 1 , α 2 , α 4 , α 7 , and α 8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C and vitronectin were observed and functional blocking of integrin heterodimer α 5 β 1 , α 9 β 1 , or α V β 1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α 6 β 1 heterodimer?mediated adhesion to laminin was detected. These results demonstrate that active α 5 β 1 , α 9 β 1 , and α V β 1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α 3 (presumed) and α 6 subunits. This article is protected by copyright. All rights reserved.

Oxygenated hemoglobin diffuse reflectance ratio for in vitro detection of human gastric pre-cancer

NASA Astrophysics Data System (ADS)

Li, L. Q.; Wei, H. J.; Guo, Z. Y.; Yang, H. Q.; Wu, G. Y.; Xie, S. S.; Zhong, H. Q.; Li, X. Y.; Zhao, Q. L.; Guo, X.

2010-07-01

Oxygenated hemoglobin diffuse reflectance (DR) ratio (R540/R575) method based on DR spectral signatures is used for early diagnosis of malignant lesions of human gastric epithelial tissues in vitro. The DR spectra for four different kinds of gastric epithelial tissues were measured using a spectrometer with an integrating sphere detector in the spectral range from 400 to 650 nm. The results of measurement showed that the average DR spectral intensity for the epithelial tissues of normal stomach is higher than that for the epithelial tissues of chronic and malignant stomach and that for the epithelial tissues of chronic gastric ulcer is higher than that for the epithelial tissues of malignant stomach. The average DR spectra for four different kinds of gastric epithelial tissues show dips at 542 and 577 nm owing to absorption from oxygenated Hemoglobin (HbO2). The differences in the mean R540/R575 ratios of HbO2 bands are 6.84% between the epithelial tissues of normal stomach and chronic gastric ulcer, 14.7% between the epithelial tissues of normal stomach and poorly differentiated gastric adenocarcinoma and 22.6% between the epithelial tissues of normal stomach and undifferentiated gastric adenocarcinoma. It is evident from results that there were significant differences in the mean R540/R575 ratios of HbO2 bands for four different kinds of gastric epithelial tissues in vitro ( P < 0.01).

Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination.

PubMed

Merchant-Larios, H; Díaz-Hernández, V; Marmolejo-Valencia, A

2010-01-01

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination. (c) 2010 S. Karger AG, Basel.

The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells.

PubMed

Ogier-Denis, E; Codogno, P; Chantret, I; Trugnan, G

1988-05-05

Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.

Fine needle aspiration in the diagnosis and classification of hepatoblastoma: Analysis of 21 New Cases.

PubMed

Vlajnic, Tatjana; Brisse, Hervé J; Aerts, Isabelle; Fréneaux, Paul; Cellier, Cécile; Fabre, Monique; Klijanienko, Jerzy

2017-02-01

Diagnosis of hepatoblastoma (HBL) is based on characteristic clinical and radiological presentation, young age and marked elevation of serum α-fetoprotein (aFP). Fine needle aspiration (FNA) technique is successfully used in the diagnosis of hepatoblastoma. To evaluate the value of FNA in the diagnosis and subtyping of HBL, we report our experience correlated to histological sections (core needle biopsy, CNB). From 1991 to 2015, 21 cases from 20 patients were cytologically diagnosed as HBL. The patients were 15 males and 5 females, mean age being 3 years, and median age being 2 years and 4 months. Serum aFP levels ranged from negative to 1,285,000 ng/ml. We defined cytological criteria to diagnose fetal, embryonal, mesenchymal, and small cell undifferentiated components. The accurate cytological diagnosis of HBL was made in all cases; 8 cases exhibited a single component and 13 cases exhibited two or more components. Fetal and embryonal components were seen in 18 and 13 cases, respectively, and small cell undifferentiated component was seen in one case. Mesenchymal component was seen in 12 cases. Comparing cytology and histology, identical components were identified on both, FNA and CNB in 14 cases. When analyzing only the presence of epithelial components, 17 cases were concordant in both techniques. FNA allows to accurately diagnose HBL and recognize its histological subtypes. On the basis of high concordance between cytological and histological diagnosis, FNA is validated as an alternative diagnostic method to CNB. Diagn. Cytopathol. 2017;45:91-100. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

PubMed

Imran, Saima; Ferretti, Patrizia; Vrzal, Radim

2015-01-01

Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

MiR-205 and MiR-373 Are Associated with Aggressive Human Mucinous Colorectal Cancer.

PubMed

Eyking, Annette; Reis, Henning; Frank, Magdalena; Gerken, Guido; Schmid, Kurt W; Cario, Elke

2016-01-01

Mucinous adenocarcinoma (MAC) represents a distinct histopathological entity of colorectal cancer (CRC), which is associated with disease progression and poor prognosis. Here, we found that expression levels of miR-205 and miR-373 were specifically upregulated only in patients with mucinous colon cancers, but not in CRC that lack mucinous components. To investigate the effects of miR-205 and miR-373 on intestinal epithelial cell (IEC) biology by gain- and loss-of-function experiments in a proof-of-concept approach, we chose previously established in-vitro human Caco-2-based models of differentiated, non-invasive (expressing TLR4 wild-type; termed Caco-2[WT]) versus undifferentiated, invasive (expressing TLR4 mutant D299G; termed Caco-2[D299G]) IEC. Enterocyte-like Caco-2[WT] showed low levels of miR-205 and miR-373 expression, while both miRNAs were significantly upregulated in colorectal carcinoma-like Caco-2[D299G], thus resembling the miRNA expression pattern of paired normal versus tumor samples from MAC patients. Using stable transfection, we generated miR-205- or miR-373-expressing and miR-205- or miR-373-inhibiting subclones of these IEC lines. We found that introduction of miR-205 into Caco-2[WT] led to expansion of mucus-secreting goblet cell-like cells, which was associated with induction of KLF4, MUC2 and TGFβ1 expression. Activation of miR-205 in Caco-2[WT] induced chemoresistance, while inhibition of miR-205 in Caco-2[D299G] promoted chemosensitivity. Caco-2[WT] overexpressing miR-373 showed mitotic abnormalities and underwent morphologic changes (loss of epithelial polarity, cytoskeletal reorganization, and junctional disruption) associated with epithelial-mesenchymal transition and progression to inflammation-associated colonic carcinoma, which correlated with induction of phosphorylated STAT3 and N-CADHERIN expression. Functionally, introduction of miR-373 into Caco-2[WT] mediated loss of cell-cell adhesion and increased proliferation and invasion. Reversely, inhibition of miR-373 allowed mesenchymal IEC to regain epithelial properties, which correlated with absence of neoplastic progression. Using xenografts in mice demonstrated miR-373-mediated acceleration of malignant intestinal tumor growth. In conclusion, our results provide first evidence that miR-205 and miR-373 may differentially contribute to the aggressive phenotype of MAC in CRC.

Olaparib or Cediranib Maleate and Olaparib Compared With Standard Platinum-Based Chemotherapy in Treating Patients With Recurrent Platinum-Sensitive Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-11

BRCA Rearrangement; Deleterious BRCA1 Gene Mutation; Deleterious BRCA2 Gene Mutation; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Tumor; Ovarian Seromucinous Carcinoma; Ovarian Serous Tumor; Ovarian Transitional Cell Carcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Differential expression of syndecan isoforms during mouse incisor amelogenesis.

PubMed

Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

2007-08-01

Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

[Undifferentiated blastic cell crisis of chronic myelogenous leukemia with myeloblastic tumor in the skin].

PubMed

Kawakami, K; Kiyosaki, M; Amaya, H; Nakamaki, T; Hino, K; Tomoyasu, S

2000-04-01

A 54-year-old female, who had been treated for 4 years in the chronic phase of chronic myelogenous leukemia (CML) was admitted for management of a CML blastic crisis. Blast cells showed strong positive expression of CD7 and HLA-DR, and weakly expressed CD2, CD5 and CD10, as well. The cells were peroxidase negative in peripheral blood and bone marrow. An undifferentiated blastic crisis was diagnosed and she was treated with Interferon-alpha and VP(vincristine 2 mg/week; prednisolone 30 mg/day). A 5-7 mm in diameter tumor in the skin of the anterior right chest appeared one week after VP therapy. The tumor consisted of blasts which were CD13, CD33 and peroxidase positive, unlike the peripheral undifferentiated blasts. This is a rare case of mixed blast crisis with an increase in undifferentiated blasts in peripheral blood and bone marrow, and myeloblastic tumor formation in the skin.

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

PubMed

Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

1992-12-01

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development

PubMed Central

Chen, Liang-Yu; Willis, William D.; Eddy, Edward M.

2016-01-01

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo. PMID:26831079

Isolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis).

PubMed

Huang, Ben; Xie, Ti-San; Shi, De-Shun; Li, Tong; Wang, Xiao-Li; Mo, Yi; Wang, Zhi-Qiang; Li, Meng-Mei

2007-10-01

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.

gone early, a Novel Germline Factor, Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary

PubMed Central

Matsuoka, Shinya; Gupta, Swati; Suzuki, Emiko; Hiromi, Yasushi; Asaoka, Miho

2014-01-01

In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment. PMID:25420147

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory

PubMed Central

Kulkarni, Prakash; Levine, Herbert

2017-01-01

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways. PMID:28640191

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

PubMed Central

Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

2011-01-01

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

In situ label-free quantification of human pluripotent stem cells with electrochemical potential.

PubMed

Yea, Cheol-Heon; Jeong, Ho-Chang; Moon, Sung-Hwan; Lee, Mi-Ok; Kim, Kyeong-Jun; Choi, Jeong-Woo; Cha, Hyuk-Jin

2016-01-01

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein, we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs, their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs, which can assess the risk of teratoma formation efficiently and economically. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of Tissue Factor in Epithelial Ovarian Carcinoma Is Involved in the Development of Venous Thromboembolism.

PubMed

Sakurai, Manabu; Matsumoto, Koji; Gosho, Masahiko; Sakata, Akiko; Hosokawa, Yoshihiko; Tenjimbayashi, Yuri; Katoh, Takashi; Shikama, Ayumi; Komiya, Haruna; Michikami, Hiroo; Tasaka, Nobutaka; Akiyama-Abe, Azusa; Nakao, Sari; Ochi, Hiroyuki; Onuki, Mamiko; Minaguchi, Takeo; Yoshikawa, Hiroyuki; Satoh, Toyomi

2017-01-01

Our 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor (TF) in the development of venous thromboembolism (VTE) before treatment, especially in clear cell carcinoma (CCC). This follow-up study further investigated this possibility in a larger cohort. We investigated the intensity of TF expression (ITFE) and other variables for associations with VTE using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between November 2004 and December 2010, none of whom had received neoadjuvant chemotherapy. Before starting treatment, all patients were ultrasonographically screened for VTE. The ITFE was graded based on immunostaining of surgical specimens. Histological types were serous carcinoma (n = 42), CCC (n = 12), endometrioid carcinoma (n = 15), mucinous carcinoma (n = 53), and undifferentiated carcinoma (n = 6). The prevalence of VTE was significantly higher in CCC (34%) than in non-CCC (17%, P = 0.03). As ITFE increased, the frequencies of CCC and VTE increased significantly (P < 0.001 and P = 0.014, respectively). Multivariate analysis identified TF expression and pretreatment dimerized plasmin fragment D level as significant independent risk factors for VTE development. These factors showed particularly strong impacts on advanced-stage disease (P = 0.021). The 2007 cohort was small, preventing multivariate analysis. This study of a larger cohort yielded stronger evidence that the development of VTE in epithelial ovarian cancer may involve TF expression in cancer tissues.

Chronic idiopathic myelofibrosis terminating in extramedullary anaplastic plasmacytoma.

PubMed

Liu, Min-Ling; Kallakury, Bhaskar; Kessler, Craig; Hartmann, Dan-Paul; Azumi, Norio; Ozdemirli, Metin

2006-02-01

Chronic idiopathic myelofibrosis (CIMF) is a chronic myeloproliferative disorder (CMPD) with progressive fibrosis and extramedullary hematopoiesis. Similar to other CMPDs, the stem cell in CIMF has the potential to differentiate into myeloid or lymphoid lineages, and thus CIMF can culminate in acute leukemia of myeloid or, rarely, lymphoid lineage. We describe an unusual case of CIMF terminating in extramedullary anaplastic plasmacytoma. The patient was a 61-year-old male with an 11-year history of CIMF. His course was complicated by rapidly growing abdominal and inguinal lymphadenopathy. Lymph node biopsy revealed a diffuse undifferentiated infiltrate in the background of extramedullary hematopoiesis. Flow cytometric and immunohistochemical analysis demonstrated plasma cell-related antigens (CD138, CD38, cytoplasmic kappa light chain), epithelial membrane antigen and CD43 in the tumor cells. The myeloid, B-cell or T-cell markers were negative. A clonal immunoglobulin heavy chain gene rearrangement was identified by polymerase chain reaction. The plasma cell origin was further confirmed by electron microscopic examination, which revealed stacks of rough endoplasmic reticulum. Monoclonal gammopathy may occur in CIMF, and rare cases of simultaneous plasma cell myeloma and CIMF have been reported in the literature. However, to the best of our knowledge, this is the first report of CIMF terminating in extramedullary anaplastic plasmacytoma.

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells.

PubMed

Park, Jongjin; Son, Yeonsung; Lee, Na Geum; Lee, Kyungmin; Lee, Dong Gwang; Song, Jinhoi; Lee, Jaemin; Kim, Seokho; Cho, Min Ji; Jang, Ju-Hong; Lee, Jangwook; Park, Jong-Gil; Kim, Yeon-Gu; Kim, Jang-Seong; Lee, Jungwoon; Cho, Yee Sook; Park, Young-Jun; Han, Baek Soo; Bae, Kwang-Hee; Han, Seungmin; Kang, Byunghoon; Haam, Seungjoo; Lee, Sang-Hyun; Lee, Sang Chul; Min, Jeong-Ki

2018-06-08

Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of β-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Acute erythroblastic leukemia presenting as acute undifferentiated leukemia: a report of two cases with ultrastructural features.

PubMed

Reiffers, J; Bernard, P; Larrue, J; Dachary, D; David, B; Boisseau, M; Broustet, A

1985-01-01

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

Cediranib Maleate and Olaparib or Standard Chemotherapy in Treating Patients With Recurrent Platinum-Resistant or -Refractory Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-15

Deleterious BRCA1 Gene Mutation; Deleterious BRCA2 Gene Mutation; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Trisomy 4 in a case of acute undifferentiated myeloblastic leukemia with hand-mirror cells.

PubMed

Kao, Y S; McCormick, C; Vial, R

1990-04-01

A case of acute undifferentiated myelocytic leukemic with trisomy 4 is described. The patient is a 61-year-old woman who developed leukemia 4 1/2 years after receiving radiation therapy for uterine carcinoma. Many leukemic cells exhibited hand-mirror configuration after the bone marrow aspirate was left at room temperature overnight. The relationship between trisomy 4 and hand-mirror cells in acute myelocytic leukemia is unknown.

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

PubMed

Tsao, C J; Cheng, T Y; Chang, S L; Su, W J; Tseng, J Y

1992-05-01

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Pembrolizumab, Bevacizumab, and Cyclophosphamide in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-05-03

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.

PubMed

Özdemir, Berna C; Pentcheva-Hoang, Tsvetelina; Carstens, Julienne L; Zheng, Xiaofeng; Wu, Chia-Chin; Simpson, Tyler R; Laklai, Hanane; Sugimoto, Hikaru; Kahlert, Christoph; Novitskiy, Sergey V; De Jesus-Acosta, Ana; Sharma, Padmanee; Heidari, Pedram; Mahmood, Umar; Chin, Lynda; Moses, Harold L; Weaver, Valerie M; Maitra, Anirban; Allison, James P; LeBleu, Valerie S; Kalluri, Raghu

2014-06-16

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC. Copyright © 2014 Elsevier Inc. All rights reserved.

Behavior of Xeno-Transplanted Undifferentiated Human Induced Pluripotent Stem Cells Is Impacted by Microenvironment Without Evidence of Tumors.

PubMed

Martínez-Cerdeño, Veronica; Barrilleaux, Bonnie L; McDonough, Ashley; Ariza, Jeanelle; Yuen, Benjamin T K; Somanath, Priyanka; Le, Catherine T; Steward, Craig; Horton-Sparks, Kayla; Knoepfler, Paul S

2017-10-01

Human pluripotent stem cells (hPSC) have great clinical potential through the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. In this study, we sought to measure how undifferentiated hPSC behave following xenotransplant. We directly transplanted undifferentiated human induced pluripotent stem cells (hIPSC) and human embryonic stem cells (hESC) into the adult mouse brain ventricle and analyzed their fates. No tumors or precancerous lesions were present at more than one year after transplantation. This result differed with the tumorigenic capacity we observed after allotransplantation of mouse ESC into the mouse brain. A substantial population of cellular derivatives of undifferentiated hESC and hIPSC engrafted, survived, and migrated within the mouse brain parenchyma. Within brain structures, transplanted cell distribution followed a very specific pattern, suggesting the existence of distinct microenvironments that offer different degrees of permissibility for engraftment. Most of the transplanted hESC and hIPSC that developed into brain cells were NeuN+ neuronal cells, and no astrocytes were detected. Substantial cell and nuclear fusion occurred between host and transplanted cells, a phenomenon influenced by microenvironment. Overall, hIPSC appear to be largely functionally equivalent to hESC in vivo. Altogether, these data bring new insights into the behavior of stem cells without prior differentiation following xenotransplantation into the adult brain.

Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas

PubMed Central

Moreno-Bueno, Gema; Salvador, Fernando; Martín, Alberto; Floristán, Alfredo; Cuevas, Eva P; Santos, Vanesa; Montes, Amalia; Morales, Saleta; Castilla, Maria Angeles; Rojo-Sebastián, Alejandro; Martínez, Alejandra; Hardisson, David; Csiszar, Katalin; Portillo, Francisco; Peinado, Héctor; Palacios, José; Cano, Amparo

2011-01-01

Basal-like breast carcinoma is characterized by the expression of basal/myoepithelial markers, undifferentiated phenotype, highly aggressive behaviour and frequent triple negative status (ESR−, PR−, Her2neu−). We have previously shown that epithelial–mesenchymal transition (EMT) occurs in basal-like breast tumours and identified Lysyl-oxidase-like 2 (LOXL2) as an EMT player and poor prognosis marker in squamous cell carcinomas. We now show that LOXL2 mRNA is overexpressed in basal-like human breast carcinomas. Breast carcinoma cell lines with basal-like phenotype show a specific cytoplasmic/perinuclear LOXL2 expression, and this subcellular distribution is significantly associated with distant metastatic incidence in basal-like breast carcinomas. LOXL2 silencing in basal-like carcinoma cells induces a mesenchymal-epithelial transition (MET) associated with a decrease of tumourigenicity and suppression of metastatic potential. Mechanistic studies indicate that LOXL2 maintains the mesenchymal phenotype of basal-like carcinoma cells by a novel mechanism involving transcriptional downregulation of Lgl2 and claudin1 and disorganization of cell polarity and tight junction complexes. Therefore, intracellular LOXL2 is a new candidate marker of basal-like carcinomas and a target to block metastatic dissemination of this aggressive breast tumour subtype. PMID:21732535

Primary cilia in gastric Gastrointestinal Stromal Tumours (GISTs): an ultrastructural study

PubMed Central

Castiella, Tomás; Muñoz, Guillermo; Luesma, María José; Santander, Sonia; Soriano, Mario; Junquera, Concepción

2013-01-01

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal (non-epithelial) neoplasms of the human gastrointestinal (GI) tract. They are thought to derive from interstitial cells of Cajal (ICCs) or an ICC progenitor based on immunophenotypical and ultrastructural similarities. Because ICCs show primary cilium, our hypothesis is based on the possibility that some of these neoplastic cells could also present it. To determine this, an exhaustive ultrastructural study has been developed on four gastric GISTs. Previous studies had demonstrated considerable variability in tumour cells with two dominating phenotypes, spindly and epithelioid. In addition to these two types, we have found another cell type reminiscent of adult ICCs with a voluminous nucleus surrounded by narrow perinuclear cytoplasm with long slender cytoplasmic processes. We have also noted the presence of small undifferentiated cells. In this study, we report for the first time the presence of primary cilia (PCs) in spindle and epithelioid tumour cells, an ultrastructural feature we consider of special interest that has hitherto been ignored in the literature dealing with the ultrastructure of GISTs. We also point out the frequent occurrence of multivesicular bodies (MVBs). The ultrastructural findings described in gastric GISTs in this study appear to be relevant considering the critical roles played by PCs and MVBs recently demonstrated in tumourigenic processes. PMID:23672577

The ultrastructure of the sweat glands of the ox, sheep and goat during sweating and recovery.

PubMed Central

Jenkinson, D M; Montgomery, I; Elder, H Y

1979-01-01

The ultrastructure of the sweat glands of cattle, sheep and goats was studied before, during, and after, exposure of the animals to controlled warm environments. In cattle, sweating induced little ultrastructural change in the gland, although fluid-filled spaces appeared between the myo- and secretory epithelial layers. The mechanism appears to be one of fluid transport and exocytosis of secretory vesicles, which in this species seem to be derived from the Golgi apparatus and/or mitochondria. The glands of the sheep and goat also displayed signs of vesicle exocytosis and of fluid transport during sweating. The sweating 'fatigue' in these species was apparently due to failure of the secretory cells, some of which ruptured and were extruded into the lumen. The evidence during subsequent recovery indicates that neighbouring cells spread to make contact, encase remnants of atretic cells between them and the underlying myoepithelium, and engulf them. Sweat in these species appears to be formed (a) by secretion and (b) from cells which can no longer meet the demands of stimulation. The role in sweating of cell replacement, and of undifferentiated cells found between the myo- and secretory epithelia, is discussed. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:511758

Effect of Storage Temperature on the Phenotype of Cultured Epidermal Cells Stored in Xenobiotic-Free Medium.

PubMed

Jackson, Catherine; Eidet, Jon R; Reppe, Sjur; Aass, Hans Christian D; Tønseth, Kim A; Roald, Borghild; Lyberg, Torstein; Utheim, Tor P

2016-06-01

Cultured epidermal cell sheets (CECS) are used in the treatment of large area burns to the body and have potential to treat limbal stem cell deficiency (LSCD) as shown in animal studies. Despite widespread use, storage options for CECS are limited. Short-term storage allows flexibility in scheduling surgery, quality control and improved transportation to clinics worldwide. Recent evidence points to the phenotype of cultured epithelial cells as a critical predictor of post-operative success following transplantation of CECS in burns and in transplantation of cultured epithelial cells in patients with LSCD. This study, therefore assessed the effect of a range of temperatures, spanning 4-37 °C, on the phenotype of CECS stored over a 2-week period in a xenobiotic-free system. Progenitor cell (p63, ΔNp63α and ABCG2) and differentiation (C/EBPδ and CK10) associated marker expression was assessed using immunocytochemistry. Immunohistochemistry staining of normal skin for the markers p63, ABCG2 and C/EBPδ was also carried out. Assessment of progenitor cell side population (SP) was performed using JC1 dye by flow cytometry. P63 expression remained relatively constant throughout the temperature range but was significantly lower compared to control between 20 and 28 °C (p < 0.05). High C/EBPδ together with low p63 suggested more differentiation beginning at 20 °C and above. Lower CK10 and C/EBPδ expression most similar to control was seen at 12 °C. The percentage of ABCG2 positive cells was most similar to control between 8 and 24 °C. Between 4 and 24 °C, the SP fluctuated, but was not significantly different compared to control. Results were supported by staining patterns indicating differentiation status associated with markers in normal skin sections. Lower storage temperatures, and in particular 12 °C, merit further investigation as optimal storage temperature for maintenance of undifferentiated phenotype in CECS.

Pegylated Liposomal Doxorubicin Hydrochloride With Atezolizumab and/or Bevacizumab in Treating Patients With Recurrent Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-20

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; High Grade Fallopian Tube Serous Adenocarcinoma; High Grade Ovarian Serous Adenocarcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Primary Peritoneal High Grade Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Ultrastructural study on the embryonic development of the orthokeratinized epithelium and its cornified layer (lingual nail) on the ventral surface of the lingual apex in the domestic duck (Anas platyrhynchos f. domestica).

PubMed

Skieresz-Szewczyk, Kinga; Jackowiak, Hanna; Ratajczak, Marlena

2018-02-01

The lingual nail as the cornified layer of the orthokeratinized epithelium in birds is responsible for the collection of solid food by pecking. The aim of the present study is to determine the manner of orthokeratinized epithelium development and assess the degree of readiness of the epithelium to fulfill its mechanical function at hatching. Three developmental phases are distinguished, i.e. embryonic, transformation and pre-hatching stage. In the embryonic stage lasting until day 13 of incubation the epithelium is composed of several layers of undifferentiated cells. During the transformation stage, from day 14 to 20 of incubation, the epithelium becomes differentiated to form three layers. A characteristic feature is the formation of osmophilic granules in the superficial layer, referred to as periderm granules. Until the pre-hatching stage the fibrous cytoskeleton of epithelial cells and an impermeable epithelial barrier are gradually developed. In the pre-hatching stage, a cornified lingual nail is formed, while the periderm is exfoliated. At hatching the orthokeratinized epithelium and lingual nail are fully developed and ready to perform feeding activities. The presence of periderm, similarly as in the epidermis, indicates the ectodermal derivation of the oral cavity epithelium. Moreover, occurrence of osmophilic granules may be considered as evidence for the phylogenetic affinity of birds and reptiles. Copyright © 2018 Elsevier GmbH. All rights reserved.

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

PubMed

Wang, Kui; Kievit, Forrest M; Erickson, Ariane E; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

2016-12-01

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

PubMed

Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

2014-07-04

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells

PubMed Central

Guarnerio, Jlenia; Riccardi, Luisa; Taulli, Riccardo; Maeda, Takahiro; Wang, Guocan; Hobbs, Robin M.; Song, Min Sup; Sportoletti, Paolo; Bernardi, Rosa; Bronson, Roderick T.; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Lunardi, Andrea; Pandolfi, Pier Paolo

2015-01-01

The regulatory factors governing adult mesenchymal stem cells (MSCs) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal form of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSCs transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF-DLK1-SOX9 pathway in the pathogenesis of undifferentiated sarcoma with important therapeutic implications. PMID:25614485

The Role of Integrin α6 (CD49f) in Stem Cells: More than a Conserved Biomarker.

PubMed

Krebsbach, Paul H; Villa-Diaz, Luis G

2017-08-01

Stem cells have the capacity for self-renewal and differentiation into specialized cells that form and repopulated all tissues and organs, from conception to adult life. Depending on their capacity for differentiation, stem cells are classified as totipotent (ie, zygote), pluripotent (ie, embryonic stem cells), multipotent (ie, neuronal stem cells, hematopoietic stem cells, epithelial stem cells, etc.), and unipotent (ie, spermatogonial stem cells). Adult or tissue-specific stem cells reside in specific niches located in, or nearby, their organ or tissue of origin. There, they have microenvironmental support to remain quiescent, to proliferate as undifferentiated cells (self-renewal), and to differentiate into progenitors or terminally differentiated cells that migrate from the niche to perform specialized functions. The presence of proteins at the cell surface is often used to identify, classify, and isolate stem cells. Among the diverse groups of cell surface proteins used for these purposes, integrin α6, also known as CD49f, may be the only biomarker commonly found in more than 30 different populations of stem cells, including some cancer stem cells. This broad expression among stem cell populations indicates that integrin α6 may play an important and conserved role in stem cell biology, which is reaffirmed by recent demonstrations of its role maintaining self-renewal of pluripotent stem cells and breast and glioblastoma cancer stem cells. Therefore, this review intends to highlight and synthesize new findings on the importance of integrin α6 in stem cell biology.

Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in CaR protein expression, occurring at a translational level during their differentiation into cells with a monocyte/macrophage phenotype in response to treatment with PMA or 1, 25(OH)(2)D(3), which is functionally linked to activation of a nonselective cation channel.

Ultrastructure of the labrum and foregut of Derocheilocaris remanei (Crustacea, Mystacocarida).

PubMed

Herrera-Alvarez, Letizia; Fernández, Isabel; Benito, Jesús; Pardos, Fernando

1996-11-01

The cuticle-lined foregut of Derocheilocaris remanei consists of the mouth with its associated labrum, and an undifferentiated esophagus. It is separated from the midgut by an esophageal valve. The labrum is a conspicuous structure moved by five pairs of muscles (four dorsoventral and one longitudinal). Four pairs of subcuticular glands open to its inner face forming two longitudinal, lateral rows of cuticular pores. Each secretory unit is composed of a glandular component (with one or two secretory cells), a neck cell, and a duct cell. In addition, a single gland cell opens mesially into the buccal cavity. The ventrally located mouth is a complex structure characterized by a filter-like system, a sensory organ, and epithelial cells with highly developed microvilli. The esophagus is a simple tube with a characteristic curvature following the mouth. It has a rounded cross section and a triradiate lumen. A layer of circular musculature surrounds this region. The end of the esophagus protrudes into the midgut lumen forming the so-called esophageal valve. The ultrastructural features of the foregut, with the presence of a mucus-trapping mechanism, a relatively well-developed filter system and associated structures and an esophagus lacking glands confirm the microphagic feeding habits of mystacocarids. © 1996 Wiley-Liss, Inc. Copyright © 1996 Wiley-Liss, Inc.

Cellular Location and Expression of Na+, K+-ATPase α Subunits Affect the Anti-Proliferative Activity of Oleandrin

PubMed Central

Yang, Peiying; Cartwright, Carrie; Efuet, Ekem; Hamilton, Stanley R.; Wistuba, Ignacio Ivan; Menter, David; Addington, Crandell; Shureiqi, Imad; Newman, Robert A.

2015-01-01

The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. PMID:23073998

EMT and induction of miR-21 mediate metastasis development in Trp53-deficient tumours

PubMed Central

Bornachea, Olga; Santos, Mirentxu; Martínez-Cruz, Ana Belén; García-Escudero, Ramón; Dueñas, Marta; Costa, Clotilde; Segrelles, Carmen; Lorz, Corina; Buitrago, Agueda; Saiz-Ladera, Cristina; Agirre, Xabier; Grande, Teresa; Paradela, Beatriz; Maraver, Antonio; Ariza, José M.; Prosper, Felipe; Serrano, Manuel; Sánchez-Céspedes, Montse; Paramio, Jesús M.

2012-01-01

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia. PMID:22666537

A case of two different tumors in the heart of a dog.

PubMed

Grieco, Valeria; Locatelli, Chiara; Riccardi, Elena; Brambilla, Paola

2008-05-01

A 9-year-old, spayed, female Maremmano shepherd had a bilateral mastectomy for multiple mammary adenocarcinomas 2 years previous and was referred to the Cardiology Service of the School of Veterinary Medicine of Milan after an acute episode of cardiogenic collapse. Because of severe cardiovascular symptoms and poor prognosis, the dog was euthanized. Necropsy examination revealed the presence of multiple firm grayish neoplastic nodules in the myocardium of the left ventricle and scattered in the pulmonary parenchyma. Neoplastic nodules were also detected in the spleen, pancreas, liver, kidneys, and omentum. Histological examination revealed the coexistence of tubular adenocarcinoma and an undifferentiated sarcoma in the myocardium. Immunohistochemical staining of the sarcoma cells was negative for cytokeratin, desmin, and smooth muscle myosin, thus excluding their epithelial or myoepithelial origin, as well as an origin from smooth muscles cells. These findings, together with the coexpression of vimentin and alpha-smooth muscle actin, suggested that the sarcoma was derived from myofibroblasts. To the authors' knowledge, this is the first report describing cardiac sarcoma of presumptive myofibroblastic origin in a dog with simultaneous occurrence of cardiac metastasis of mammary gland adenocarcinoma.

Equine nasal and paranasal sinus tumours: part 2: a contribution of 28 case reports.

PubMed

Dixon, P M; Head, K W

1999-05-01

The clinical and pathological findings of 28 cases (27 horses, 1 donkey) of equid sinonasal tumours examined at the Edinburgh Veterinary School are presented and include: seven cases of squamous cell carcinoma (SCC); five adenocarcinomas; three undifferentiated carcinomas; two adenomas; five fibro-osseous and bone tumours; and single cases of ameloblastoma, fibroma, fibrosarcoma, undifferentiated sarcoma, melanoma and lymphosarcoma. The median ages of animals affected with epithelial, and fibro-osseous/bone tumours were 14 and 4 years, respectively. Unilateral purulent or mucopurulent nasal discharge (81% of cases) and gross facial swellings (82% of cases) were the most common presenting signs with sinonasal tumours, with epistaxis recorded in just 23% of cases. Radiology and endoscopy were the most useful ancillary diagnostic techniques. The maxillary area was the most common site of tumour origin, and only three cases were definitively identified as originating in the nasal cavity. Four of the maxillary SCC lesions originated within the nasal cavities or maxillary sinuses, while two originated in the oral cavity. Fourteen of 15 carcinomas, but only two of the 13 remaining tumours, spread to other sites in the head. Only three cases of sinonasal tumour had lymph node metastases, and none had distant metastases. In the long term, surgical treatment with seven malignant tumours was unsuccessful (6 months median survival post-operatively), but was successful with four out of five benign tumours (no regrowth at a median of 4 years post-operatively).

MiR-205 and MiR-373 Are Associated with Aggressive Human Mucinous Colorectal Cancer

PubMed Central

Eyking, Annette; Reis, Henning; Frank, Magdalena; Gerken, Guido; Schmid, Kurt W.; Cario, Elke

2016-01-01

Mucinous adenocarcinoma (MAC) represents a distinct histopathological entity of colorectal cancer (CRC), which is associated with disease progression and poor prognosis. Here, we found that expression levels of miR-205 and miR-373 were specifically upregulated only in patients with mucinous colon cancers, but not in CRC that lack mucinous components. To investigate the effects of miR-205 and miR-373 on intestinal epithelial cell (IEC) biology by gain- and loss-of-function experiments in a proof-of-concept approach, we chose previously established in-vitro human Caco-2-based models of differentiated, non-invasive (expressing TLR4 wild-type; termed Caco-2[WT]) versus undifferentiated, invasive (expressing TLR4 mutant D299G; termed Caco-2[D299G]) IEC. Enterocyte-like Caco-2[WT] showed low levels of miR-205 and miR-373 expression, while both miRNAs were significantly upregulated in colorectal carcinoma-like Caco-2[D299G], thus resembling the miRNA expression pattern of paired normal versus tumor samples from MAC patients. Using stable transfection, we generated miR-205- or miR-373-expressing and miR-205- or miR-373-inhibiting subclones of these IEC lines. We found that introduction of miR-205 into Caco-2[WT] led to expansion of mucus-secreting goblet cell-like cells, which was associated with induction of KLF4, MUC2 and TGFβ1 expression. Activation of miR-205 in Caco-2[WT] induced chemoresistance, while inhibition of miR-205 in Caco-2[D299G] promoted chemosensitivity. Caco-2[WT] overexpressing miR-373 showed mitotic abnormalities and underwent morphologic changes (loss of epithelial polarity, cytoskeletal reorganization, and junctional disruption) associated with epithelial-mesenchymal transition and progression to inflammation-associated colonic carcinoma, which correlated with induction of phosphorylated STAT3 and N-CADHERIN expression. Functionally, introduction of miR-373 into Caco-2[WT] mediated loss of cell-cell adhesion and increased proliferation and invasion. Reversely, inhibition of miR-373 allowed mesenchymal IEC to regain epithelial properties, which correlated with absence of neoplastic progression. Using xenografts in mice demonstrated miR-373-mediated acceleration of malignant intestinal tumor growth. In conclusion, our results provide first evidence that miR-205 and miR-373 may differentially contribute to the aggressive phenotype of MAC in CRC. PMID:27271572

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

PubMed

Grassi, Elisa Stellaria; Vezzoli, Valeria; Negri, Irene; Lábadi, Árpád; Fugazzola, Laura; Vitale, Giovanni; Persani, Luca

2015-11-03

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

High-Density ZnO Nanowires as a Reversible Myogenic-Differentiation Switch.

PubMed

Errico, Vito; Arrabito, Giuseppe; Fornetti, Ersilia; Fuoco, Claudia; Testa, Stefano; Saggio, Giovanni; Rufini, Stefano; Cannata, Stefano; Desideri, Alessandro; Falconi, Christian; Gargioli, Cesare

2018-04-25

Mesoangioblasts are outstanding candidates for stem-cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells because growth is typically accompanied by differentiation. Here reversible myogenic-differentiation switching during proliferation is achieved by functionalizing the glass substrate with high-density ZnO nanowires (NWs). Specifically, mesoangioblasts grown on ZnO NWs present a spherical viable undifferentiated cell state without lamellopodia formation during the entire observation time (8 days). Consistently, the myosin heavy chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, NWs do not induce any damage while they reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cell removal from the NW-functionalized substrate and replating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch that does not affect the viability. These results can be the first step toward for the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell-based therapy and tissue engineering.

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

PubMed Central

Tint, I S; Bonder, E M; Feder, H H; Reboulleau, C P; Vasiliev, J M; Gelfand, I M

1992-01-01

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue. Images PMID:1518842

TGF-β1-Induced Epithelial–Mesenchymal Transition Promotes Monocyte/Macrophage Properties in Breast Cancer Cells

PubMed Central

Johansson, Joel; Tabor, Vedrana; Wikell, Anna; Jalkanen, Sirpa; Fuxe, Jonas

2015-01-01

Breast cancer progression toward metastatic disease is linked to re-activation of epithelial–mesenchymal transition (EMT), a latent developmental process. Breast cancer cells undergoing EMT lose epithelial characteristics and gain the capacity to invade the surrounding tissue and migrate away from the primary tumor. However, less is known about the possible role of EMT in providing cancer cells with properties that allow them to traffic to distant sites. Given the fact that pro-metastatic cancer cells share a unique capacity with immune cells to traffic in-and-out of blood and lymphatic vessels we hypothesized that tumor cells undergoing EMT may acquire properties of immune cells. To study this, we performed gene-profiling analysis of mouse mammary EpRas tumor cells that had been allowed to adopt an EMT program after long-term treatment with TGF-β1 for 2 weeks. As expected, EMT cells acquired traits of mesenchymal cell differentiation and migration. However, in addition, we found another cluster of induced genes, which was specifically enriched in monocyte-derived macrophages, mast cells, and myeloid dendritic cells, but less in other types of immune cells. Further studies revealed that this monocyte/macrophage gene cluster was enriched in human breast cancer cell lines displaying an EMT or a Basal B profile, and in human breast tumors with EMT and undifferentiated (ER−/PR−) characteristics. The results identify an EMT-induced monocyte/macrophage gene cluster, which may play a role in breast cancer cell dissemination and metastasis. PMID:25674539

Primary undifferentiated small round cell sarcoma of the deep abdominal wall with a novel variant of t(10;19) CIC-DUX4 gene fusion.

PubMed

Tsukamoto, Yoshitane; Futani, Hiroyuki; Yoshiya, Shinichi; Watanabe, Takahiro; Kihara, Takako; Matsuo, Shohei; Hirota, Seiichi

2017-10-01

We experienced a 38-year-old Japanese male with t(10;19) CIC-DUX4 -positive undifferentiated small round cell sarcoma in the deep abdominal wall. Three months before his first visit to our hospital, he noticed a mass in his right abdominal wall. Computed tomography on admission revealed a solid abdominal tumor 70×53mm in size and multiple small tumors in both lungs. The biopsy of the abdominal tumor revealed undifferentiated small round cell sarcoma, suggestive of Ewing sarcoma. Under the clinical diagnosis of Ewing-like sarcoma of the abdominal wall with multiple lung metastases, several cycles of ICE (ifosfamide, carboplatin and etoposide) therapy were performed. After the chemotherapy, the lung metastases disappeared, while the primary lesion rapidly grew. Additional VDC (vincristine, doxorubicin and cyclophosphamide) therapy was carried out without apparent effect. Although the surgical removal of the primary lesion was done, peritoneal dissemination and a huge metastatic liver tumor appeared thereafter. The patient died of disease progression two months after the surgery. The total clinical course was approximately one year, showing that the tumor was extremely aggressive. The tumor cells of the surgical specimen were positive for CD99, WT1, calretinin, INI1, ERG and Fli1 by immunohistochemistry. Fusion gene analyses using the frozen surgical material revealed negativity for EWSR1-Fli1, EWSR1-ERG and t(4;19) CIC-DUX4 fusions, but positivity for t(10;19) CIC-DUX4 fusion. Thus, we made a final pathological diagnosis of t(10;19) CIC-DUX4-positive undifferentiated small round cell sarcoma. To our knowledge, this is the 13th case of t(10;19) CIC-DUX4 undifferentiated small round cell sarcoma with precise clinicopathological information. Especially in our case, two types of t(10;19) CIC-DUX4 fusion transcripts were observed, both of which are in-frame and novel. Copyright © 2017 Elsevier GmbH. All rights reserved.

β2-Microglobulin as a potential factor for the expansion of mesenchymal stem cells

PubMed Central

Zhu, Ying; Su, Yongping; Cheng, Tianmin; Chung, Leland W. K.

2010-01-01

Multipotent mesenchymal stem cells (MSCs) hold great promise in regenerative medicine, but one of the biggest challenges facing for their application is the ex vivo expansion to obtain enough undifferentiated cells. Fetal bovine serum (FBS), which can elicit possible contaminations of prion, virus, zoonosis or immunological reaction against xenogenic serum antigens, still remains essential to the culture formulations. There is an urgent need to identify potential factors for the undifferentiated expansion of MSCs to reduce the use of FBS or eventually replace it. A previously recognized housekeeping gene, β2-microglobulin (β2M), is demonstrated to act as a novel growth factor to stimulate the undifferentiated ex vivo expansion and preserve the pluripotency of adult MSCs from various sources. The use of β2M might have promising implications for future clinical application of MSCs. PMID:19466557

FMR1 Epigenetic Silencing Commonly Occurs in Undifferentiated Fragile X-Affected Embryonic Stem Cells

PubMed Central

Avitzour, Michal; Mor-Shaked, Hagar; Yanovsky-Dagan, Shira; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Levy-Lahad, Ephrat; Epsztejn-Litman, Silvina; Eiges, Rachel

2014-01-01

Summary Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases. PMID:25418717

FMR1 epigenetic silencing commonly occurs in undifferentiated fragile X-affected embryonic stem cells.

PubMed

Avitzour, Michal; Mor-Shaked, Hagar; Yanovsky-Dagan, Shira; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Levy-Lahad, Ephrat; Epsztejn-Litman, Silvina; Eiges, Rachel

2014-11-11

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5'-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction ofmore » A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.« less

Induction of a menopausal state alters the growth and histology of ovarian tumors in a mouse model of ovarian cancer.

PubMed

Laviolette, Laura A; Ethier, Jean-François; Senterman, Mary K; Devine, Patrick J; Vanderhyden, Barbara C

2011-05-01

Ovarian cancer is often diagnosed in women after menopause when the levels of the serum gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are increased because of the depletion of growing follicles within the ovary. The ability of FSH and LH to modulate the disease has not been well studied owing to a lack of physiologically relevant models of ovarian cancer. In this study, 4-vinylcyclohexene diepoxide (VCD) was used to deplete ovarian follicles and increase the levels of circulating FSH and LH in the tgCAG-LS-TAg mouse model of ovarian cancer. VCD-induced follicle depletion was performed either before or after induction of the oncogene SV40 large and small T-antigens in the ovarian surface epithelial cells of tgCAG-LS-TAg mice, which was mediated by the intrabursal delivery of an adenovirus expressing Cre recombinase (AdCre). tgCAG-LS-TAg mice injected with AdCre developed undifferentiated ovarian tumors with mixed epithelial and stromal components and some features of sex cord stromal tumors. Treatment with VCD before or after AdCre injection yielded tumors of similar histology, but with the unique appearance of Sertoli cell nests. In mice treated with VCD before the induction of tumorigenesis, the ovarian tumors tended to grow more slowly. The human ovarian cancer cell lines SKOV3 and OVCAR3 responded similarly to increased levels of gonadotropins in a second model of menopause, growing more slowly in ovariectomized mice compared with cycling controls. These results suggest that follicle depletion and increased gonadotropin levels can alter the histology and the rate of growth of ovarian tumors.

A reappraisal of hemangiopericytoma of bone; analysis of cases reclassified as synovial sarcoma and solitary fibrous tumor of bone.

PubMed

Verbeke, Sofie L J; Fletcher, Christopher D M; Alberghini, Marco; Daugaard, Søren; Flanagan, Adrienne M; Parratt, Tim; Kroon, Herman M; Hogendoorn, Pancras C W; Bovée, Judith V M G

2010-06-01

Hemangiopericytoma (HPC) was first described as a neoplasm with distinct morphologic features, presumably composed of pericytes. In soft tissue, it is accepted that most such lesions are solitary fibrous tumors (SFTs), monophasic synovial sarcomas (SSs), or myofibromatoses. It is unclear whether HPC of bone exists. We reviewed 9 primary "HPC" of bone from 4 institutions diagnosed between 1952 and 2002. Immunohistochemistry was performed for CD31, CD34, von Willebrand factor, smooth muscle actin, keratin AE1/AE3, and epithelial membrane antigen. There were 4 male and 5 female patients between 21 and 73 years. All tumors were located within bone, either sited within spine or extremities. All tumors showed thin-walled branching vessels surrounded by undifferentiated spindle or round cells. These cells showed variation in their morphologic pattern: 6 tumors showed a pattern-less architecture and varying cellularity, consistent with SFT; 3 of 5 cases examined were CD34-positive. Three tumors showed more densely packed sheets and fascicles of poorly differentiated cells, resembling SS, of which 2 showed focal staining for keratin AE1/AE3 or epithelial membrane antigen. Fluorescent in-situ hybridization confirmed the presence of SS18 rearrangement in 1 of 2 tumors examined. In conclusion, similar to their soft-tissue counterpart, HPC-like features in bone are a nonspecific growth pattern rather than a true diagnosis. We confirm the existence of 2 entities: SFT and SS of bone. Both are characterized by distinct morphology and immunohistochemical profile. SFT of bone is located within spine and has a better prognosis, whereas SS of bone is located within long bones having a poor prognosis.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

PubMed

Otto, Benjamin; Gruner, Katharina; Heinlein, Christina; Wegwitz, Florian; Nollau, Peter; Ylstra, Bauke; Pantel, Klaus; Schumacher, Udo; Baumbusch, Lars O; Martin-Subero, José Ignacio; Siebert, Reiner; Wagener, Christoph; Streichert, Thomas; Deppert, Wolfgang; Tolstonog, Genrich V

2013-03-15

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling. Copyright © 2012 UICC.

Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

PubMed

Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

2015-11-17

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

BCOR-CCNB3 Fusions Are Frequent in Undifferentiated Sarcomas of Male Children

PubMed Central

Peters, Tricia L.; Kumar, Vijetha; Polikepahad, Sumanth; Lin, Frank Y.; Sarabia, Stephen F.; Liang, Yu; Wang, Wei-Lien; Lazar, Alexander J.; Doddapaneni, Harsha Vardhan; Chao, Hsu; Muzny, Donna M.; Wheeler, David A.; Okcu, M. Fatih; Plon, Sharon E.; Hicks, M. John; López-Terrada, Dolores; Parsons, D. Williams; Roy, Angshumoy

2014-01-01

The BCOR-CCNB3 fusion gene, resulting from a chromosome X paracentric inversion, was recently described in translocation-negative ‘Ewing-like’ sarcomas arising in bone and soft tissue. Genetic subclassification of undifferentiated unclassified sarcomas may potentially offer markers for reproducible diagnosis and substrates for therapy. Using whole transcriptome paired end RNA sequencing (RNA-seq) we unexpectedly identified BCOR-CCNB3 fusion transcripts in an undifferentiated spindle cell sarcoma. RNA-seq results were confirmed through direct RT-PCR of tumor RNA and cloning of the genomic breakpoints from tumor DNA. Five additional undifferentiated sarcomas with BCOR-CCNB3 fusions were identified in a series of 42 pediatric and adult unclassified sarcomas. Genomic breakpoint analysis demonstrated unique breakpoint locations in each case at the DNA level even though the resulting fusion mRNA was identical in all cases. All patients with BCOR-CCNB3 sarcoma were males diagnosed in mid-childhood (7-13 years of age). Tumors were equally distributed between axial and extra-axial locations. Five of the six tumors were soft tissue lesions with either predominant spindle cell morphology or spindle cell areas interspersed with ovoid to round cells. CCNB3 immunohistochemistry showed strong nuclear positivity in 5 tumors prior to oncologic therapy, but was patchy to negative in post-treatment tumor samples. An RT-PCR assay developed to detect the fusion transcript in archival formalin-fixed tissue was positive in all 6 cases, with high sensitivity and specificity in both pre- and post-treated samples. This study adds to recent reports on the clinicopathologic spectrum of BCOR-CCNB3 fusion-positive sarcomas, a newly-emerging entity within the undifferentiated unclassified sarcoma category, and describes a simple RT-PCR assay that in conjunction with CCNB3 immunohistochemistry can be useful in diagnosing these tumors. PMID:25360585

Changes in Brain Function in Patients With Stage I, Stage II, Stage III, or Stage IV Ovarian, Primary Peritoneal, or Fallopian Tube Cancer Who Are Receiving Chemotherapy

ClinicalTrials.gov

2018-04-11

Cognitive Side Effects of Cancer Therapy; Malignant Ovarian Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Carcinosarcoma; Ovarian Choriocarcinoma; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Dysgerminoma; Ovarian Embryonal Carcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mixed Germ Cell Tumor; Ovarian Mucinous Cystadenocarcinoma; Ovarian Polyembryoma; Ovarian Sarcoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Teratoma; Ovarian Yolk Sac Tumor; Stage I Ovarian Cancer; Stage IA Fallopian Tube Cancer; Stage IA Ovarian Cancer; Stage IA Ovarian Germ Cell Tumor; Stage IB Fallopian Tube Cancer; Stage IB Ovarian Cancer; Stage IB Ovarian Germ Cell Tumor; Stage IC Fallopian Tube Cancer; Stage IC Ovarian Cancer; Stage IC Ovarian Germ Cell Tumor; Stage II Ovarian Cancer; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIA Ovarian Germ Cell Tumor; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIB Ovarian Germ Cell Tumor; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIC Ovarian Germ Cell Tumor; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

Clonal yeast biofilms can reap competitive advantages through cell differentiation without being obligatorily multicellular

PubMed Central

Hanghøj, Kristian Ebbesen; Andersen, Kaj Scherz; Boomsma, Jacobus J.

2016-01-01

How differentiation between cell types evolved is a fundamental question in biology, but few studies have explored single-gene phenotypes that mediate first steps towards division of labour with selective advantage for groups of cells. Here, we show that differential expression of the FLO11 gene produces stable fractions of Flo11+ and Flo11− cells in clonal Saccharomyces cerevisiae biofilm colonies on medium with intermediate viscosity. Differentiated Flo11+/− colonies, consisting of adhesive and non-adhesive cells, obtain a fourfold growth advantage over undifferentiated colonies by overgrowing glucose resources before depleting them, rather than depleting them while they grow as undifferentiated Flo11− colonies do. Flo11+/− colonies maintain their structure and differentiated state by switching non-adhesive cells to adhesive cells with predictable probability. Mixtures of Flo11+ and Flo11− cells from mutant strains that are unable to use this epigenetic switch mechanism produced neither integrated colonies nor growth advantages, so the condition-dependent selective advantages of differentiated FLO11 expression can only be reaped by clone-mate cells. Our results show that selection for cell differentiation in clonal eukaryotes can evolve before the establishment of obligate undifferentiated multicellularity, and without necessarily leading to more advanced organizational complexity. PMID:27807261

Spaceflight Transcriptomes: Unique Responses to a Novel Environment

PubMed Central

Paul, Anna-Lisa; Zupanska, Agata K.; Ostrow, Dejerianne T.; Zhang, Yanping; Sun, Yijun; Li, Jian-Liang; Shanker, Savita; Farmerie, William G.; Amalfitano, Claire E.

2012-01-01

Abstract The spaceflight environment presents unique challenges to terrestrial biology, including but not limited to the direct effects of gravity. As we near the end of the Space Shuttle era, there remain fundamental questions about the response and adaptation of plants to orbital spaceflight conditions. We address a key baseline question of whether gene expression changes are induced by the orbital environment, and then we ask whether undifferentiated cells, cells presumably lacking the typical gravity response mechanisms, perceive spaceflight. Arabidopsis seedlings and undifferentiated cultured Arabidopsis cells were launched in April, 2010, as part of the BRIC-16 flight experiment on STS-131. Biologically replicated DNA microarray and averaged RNA digital transcript profiling revealed several hundred genes in seedlings and cell cultures that were significantly affected by launch and spaceflight. The response was moderate in seedlings; only a few genes were induced by more than 7-fold, and the overall intrinsic expression level for most differentially expressed genes was low. In contrast, cell cultures displayed a more dramatic response, with dozens of genes showing this level of differential expression, a list comprised primarily of heat shock–related and stress-related genes. This baseline transcriptome profiling of seedlings and cultured cells confirms the fundamental hypothesis that survival of the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression. The comparison of intact plants with cultures of undifferentiated cells confirms a second hypothesis: undifferentiated cells can detect spaceflight in the absence of specialized tissue or organized developmental structures known to detect gravity. Key Words: Tissue culture—Microgravity—Low Earth orbit—Space Shuttle—Microarray. Astrobiology 12, 40–56. PMID:22221117

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study.

PubMed

Song, Zheng-Mei; Chen, Ni; Liu, Jia-Hui; Tang, Huan; Deng, Xiaoyong; Xi, Wen-Song; Han, Kai; Cao, Aoneng; Liu, Yuanfang; Wang, Haifang

2015-10-01

Titanium dioxide nanoparticles (TiO2 NPs) are widely found in food-related consumer products. Understanding the effect of TiO2 NPs on the intestinal barrier and absorption is essential and vital for the safety assessment of orally administrated TiO2 NPs. In this study, the cytotoxicity and translocation of two native TiO2 NPs, and these two TiO2 NPs pretreated with the digestion simulation fluid or bovine serum albumin were investigated in undifferentiated Caco-2 cells, differentiated Caco-2 cells and Caco-2 monolayer. TiO2 NPs with a concentration less than 200 µg ml(-1) did not induce any toxicity in differentiated cells and Caco-2 monolayer after 24 h exposure. However, TiO2 NPs pretreated with digestion simulation fluids at 200 µg ml(-1) inhibited the growth of undifferentiated Caco-2 cells. Undifferentiated Caco-2 cells swallowed native TiO2 NPs easily, but not pretreated NPs, implying the protein coating on NPs impeded the cellular uptake. Compared with undifferentiated cells, differentiated ones possessed much lower uptake ability of these TiO2 NPs. Similarly, the traverse of TiO2 NPs through the Caco-2 monolayer was also negligible. Therefore, we infer the possibility of TiO2 NPs traversing through the intestine of animal or human after oral intake is quite low. This study provides valuable information for the risk assessment of TiO2 NPs in food. Copyright © 2015 John Wiley & Sons, Ltd.

Hematopoiesis and hematopoietic organs in arthropods.

PubMed

Grigorian, Melina; Hartenstein, Volker

2013-03-01

Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.

Biogenic amines as regulators of the proliferative activity of normal and neoplastic intestinal epithelial cells (review).

PubMed

Tutton, P J; Barkla, D H

1987-01-01

The role of extracellular amines such as noradrenaline and serotonin and their interaction with cyclic nucleotides and intracellular polyamines in the regulation of intestinal epithelial cell proliferation is reviewed with particular reference to the differences between normal and neoplastic cells. In respect to the normal epithelium of the small intestine there is a strong case to support the notion that cell proliferation is controlled by, amongst other things, sympathetic nerves. In colonic carcinomas, antagonists for certain serotonin receptors, for histamine H2 receptors and for dopamine D2 receptors inhibit both cell division and tumour growth. Because of the reproducible variations between tumour lines in the response to these antagonists, this inhibition appears to be due to a direct effect on the tumour cells rather than an indirect effect via the tumour host or stroma. This conclusion is supported by the cytocidal effects of toxic congeners of serotonin on the tumour cells. The most salient difference between the amine responses of normal and neoplastic cells relates to the issue of amine uptake. Proliferation of crypt cells is promoted by amine uptake inhibitors, presumably because they block amine re-uptake by the amine secreting cells--sympathetic neurones and enteroendocrine cells. However, tumour cell proliferation is strongly inhibited by amine uptake inhibitors, suggesting that neoplastic cells can, and need to take up the amine before being stimulated by it. Recent revelations in the field of oncogenes also support an important association between amines, cyclic nucleotides and cell division. The ras oncogenes code for a protein that is a member of a family of molecules which relay information from extracellular regulators, such as biogenic amines, to the intracellular regulators, including cyclic nucleotides. Evidence is presented suggesting that enteroendocrine cells, enterocytes, carcinoid tumour cells and adenocarcinoma cells all have the same embryonic origin and that cells exhibiting an admixture of endocrine and proliferative properties exist in colonic tumours, but not in the normal intestinal epithelium. Thus, it appears that in the normal intestine a clear structural and functional distinction exists between the regulating cells (i.e. the sympathetic neurones and enteroendocrine cells) and the regulated cells (i.e. the undifferentiated crypt cells): cells that have acquired a regulating role are no longer able to divide and cells which are able to divide do not take up or store amines.(ABSTRACT TRUNCATED AT 400 WORDS)

PRMT5 as a druggable target for glioblastoma therapy.

PubMed

Banasavadi-Siddegowda, Yeshavanth Kumar; Welker, Alessandra M; An, Min; Yang, Xiaozhi; Zhou, Wei; Shi, Guqin; Imitola, Jaime; Li, Chenglong; Hsu, Sigmund; Wang, Jiang; Phelps, Mitch; Zhang, Jianying; Beattie, Christine E; Baiocchi, Robert; Kaur, Balveen

2018-05-18

In spite of standard multimodal therapy consisting of surgical resection followed by radiation and concurrent chemotherapy, prognosis for glioblastoma (GBM) patients remains poor. The identification of both differentiated and undifferentiated "stem cell like" populations in the tumor highlights the significance of finding novel targets that affect the heterogeneous tumor cell population. Protein arginine methyltransferase 5 (PRMT5) is one such candidate gene whose nuclear expression correlates with poor survival and has been reported to be required for survival of differentiated GBM cells and self-renewal of undifferentiated GBM cells. In the current study we screened the specificity and efficacy of 4 novel PRMT5 inhibitors in the treatment of GBM. Efficacies of these inhibitors were screened using an in vitro GBM neurosphere model and an in vivo intracranial zebrafish model of glioma. Standard molecular biology methods were employed to investigate changes in cell cycle, growth, and senescence. In vitro and in vivo studies revealed that among the 4 PRMT5 inhibitors, treatment of GBM cells with compound 5 (CMP5) mirrored the effects of PRMT5 knockdown wherein it led to apoptosis of differentiated GBM cells and drove undifferentiated primary patient derived GBM cells into a nonreplicative senescent state. In vivo antitumor efficacy combined with the specificity of CMP5 underscores the importance of developing it for translation.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP.

PubMed

Bodewei, R; Hering, S; Schubert, B; Wollenberger, A

1985-04-01

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

PubMed Central

Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

2013-01-01

Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Assessing the risk of pelvic and para-aortic nodal involvement in apparent early-stage ovarian cancer: A predictors- and nomogram-based analyses.

PubMed

Bogani, Giorgio; Tagliabue, Elena; Ditto, Antonino; Signorelli, Mauro; Martinelli, Fabio; Casarin, Jvan; Chiappa, Valentina; Dondi, Giulia; Leone Roberti Maggiore, Umberto; Scaffa, Cono; Borghi, Chiara; Montanelli, Luca; Lorusso, Domenica; Raspagliesi, Francesco

2017-10-01

To estimate the prevalence of lymph node involvement in early-stage epithelial ovarian cancer in order to assess the prognostic value of lymph node dissection. Data of consecutive patients undergoing staging for early-stage epithelial ovarian cancer were retrospectively evaluated. Logistic regression and a nomogram-based analysis were used to assess the risk of lymph node involvement. Overall, 290 patients were included. All patients had lymph node dissection including pelvic and para-aortic lymphadenectomy. Forty-two (14.5%) patients were upstaged due to lymph node metastatic disease. Pelvic and para-aortic nodal metastases were observed in 22 (7.6%) and 42 (14.5%) patients. Lymph node involvement was observed in 18/95 (18.9%), 1/37 (2.7%), 4/29 (13.8%), 11/63 (17.4%), 3/41 (7.3%) and 5/24 (20.8%) patients with high-grade serous, low-grade-serous, endometrioid G1, endometrioid G2&3, clear cell and undifferentiated, histology, respectively (p=0.12, Chi-square test). We observed that high-grade serous histology was associated with an increased risk of pelvic node involvement; while, histology rather than low-grade serous and bilateral tumors were independently associated with para-aortic lymph node involvement (p<0.05). Nomograms displaying the risk of nodal involvement in the pelvic and para-aortic areas were built. High-grade serous histology and bilateral tumors are the main characteristics suggesting lymph node positivity. Our data suggested that high-grade serous and bilateral early-stage epithelial ovarian cancer are at high risk of having disease harboring in the lymphatic tissues of both pelvic and para-aortic area. After receiving external validation, our data will help to identify patients deserving comprehensive retroperitoneal staging. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

PubMed

Schroeter, Christina B; Koehler, Sybille; Kann, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T; Rinschen, Markus M

2018-04-25

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

PubMed Central

Wang, Yu-Chieh; Stein, Jason W.; Lynch, Candace L.; Tran, Ha T.; Lee, Chia-Yao; Coleman, Ronald; Hatch, Adam; Antontsev, Victor G.; Chy, Hun S.; O’Brien, Carmel M.; Murthy, Shashi K.; Laslett, Andrew L.; Peterson, Suzanne E.; Loring, Jeanne F.

2015-01-01

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity. PMID:26304831

Convective exosome-tracing microfluidics for analysis of cell-non-autonomous neurogenesis.

PubMed

Oh, Hyun Jeong; Shin, Yoojin; Chung, Seok; Hwang, Do Won; Lee, Dong Soo

2017-01-01

The effective role of exosome delivering neurogenic microRNA (miRNA) enables to induce efficient differentiation process during neurogenesis. The microfludic system capable of visualizing the exosomal behavior such as secretion, migration, and uptake of individual exosomes can be used as a robust technique to understand the exosome-mediated change of cellular behavior. Here, we developed the exosome-tracing microfluidic system to visualize exosomal transport carrying the neurogenic miRNA from leading to neighboring cells, and found a new mode of exosome-mediated cell-non-autonomous neurogenesis. The miR-193a facilitated neurogenesis in F11 cells by blocking proliferation-related target genes. In addition to time-lapse live-cell imaging using microfluidics visualized the convective transport of exosomes from differentiated to undifferentiated cells. Individual exosomes containing miR-193a from differentiated donor cells were taken up by undifferentiated cells to lead them to neurogenesis. Induction of anti-miR-193a was sufficient to block neurogenesis in F11 cells. Inhibition of the exosomal production by manumycin-A and treatment of anti-miR-193a in the differentiated donor cells failed to induce neurogenesis in undifferentiated recipient cells. These findings indicate that exosomes of neural progenitors and neurogenic miRNA within these exosomes propagate cell-non-autonomous differentiation to neighboring progenitors, to delineate the roles of exosome mediating neurogenesis of population of homologous neural progenitor cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

CD24-Positive Cells from Normal Adult Mouse Liver Are Hepatocyte Progenitor Cells

PubMed Central

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M.; Rao, Pulivarthi H.

2011-01-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45−, Ter119−) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes. PMID:21361791

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

PubMed

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M; Rao, Pulivarthi H; Darlington, Gretchen J

2011-12-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.

Effect of aqueous and lipophilic mullet (Mugil cephalus) Bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells.

PubMed

Rosa, Antonella; Atzeri, Angela; Deiana, Monica; Melis, M Paola; Loru, Debora; Incani, Alessandra; Cabboi, Barbara; Dessì, M Assunta

2011-03-09

The importance of n-3 polyunsaturated fatty acid (n-3 PUFA) intake has long been recognized in human nutrition. Although health benefits, n-3 PUFA are subject to rapid and/or extensive oxidation during processing and storage, resulting in potential alteration in nutritional composition and quality of food. Bottarga, a salted and semi-dried mullet ( Mugil cephalus ) ovary product, is proposed as an important source of n-3 PUFA, having high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we investigated the extent of lipid oxidation of grated bottarga samples during 7 months of storage at -20 °C and room temperature under light exposure. Cell viability, lipid composition, and lipid peroxidation were measured in intestinal differentiated Caco-2 cell monolayers after 6-48 h of incubation with lipid and hydrophilic extracts obtained from bottarga samples at different storage conditions. The storage of bottarga did not affect the n-3 PUFA level, but differences were observed in hydroperoxide levels in samples from different storage conditions. All tested bottarga extracts did not show a toxic effect on cell viability of differentiated Caco-2 cells. Epithelial cells incubated with bottarga oil had significant changes in fatty acid composition but not in cholesterol levels with an accumulation of EPA, DHA, and 22:5. Cell hydroperoxides were higher in treated cells, in relation to the oxidative status of bottarga oil. Moreover, the bottarga lipid extract showed an in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells).

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

PubMed

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

Myeloblastic and lymphoblastic markers in acute undifferentiated leukemia and chronic myelogenous leukemia in blast crisis.

PubMed

Shumak, K H; Baker, M A; Taub, R N; Coleman, M S

1980-11-01

Blast cells were obtained from 17 patients with acute undifferentiated leukemia and 13 patients with chronic myelogenous leukemia in blast crisis. The blasts were tested with anti-i serum in cytotoxicity tests and with antisera to myeloblastic leukemia-associated antigens in immunofluorescence tests. The terminal deoxynucleotidyl transferase (TDT) content of the blasts was also measured. Lymphoblasts react strongly with anti-i, do not react with anti-myeloblast serum, and have high levels of TDT; myeloblasts react weakly with anti-i, do not react with anti-myeloblast serum, and have very low levels of TDT. Of the 17 patients with acute undifferentiated leukemia, there were six with blasts which reacted like lymphoblasts, six with blasts which reacted like myeloblasts, and five with blasts bearing different combinations of these lymphoblastic and myeloblastic markers. Eight of the 11 patients with lymphoblastic or mixed lymphoblastic-myeloblastic markers, but only one of the six with myeloblastic markers, achieved complete or partial remission in response to therapy. Thus, in acute undifferentiated leukemia, classification of blasts with these markers may be of prognostic value. Of the 13 patients with chronic myelogenous leukemia in blast crises, the markers were concordant (for myeloblasts) in only two cases. Three of the 13 patients had TDT-positive blasts, but the reactions of these cells with anti-i and with anti-myeloblast serum differed from those seen with lymphoblasts from patients with acute lymphoblastic leukemia. Although the cell involved in "lymphoid" blast crisis of chronic myelogenous leukemia is similar in many respects to that involved in acute lymphoblastic leukemia, these cells are not identical.

Contrast-enhanced CT features of hepatoblastoma: Can we predict histopathology?

PubMed

Baheti, Akshay D; Luana Stanescu, A; Li, Ning; Chapman, Teresa

Hepatoblastoma is the most common hepatic malignancy occurring in the pediatric population. Intratumoral cellular behavior varies, and the small-cell undifferentiated histopathology carries a poorer prognosis than other tissue subtypes. Neoadjuvant chemotherapy is recommended for this tumor subtype prior to surgical resection in most cases. Early identification of tumors with poor prognosis could have a significant clinical impact. Objective The aim of this work was to identify imaging features of small-cell undifferentiated subtype hepatoblastoma that can help distinguish this subtype from more favorable tumors and potentially guide the clinical management. We also sought to characterize contrast-enhanced CT (CECT) features of hepatoblastoma that correlate with metastatic disease and patient outcome. Our study included 34 patients (24 males, 10 females) with a mean age of 16months (range: 0-46months) with surgically confirmed hepatoblastoma and available baseline abdominal imaging by CECT. Clinical data and CT abdominal images were retrospectively analyzed. Five tumors with small-cell undifferentiated components were identified. All of these tumors demonstrated irregular margins on CT imaging. Advanced PRETEXT stage, vascular invasion and irregular margins were associated with metastatic disease and decreased survival. Capsular retraction was also significantly associated with decreased survival. Irregular tumor margins demonstrated statistically significant association with the presence of small-cell undifferentiated components. No other imaging feature showed statistically significant association. Tumor margin irregularity, vascular invasion, capsular retraction, and PRETEXT stage correlate with worse patient outcomes. Irregular tumor margin was the only imaging feature significantly associated with more aggressive tumor subtype. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessing the genetic architecture of epithelial ovarian cancer histological subtypes.

PubMed

Cuellar-Partida, Gabriel; Lu, Yi; Dixon, Suzanne C; Fasching, Peter A; Hein, Alexander; Burghaus, Stefanie; Beckmann, Matthias W; Lambrechts, Diether; Van Nieuwenhuysen, Els; Vergote, Ignace; Vanderstichele, Adriaan; Doherty, Jennifer Anne; Rossing, Mary Anne; Chang-Claude, Jenny; Rudolph, Anja; Wang-Gohrke, Shan; Goodman, Marc T; Bogdanova, Natalia; Dörk, Thilo; Dürst, Matthias; Hillemanns, Peter; Runnebaum, Ingo B; Antonenkova, Natalia; Butzow, Ralf; Leminen, Arto; Nevanlinna, Heli; Pelttari, Liisa M; Edwards, Robert P; Kelley, Joseph L; Modugno, Francesmary; Moysich, Kirsten B; Ness, Roberta B; Cannioto, Rikki; Høgdall, Estrid; Høgdall, Claus; Jensen, Allan; Giles, Graham G; Bruinsma, Fiona; Kjaer, Susanne K; Hildebrandt, Michelle A T; Liang, Dong; Lu, Karen H; Wu, Xifeng; Bisogna, Maria; Dao, Fanny; Levine, Douglas A; Cramer, Daniel W; Terry, Kathryn L; Tworoger, Shelley S; Stampfer, Meir; Missmer, Stacey; Bjorge, Line; Salvesen, Helga B; Kopperud, Reidun K; Bischof, Katharina; Aben, Katja K H; Kiemeney, Lambertus A; Massuger, Leon F A G; Brooks-Wilson, Angela; Olson, Sara H; McGuire, Valerie; Rothstein, Joseph H; Sieh, Weiva; Whittemore, Alice S; Cook, Linda S; Le, Nhu D; Blake Gilks, C; Gronwald, Jacek; Jakubowska, Anna; Lubiński, Jan; Kluz, Tomasz; Song, Honglin; Tyrer, Jonathan P; Wentzensen, Nicolas; Brinton, Louise; Trabert, Britton; Lissowska, Jolanta; McLaughlin, John R; Narod, Steven A; Phelan, Catherine; Anton-Culver, Hoda; Ziogas, Argyrios; Eccles, Diana; Campbell, Ian; Gayther, Simon A; Gentry-Maharaj, Aleksandra; Menon, Usha; Ramus, Susan J; Wu, Anna H; Dansonka-Mieszkowska, Agnieszka; Kupryjanczyk, Jolanta; Timorek, Agnieszka; Szafron, Lukasz; Cunningham, Julie M; Fridley, Brooke L; Winham, Stacey J; Bandera, Elisa V; Poole, Elizabeth M; Morgan, Terry K; Goode, Ellen L; Schildkraut, Joellen M; Pearce, Celeste L; Berchuck, Andrew; Pharoah, Paul D P; Webb, Penelope M; Chenevix-Trench, Georgia; Risch, Harvey A; MacGregor, Stuart

2016-07-01

Epithelial ovarian cancer (EOC) is one of the deadliest common cancers. The five most common types of disease are high-grade and low-grade serous, endometrioid, mucinous and clear cell carcinoma. Each of these subtypes present distinct molecular pathogeneses and sensitivities to treatments. Recent studies show that certain genetic variants confer susceptibility to all subtypes while other variants are subtype-specific. Here, we perform an extensive analysis of the genetic architecture of EOC subtypes. To this end, we used data of 10,014 invasive EOC patients and 21,233 controls from the Ovarian Cancer Association Consortium genotyped in the iCOGS array (211,155 SNPs). We estimate the array heritability (attributable to variants tagged on arrays) of each subtype and their genetic correlations. We also look for genetic overlaps with factors such as obesity, smoking behaviors, diabetes, age at menarche and height. We estimated the array heritabilities of high-grade serous disease ([Formula: see text] = 8.8 ± 1.1 %), endometrioid ([Formula: see text] = 3.2 ± 1.6 %), clear cell ([Formula: see text] = 6.7 ± 3.3 %) and all EOC ([Formula: see text] = 5.6 ± 0.6 %). Known associated loci contributed approximately 40 % of the total array heritability for each subtype. The contribution of each chromosome to the total heritability was not proportional to chromosome size. Through bivariate and cross-trait LD score regression, we found evidence of shared genetic backgrounds between the three high-grade subtypes: serous, endometrioid and undifferentiated. Finally, we found significant genetic correlations of all EOC with diabetes and obesity using a polygenic prediction approach.

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

PubMed

Cheung, Yuen-Ting; Lau, Way Kwok-Wai; Yu, Man-Shan; Lai, Cora Sau-Wan; Yeung, Sze-Chun; So, Kwok-Fai; Chang, Raymond Chuen-Chung

2009-01-01

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Dalantercept in Treating Patients With Recurrent or Persistent Endometrial Cancer

ClinicalTrials.gov

2018-02-13

Endometrial Adenocarcinoma; Endometrial Clear Cell Adenocarcinoma; Endometrial Mixed Adenocarcinoma; Endometrial Mucinous Adenocarcinoma; Endometrial Serous Adenocarcinoma; Endometrial Squamous Cell Carcinoma; Endometrial Transitional Cell Carcinoma; Endometrial Undifferentiated Carcinoma; Recurrent Uterine Corpus Carcinoma

Brivanib Alaninate in Treating Patients With Recurrent or Persistent Endometrial Cancer

ClinicalTrials.gov

2017-11-02

Endometrial Adenocarcinoma; Endometrial Clear Cell Adenocarcinoma; Endometrial Mixed Adenocarcinoma; Endometrial Mucinous Adenocarcinoma; Endometrial Serous Adenocarcinoma; Endometrial Squamous Cell Carcinoma; Endometrial Transitional Cell Carcinoma; Endometrial Undifferentiated Carcinoma; Recurrent Uterine Corpus Carcinoma

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

PubMed Central

He, Min; Storr-Paulsen, Thomas; Wang, Annie L.; Ghezzi, Chiara E.; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P.; Islam, Rakibul; Griffith, May; Islam, M. Mirazul; Hodges, Robin R.; Wnek, Gary E.; Kaplan, David L.; Dartt, Darlene A.

2016-01-01

Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study. PMID:27832279

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion.

PubMed

He, Min; Storr-Paulsen, Thomas; Wang, Annie L; Ghezzi, Chiara E; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P; Islam, Rakibul; Griffith, May; Islam, M Mirazul; Hodges, Robin R; Wnek, Gary E; Kaplan, David L; Dartt, Darlene A

2016-11-01

We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.

PubMed

Wang, Nan-Kai; Tosi, Joaquin; Kasanuki, Jennifer Mie; Chou, Chai Lin; Kong, Jian; Parmalee, Nancy; Wert, Katherine J; Allikmets, Rando; Lai, Chi-Chun; Chien, Chung-Liang; Nagasaki, Takayuki; Lin, Chyuan-Sheng; Tsang, Stephen H

2010-04-27

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

PubMed

Soncin, Francesca; Mohamet, Lisa; Eckardt, Dominik; Ritson, Sarah; Eastham, Angela M; Bobola, Nicoletta; Russell, Angela; Davies, Steve; Kemler, Rolf; Merry, Catherine L R; Ward, Christopher M

2009-09-01

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

PubMed Central

Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

2002-01-01

The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC−/ESA+ epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast. PMID:11914275

[Undifferentiated cutaneous angiosarcoma of the head: identification by the endothelial marker Ulex europaeus agglutinin I].

PubMed

Bork, K; Fries, J; Hoede, N; Korting, G W; Dienes, P

1985-06-01

Cutaneous angiosarcoma of the head is a rare tumor of the elderly and can occur in an undifferentiated form without any clinical or histological signs of the vascular origin of this tumor. In these cases, the tumor can be identified by using endothelial cell markers, such as factor-VIII-related antigen and ulex europaeus agglutinin I, in an immunofluorescence technique or a peroxidase-antiperoxidase method. A 78-year-old patient is described who died within 18 months from such a tumor, which was diagnosed using the endothelial cell marker, ulex europaeus agglutinin I.

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

PubMed

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

PubMed

Ishikawa, Masaaki; Ohnishi, Hiroe; Skerleva, Desislava; Sakamoto, Tatsunori; Yamamoto, Norio; Hotta, Akitsu; Ito, Juichi; Nakagawa, Takayuki

2017-06-01

The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

PubMed Central

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

2017-01-01

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission. PMID:28241053

RACK1 regulates mesenchymal cell recruitment during sexual and asexual reproduction of budding tunicates.

PubMed

Tatzuke, Yuki; Sunanaga, Takeshi; Fujiwara, Shigeki; Kawamura, Kaz

2012-08-15

A homolog of receptor for activated protein kinase C1 (RACK1) was cloned from the budding tunicate Polyandrocarpa misakiensis. By RT-PCR and in situ hybridization analyses, PmRACK1 showed biphasic gene expression during asexual and sexual reproduction. In developing buds, the signal was exclusively observed in the multipotent atrial epithelium and undifferentiated mesenchymal cells that contributed to morphogenesis by the mesenchymal-epithelial transition (MET). In juvenile zooids, the signal was first observable in germline precursor cells that arose as mesenchymal cell aggregated in the ventral hemocoel. In mature zooids, the germinal epithelium in the ovary and the pharynx were the most heavily stained parts. GFP reporter assay indicated that the ovarian expression of PmRACK1 was constitutive from germline precursor cells to oocytes. To elucidate the in vivo function of PmRACK1, RNA interference was challenged. When growing buds were incubated with 5 nmol/mL siRNA, most mesenchymal cells remained round and appeared to have no interactions with the extracellular matrix (ECM), causing lower activity of MET without any apparent effects on cell proliferation. The resultant zooids became growth-deficient. The dwarf zooids did not form buds or mature gonads. Prior to RNAi, buds were treated with human BMP4 that could induce PmRACK1 expression, which resulted in MET activity. We conclude that in P. misakiensis, PmRACK1 plays roles in mesenchymal cell recruitment during formation of somatic and gonad tissues, which contributes to zooidal growth and sexual and asexual reproduction. Copyright © 2012 Elsevier Inc. All rights reserved.

Bioactive nanofibers enable the identification of thrombospondin 2 as a key player in enamel regeneration.

PubMed

Huang, Zhan; Newcomb, Christina J; Lei, Yaping; Zhou, Yan; Bornstein, Paul; Amendt, Brad A; Stupp, Samuel I; Snead, Malcolm L

2015-08-01

Tissue regeneration and development involves highly synchronized signals both between cells and with the extracellular environment. Biomaterials can be tuned to mimic specific biological signals and control cell response(s). As a result, these materials can be used as tools to elucidate cell signaling pathways and candidate molecules involved with cellular processes. In this work, we explore enamel-forming cells, ameloblasts, which have a limited regenerative capacity. By exposing undifferentiated cells to a self-assembling matrix bearing RGDS epitopes, we elicited a regenerative signal at will that subsequently led to the identification of thrombospondin 2 (TSP2), an extracellular matrix protein that has not been previously recognized as a key player in enamel development and regeneration. Targeted disruption of the thrombospondin 2 gene (Thbs2) resulted in enamel formation with a disordered architecture that was highly susceptible to wear compared to their wild-type counterparts. To test the regenerative capacity, we injected the bioactive matrix into the enamel organ and discovered that the enamel organic epithelial cells in TSP-null mice failed to polarize on the surface of the artificial matrix, greatly reducing integrin β1 and Notch1 expression levels, which represent signaling pathways known to be associated with TSP2. These results suggest TSP2 plays an important role in regulating cell-matrix interactions during enamel formation. Exploiting the signaling pathways activated by biomaterials can provide insight into native signaling mechanisms crucial for tooth development and cell-based strategies for enamel regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

PubMed Central

Norrie, Jacqueline L.; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C.; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T.; Galli, Antonella; Ji, Hongkai

2016-01-01

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4. Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. PMID:27827819

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

PubMed

Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

2016-12-15

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

PubMed

Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

2011-01-01

Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

[Role of CD2-associated protein in podocyte differentiation.].

PubMed

Jiang, Hua-Jun; Chang, Ying; Zhu, Zhong-Hua; Liu, Jian-She; Deng, An-Guo; Zhang, Chun

2008-02-25

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.

Undifferentiated embryonic cell transcription factor 1 (UTF1) and deleted in azoospermia-like (DAZL) expression in the testes of donkeys.

PubMed

Lee, Y S; Jung, H J; Yoon, M J

2017-04-01

Putative markers for each specific germ cell stage can be a useful tool to study the fate and functions of these cells. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans, rats and horses. The deleted in azoospermia-like (DAZL) protein is also expressed by differentiated spermatogonia and primary spermatocytes in several species. However, whether the expression patterns of these molecular markers are identical and applicable to donkeys remains to be elucidated. The objective of this study was to investigate the expression patterns of UTF1 and DAZL in donkey testicular tissue, using immunohistochemistry (IHC). Testicular samples were collected from routine field castration of donkeys in Korea. The reproductive stages (pre- or post-puberty) of the testes were determined from the morphological characteristics of cross-sections of the seminiferous tubules. For IHC, the UTF1 and DAZL primary antibodies were diluted at 1:100 and 1:200, respectively. The immunolabelling revealed that UTF1 was expressed in approximately 50% of spermatogonia in the pre-pubertal stage, whereas its expression was limited to an early subset of spermatogonia in the post-pubertal stage. DAZL was expressed in some, but not all, spermatogonia in the pre-pubertal spermatogonia, and interestingly, its expression was also observed in spermatogonia and primary spermatocytes in the post-pubertal stage. Co-immunolabelling of the germ cells with both UTF1 and DAZL revealed three types of protein expression patterns at both reproductive stages, namely UTF1 only, DAZL only and both UTF1 and DAZL. These protein molecules were not expressed in Sertoli and Leydig cells. In conclusion, a co-immunolabelling system with UTF1 and DAZL antibodies may be used to identify undifferentiated (UTF1 only), differentiating (UTF1 and DAZL), and differentiated spermatogonia (DAZL only) in donkey testes. © 2017 Blackwell Verlag GmbH.

Enrichment of undifferentiated type a spermatogonia from goat testis using discontinuous percoll density gradient and differential plating.

PubMed

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-04-01

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes.

Enrichment of Undifferentiated Type A Spermatogonia from Goat Testis Using Discontinuous Percoll Density Gradient and Differential Plating

PubMed Central

Heidari, Banafsheh; Gifani, Minoo; Shirazi, Abolfazl; Zarnani, Amir-Hassan; Baradaran, Behzad; Naderi, Mohammad Mehdi; Behzadi, Bahareh; Borjian-Boroujeni, Sara; Sarvari, Ali; Lakpour, Niknam; Akhondi, Mohammad Mehdi

2014-01-01

Background The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Methods Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SSCs (PGP9.5 positive) in each method was compared using ANOVA and comparison between the highest percentage of corresponding value between two methods was carried out by t-test using Sigma Stat (ver. 3.5). Results The highest PGP9.5 (94.6±0.4) and the lowest c-Kit positive (25.1±0.7) in Percoll method was significantly (p ≤ 0.001) achieved in 32% percoll gradient. While the corresponding rates in differential plating method for the highest PGP9.5 positive cells (81.3±1.1) and lowest c-Kit (17.1±1.4) was achieved after 5 hr culturing (p < 0.001). The enrichment of undifferentiated type A spermatogonia using Percoll was more efficient than differential plating method (p < 0.001). Conclusion Percoll density gradient and differential plating were efficient and fast methods for enrichment of type A spermatogonial stem cells from goat testes. PMID:24834311

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds.

PubMed

Ye, Xinhai; Yin, Xiaofan; Yang, Dawei; Tan, Jian; Liu, Guangpeng

2012-07-01

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control*

PubMed Central

Bruscoli, Stefano; Velardi, Enrico; Di Sante, Moises; Bereshchenko, Oxana; Venanzi, Alessandra; Coppo, Maddalena; Berno, Valeria; Mameli, Maria Grazia; Colella, Renato; Cavaliere, Antonio; Riccardi, Carlo

2012-01-01

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis. PMID:22110132

Immunocytochemical characterization of lung tumors in fine-needle aspiration. The use of cytokeratin monoclonal antibodies for the differential diagnosis of squamous cell carcinoma and adenocarcinoma.

PubMed

Bruderman, I; Cohen, R; Leitner, O; Ronah, R; Guber, A; Griffel, B; Geiger, B

1990-10-15

In the current study, immunocytochemical typing of intermediate filaments was used for a differential diagnosis of human lung tumors from transthoracic fine-needle aspiration biopsies (TFNAB). The authors have compared the cytologic diagnosis of 53 lung cancer cases with the immunofluorescence patterns obtained using a panel of monoclonal antibodies, five of which (KG 8.13, KM 4.62, Ks B.17, KS 8.12, KK 8.60) react with specific cytokeratin polypeptides and one with vimentin (VIM 13.2). Only in six of 23 samples cytologically diagnosed as squamous cell carcinoma did the immunocytochemical typing of cytokeratins (ICTC) confirm the cytologic diagnosis. In seven cases some of the tumor cells stained positively with antibody Ks B.17 specific for simple epithelial keratin (No: 18), suggesting the presence of some cells of glandular origin. In ten additional cases the ICTC was in conflict with the cytologic diagnosis of squamous cell carcinoma (i.e., antibodies Ks 8.12 and KK 8.60 were negative, and antibody Ks B.17, positive) supporting a diagnosis of adenocarcinoma. In 14 of 18 cases cytologically diagnosed as adenocarcinoma, the ICTC confirmed the diagnosis whereas in four cases additional presence of some squamous cells was noticed. The ICTC labeling of cases cytologically diagnosed as undifferentiated and large cell carcinomas was similar to that of the group of adenocarcinomas. Thus, the application of cytokeratin typing for TFNAB samples seems to provide a vital complementation to routine cytologic study, especially for cases cytologically diagnosed as squamous carcinoma.

[Intra-abdominal desmoplastic small round cell tumour].

PubMed

Briseño-Hernández, Andrés Alejandro; Quezada-López, Deissy Roxana; Corona-Cobián, Lilia Edith; Castañeda-Chávez, Agar; Duarte-Ojeda, Alfonso Tonatiuh; Macías-Amezcua, Michel Dassaejv

2015-01-01

The desmoplastic small round cell tumour is a rare and aggressive intra-abdominal neoplasia, with only 200 cases reported, and a higher incidence in men and predilection for the second decade of life. Histologically characterized by the presence of small nests of undifferentiated tumour cells, wrapped in fibrous desmoplastic stroma. A 24 year old male started with abdominal pain of 4 weeks onset in the right upper quadrant, colic type, sporadic, self-limiting and accompanied by early satiety, decreased appetite, and involuntary weight loss of 10 kg in 3 months. At the time of admission the abdomen was globular, with decreased peristalsis, soft, depressible. Computed tomography of the abdomen showed multiple enlarged lymph nodes in the abdominal-pelvic cavity. A laparotomy was performed, with a subsequent omentum resection due to the presence of multiple tumours, which microscopically were characterised by groups of small, round, blue cells, separated by a desmoplastic stroma. The immunohistochemistry was positive for desmin (> 75%), epithelial membrane antigen (> 75%), CD99 (> 50%), and S100 (25%), concluding with an abdominal tumour of small, round, blue cells as a diagnosis. Chemotherapy treatment was initiated based on IMAP plus GM-CSF. The desmoplastic small round cell tumour is a rare neoplasia, with diagnostic complexity and a lethal course. Its clinical presentation is unspecific. Histologically, it is classified as an aggressive soft tissue sarcoma that shares similar characteristics with the family of the small and blue cells tumours. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Niche players

PubMed Central

Seandel, Marco; Falciatori, Ilaria; Shmelkov, Sergey V.; Kim, Jiyeon; James, Daylon; Rafii, Shahin

2010-01-01

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines. PMID:18256534

Development of an Aquatic Bioassay Using the Medaka (Oryzias latipes) to Assess Human Health Risks: Tumor Immunodiagnosis.

DTIC Science & Technology

1992-04-27

Skeletal muscle 5 *Undifferentiated sarcoma Peripheral nerve 3 *Undifferentiated sarcoma Peritoneum 1 Hepatocellular carcinoma Liver 2...KD) Neurofilament (3 proteins 68, 150, Detects neuronal cell origin and 200 KD) Other: Alpha-l antitrypsin Common marker for hepatocellular carcinoma Alpha... hepatocellular carcinoma from cholangiocellular carcinoma (8). 14 4 FIGURE 1. Avldln-Blotin-PeroxidSse Complex Tecbnlqu6. :4odif led from A.K.Bhan

The diagnostic and prognostic value of SALL4 in hepatoblastoma.

PubMed

Zhou, Shengmei; Venkatramani, Rajkumar; Gomulia, Ellen; Shillingford, Nick; Wang, Larry

2016-11-01

To investigate the expression of spalt-like transcription factor 4 (SALL4), a regulator of embryonal development, in three epithelial components of hepatoblastoma (HB) and the relationship between SALL4 expression levels and patients' clinicopathological features. A total of 115 specimens from 79 patients with HB were selected for immunostaining of SALL4. Nuclear staining was semi-quantified using the immunoreactive score (IS; range: 0-12). SALL4 expression was seen in all embryonal components (mean IS = 8.58) and in 41% of fetal components (mean IS = 0.78). No SALL4 expression was seen in either small cell undifferentiated or mesenchymal components of HB. Neither chemotherapy nor metastasis altered SALL4 expression significantly. High SALL4 expression levels were associated significantly with decreased overall survival (OS) (P = 0.004), event-free survival (EFS) (P = 0.003) and the presence of metastasis (P = 0.049) on univariate analysis. Multivariate analysis identified SALL4 as an independent prognostic predictor for OS (P = 0.029). SALL4 is useful for subtyping HB, and high SALL4 expression is associated with decreased survival in HB. © 2016 John Wiley & Sons Ltd.

The neuronal differentiation process involves a series of antioxidant proteins.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Hengstschläger, M; Pollak, A; Lubec, G

2005-11-01

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.

MV-NIS Infected Mesenchymal Stem Cells in Treating Patients With Recurrent Ovarian Cancer

ClinicalTrials.gov

2018-01-31

Malignant Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Ovarian Carcinoma

Relationship between ketamine-induced developmental neurotoxicity and NMDA receptor-mediated calcium influx in neural stem cell-derived neurons.

PubMed

Wang, Cheng; Liu, Fang; Patterson, Tucker A; Paule, Merle G; Slikker, William

2017-05-01

Ketamine, a noncompetitive NMDA receptor antagonist, is used as a general anesthetic and recent data suggest that general anesthetics can cause neuronal damage when exposure occurs during early brain development. To elucidate the underlying mechanisms associated with ketamine-induced neurotoxicity, stem cell-derived models, such as rodent neural stem cells harvested from rat fetuses and/or neural stem cells derived from human induced pluripotent stem cells (iPSC) can be utilized. Prolonged exposure of rodent neural stem cells to clinically-relevant concentrations of ketamine resulted in elevated NMDA receptor levels as indicated by NR1subunit over-expression in neurons. This was associated with enhanced damage in neurons. In contrast, the viability and proliferation rate of undifferentiated neural stem cells were not significantly affected after ketamine exposure. Calcium imaging data indicated that 50μM NMDA did not cause a significant influx of calcium in typical undifferentiated neural stem cells; however, it did produce an immediate elevation of intracellular free Ca 2+ [Ca 2+ ] i in differentiated neurons derived from the same neural stem cells. This paper reviews the literature on this subject and previous findings suggest that prolonged exposure of developing neurons to ketamine produces an increase in NMDA receptor expression (compensatory up-regulation) which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to neuronal cell death likely due to elevated reactive oxygen species generation. The absence of functional NMDA receptors in cultured neural stem cells likely explains why clinically-relevant concentrations of ketamine did not affect undifferentiated neural stem cell viability. Published by Elsevier B.V.

Effects of cellular differentiation, chromosomal integration and 5-aza-2'-deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines.

PubMed

Kalantari, Mina; Lee, Denis; Calleja-Macias, Itzel E; Lambert, Paul F; Bernard, Hans-Ulrich

2008-05-10

Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosomal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral life cycle, (ii) integration of the viral genome into the host chromosome events leads to an alteration in methylation patterns on the viral genome that is dependent upon the type of integration event and possibly copy number, and (iii) integration universally results in the viral DNA becoming refractory to changes in methylation state upon cellular differentiation that are observed with extrachromosomal HPV-16 genomes.

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

PubMed

Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

2016-05-06

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

PubMed

Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

2014-04-01

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

[Preinvasive lesions in gynecology -  vulva].

PubMed

Chovanec, J; Mouková, L; Feranec, R

2013-01-01

For preinvasive lesions of vulva, a common term VIN -  vulval intraepithelial neoplasia is used. VIN is a histological dia-gnosis based on abnormal squamous epithelial proliferation. There are two types of VIN apart from their association with human papillomavirus (HPV). Undifferentiated (usual) vulval intraepithelial neoplasia is commonly associated with carcinogenic genotypes of HVP, whereas differentiated vulval intraepithelial neoplasia is not associated with high-risk genotypes of HPV. The article presents an overview of VIN occurence and epidemiology, its classification system and dia-gnostics. In conclusion, VIN therapeutical possibilities are presented. It can be treated with surgical therapy (local excision, partial vulvectomy, vulvectomy, laser vaporization) or medical therapy (imiquimod).

Cell polarity proteins and spermatogenesis.

PubMed

Gao, Ying; Xiao, Xiang; Lui, Wing-Yee; Lee, Will M; Mruk, Dolores; Cheng, C Yan

2016-11-01

When the cross-section of a seminiferous tubule from an adult rat testes is examined microscopically, Sertoli cells and germ cells in the seminiferous epithelium are notably polarized cells. For instance, Sertoli cell nuclei are found near the basement membrane. On the other hand, tight junction (TJ), basal ectoplasmic specialization (basal ES, a testis-specific actin-rich anchoring junction), gap junction (GJ) and desmosome that constitute the blood-testis barrier (BTB) are also located near the basement membrane. The BTB, in turn, divides the epithelium into the basal and the adluminal (apical) compartments. Within the epithelium, undifferentiated spermatogonia and preleptotene spermatocytes restrictively reside in the basal compartment whereas spermatocytes and post-meiotic spermatids reside in the adluminal compartment. Furthermore, the heads of elongating/elongated spermatids point toward the basement membrane with their elongating tails toward the tubule lumen. However, the involvement of polarity proteins in this unique cellular organization, in particular the underlying molecular mechanism(s) by which polarity proteins confer cellular polarity in the seminiferous epithelium is virtually unknown until recent years. Herein, we discuss latest findings regarding the role of different polarity protein complexes or modules and how these protein complexes are working in concert to modulate Sertoli cell and spermatid polarity. These findings also illustrate polarity proteins exert their effects through the actin-based cytoskeleton mediated by actin binding and regulatory proteins, which in turn modulate adhesion protein complexes at the cell-cell interface since TJ, basal ES and GJ utilize F-actin for attachment. We also propose a hypothetical model which illustrates the antagonistic effects of these polarity proteins. This in turn provides a unique mechanism to modulate junction remodeling in the testis to support germ cell transport across the epithelium in particular the BTB during the epithelial cycle of spermatogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

PubMed

Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2016-01-01

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Twenty years of embryonic stem cell research in farm animals

USDA-ARS?s Scientific Manuscript database

Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase.

PubMed

Heumann, D; Losa, G; Barras, C; Morell, A; von Fliedner, V

1985-08-01

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma-GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma-GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT-blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma-GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

Metabolism of two Go alpha isoforms in neuronal cells during differentiation.

PubMed

Brabet, P; Pantaloni, C; Bockaert, J; Homburger, V

1991-07-15

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

PubMed Central

Féraud, Olivier; Valogne, Yannick; Melkus, Michael W.; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

2016-01-01

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process. PMID:26938212

Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

EPA Science Inventory

The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

PubMed

Shirogane, Yuta; Takeda, Makoto; Tahara, Maino; Ikegame, Satoshi; Nakamura, Takanori; Yanagi, Yusuke

2010-07-02

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.

Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans.

PubMed

Iskandar, Irma; Walters, John D

2011-03-01

Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 μg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.

Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

PubMed

Abeysinghe, Hima C S; Bokhari, Laita; Quigley, Anita; Choolani, Mahesh; Chan, Jerry; Dusting, Gregory J; Crook, Jeremy M; Kobayashi, Nao R; Roulston, Carli L

2015-09-29

Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E(2).

PubMed

Schlemmer, Scott R; Kaufman, David G

2012-12-01

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE(2,) we found enhanced GJIC with 1nM PGE(2). This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE(2) was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE(2) secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE(2) to the cells. Our findings show that PGE(2) may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE(2) secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Intensity-Modulated or Proton Radiation Therapy for Sinonasal Malignancy

ClinicalTrials.gov

2018-02-13

Adenoid Cystic Carcinoma; Squamous Cell Carcinoma; Sinonasal Carcinoma; Sinonasal Undifferentiated Carcinoma; Mucoepidermoid Carcinoma; Schneiderian Carcinoma; Myoepithelial Carcinoma; Esthesioneuroblastoma; Melanoma

[Incidence of anaplastic tumor in structure of other histologic forms of the thyroid gland cancer].

PubMed

Vinnik, Iu A; Gorbenko, V N; Vas'ko, A R; Kikhtenko, E V; Gargin, V V

2014-01-01

The degrees of invasiveness, proliferative activity, morphofunctional activity of nuclei in the thyroidal gland tumors were studied, while analyzing material, obtained in 1343 patients, suffering thyroidal gland cancer (THGC) and operated on in 2000-2013 yrs. Morphological point quantity of malignancy (as a criterion of the tumor progression grade) and mitotic activity in cellular population were determined in various kinds of THGC. Undifferentiated (anaplastic carcinoma) type of THGC is the most malignant one. There were determined a spindle-like, giant-cell and squamous-cell forms of undifferentiated THGC. The presence of sites of differentiated cancer in 33% of histological preparations witnesses the interrelationship with the earlier existed pathological process.

Use of electron microscopy to classify canine perivascular wall tumors.

PubMed

Palmieri, C; Avallone, G; Cimini, M; Roccabianca, P; Stefanello, D; Della Salda, L

2013-03-01

The histologic classification of canine perivascular wall tumors (PWTs) is controversial. Many PWTs are still classified as hemangiopericytomas (HEPs), and the distinction from peripheral nerve sheath tumors (PNSTs) is still under debate. A recent histologic classification of canine soft tissue sarcomas included most histologic types of PWT but omitted those that were termed undifferentiated. Twelve cases of undifferentiated canine PWTs were evaluated by transmission electron microscopy. The ultrastructural findings supported a perivascular wall origin for all cases with 4 categories of differentiation: myopericytic (n = 4), myofibroblastic (n = 1), fibroblastic (n = 2), and undifferentiated (n = 5). A PNST was considered unlikely in each case based on immunohistochemical expression of desmin and/or the lack of typical ultrastructural features, such as basal lamina. Electron microscopy was pivotal for the subclassification of canine PWTs, and the results support the hypothesis that canine PWTs represent a continuum paralleling the phenotypic plasticity of vascular mural cells. The hypothesis that a subgroup of PWTs could arise from a pluripotent mesenchymal perivascular wall cell was also considered and may explain the diverse differentiation of canine PWTs.

Adoptive TReg Cell for Suppression of aGVHD After UCB HSCT for Heme Malignancies

ClinicalTrials.gov

2018-03-26

Acute Lymphoblastic Leukemia; Burkitt Lymphoma; Natural Killer Cell Malignancies; Chronic Myelogenous Leukemia; Myelodysplastic Syndromes; Large-cell Lymphoma; Chronic Lymphocytic Leukemia; Small Lymphocytic Lymphoma; Marginal Zone B-Cell Lymphoma; Follicular Lymphoma; Lymphoplasmacytic Lymphoma; Mantle-Cell Lymphoma; Prolymphocytic Leukemia; Hodgkin Lymphoma; Multiple Myeloma; Acute Myelogenous Leukemia; Biphenotypic Leukemia; Undifferentiated Leukemia

The Cell-Surface N-Glycome of Human Embryonic Stem Cells and Differentiated Hepatic Cells thereof.

PubMed

Montacir, Houda; Freyer, Nora; Knöspel, Fanny; Urbaniak, Thomas; Dedova, Tereza; Berger, Markus; Damm, Georg; Tauber, Rudolf; Zeilinger, Katrin; Blanchard, Véronique

2017-07-04

Human embryonic stem cells (hESCs) are pluripotent stem cells that offer a wide range of applications in regenerative medicine. In addition, they have been proposed as an appropriate alternative source of hepatocytes. In this work, hESCs were differentiated into definitive endodermal cells (DECs), followed by maturation into hepatocyte-like cells (HLCs). Their cell-surface N-glycome was profiled and also compared with that of primary human hepatocytes (PHHs). Undifferentiated hESCs contained large amounts of high-mannose N-glycans. In contrast, complex-type N-glycans such as asialylated or monosialylated biantennary and triantennary N-glycans were dominant in HLCs, and fully galactosylated structures were significantly more abundant than in undifferentiated hESCs. The cell-surface N-glycosylation of PHHs was more biologically processed than that of HLCs, with bisialylated biantennary and trisialylated triantennary structures predominant. This is the first report of the cell surface N-glycome of PHHs and of HLCs being directly generated from hESCs without embryoid body formation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mice cloned from skin cells.

PubMed

Li, Jinsong; Greco, Valentina; Guasch, Géraldine; Fuchs, Elaine; Mombaerts, Peter

2007-02-20

Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar (<6% per transferred blastocyst), success rates were consistently higher for males than for females. Adult keratinocyte stem cells were better NT donors than so-called transit amplifying (TA) keratinocytes in both sexes (1.6% vs. 0% in females and 5.4% vs. 2.8% in males). Our findings reveal skin as a source of readily accessible stem cells, the nuclei of which can be reprogrammed to the pluripotent state by exposure to the cytoplasm of unfertilized oocytes.

Epithelial Cell–Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia

PubMed Central

Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A.; Zabinski, Mary C.; Yuen, Constance K.; Lung, Wing Yi; Gower, Adam C.; Belkina, Anna C.; Ramirez, Maria I.; Deng, Jane C.; Quinton, Lee J.; Jones, Matthew R.

2016-01-01

Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6GbrightCD11bbright neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia. PMID:27064756

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars.

PubMed

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-06-29

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

PubMed Central

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-01-01

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis. PMID:26160988

Multiple Cellular Responses to Serotonin Contribute to Epithelial Homeostasis

PubMed Central

Pai, Vaibhav P.; Horseman, Nelson D.

2011-01-01

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers). PMID:21390323

Epithelial Cells in Urine: MedlinePlus Lab Test Information

MedlinePlus

... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Joo-ho; Pollak, Arnold; Freilinger, Angelika; Hengstschläger, Markus; Lubec, Gert

2005-03-01

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.

Osteopontin is a Novel Marker of Pancreatic Ductal Tissues and of Undifferentiated Pancreatic Precursors in Mice

PubMed Central

Kilic, Gamze; Wang, Junfeng; Sosa-Pineda, Beatriz

2008-01-01

Matricellular proteins mediate both tissue morphogenesis and tissue homeostasis in important ways because they modulate cell-matrix and cell-cell interactions. In this study, we found that the matricellular protein osteopontin (Opn) is a novel marker of undifferentiated pancreatic precursors and pancreatic ductal tissues in mice. Our analysis also underscored a specific, dynamic profile of Opn expression in embryonic pancreatic tissues that suggests the participation of this protein’s function in processes involving cell migration, cell-cell interactions, or both. Surprisingly, our analysis of Opn-deficient pancreata did not reveal obvious alterations in the morphology or differentiation of these tissues. Therefore, in embryonic pancreatic tissues, it is possible that other proteins act redundantly to Opn or that this protein’s function is dispensable for pancreas development. Finally, the maintenance of Opn expression in pancreatic tissues of adults argues for a possible function of this protein in injury and pathologic responses. PMID:16518820

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

PubMed

Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

2016-08-01

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. © 2016 International Federation for Cell Biology.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

PubMed

Morita, Maresuke; Fujita, Naoki; Abe, Momoko; Hayashimoto, Koji; Nakagawa, Takayuki; Nishimura, Ryohei; Tsuzuki, Keiko

2018-06-01

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We foundmore » that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.« less

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Bovine rumen epithelium undergoes rapid structural adaptations during grain-induced subacute ruminal acidosis.

PubMed

Steele, Michael A; Croom, Jim; Kahler, Melissa; AlZahal, Ousama; Hook, Sarah E; Plaizier, Kees; McBride, Brian W

2011-06-01

Alterations in rumen epithelial structure and function during grain-induced subacute ruminal acidosis (SARA) are largely undescribed. In this study, four mature nonlactating dairy cattle were transitioned from a high-forage diet (HF; 0% grain) to a high-grain diet (HG; 65% grain). After feeding the HG diet for 3 wk, the cattle were transitioned back to the original HF diet, which was fed for an additional 3 wk. Continuous ruminal pH was measured on a weekly basis, and rumen papillae were biopsied during the baseline and at the first and final week of each diet. The mean, minimum, and maximum daily ruminal pH were depressed (P < 0.01) in the HG period compared with the HF period. During the HG period, SARA was diagnosed only during week 1, indicating ruminal adaptation to the HG diet. Microscopic examination of the papillae revealed a reduction (P < 0.01) in the stratum basale, spinosum, and granulosum layers, as well as total depth of the epithelium during the HG period. The highest (P < 0.05) papillae lesion scores were noted during week 1 when SARA occurred. Biopsied papillae exhibited a decline in cellular junctions, extensive sloughing of the stratum corneum, and the appearance of undifferentiated cells near the stratum corneum. Differential mRNA expression of candidate genes, including desmoglein 1 and IGF binding proteins 3, 5, and 6, was detected between diets using qRT-PCR. These results suggest that the structural integrity of the rumen epithelium is compromised during grain feeding and is associated with the differential expression of genes involved in epithelial growth and structure.

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

PubMed Central

Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

2014-01-01

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

Identifying Tumor Progenitor Cells | Center for Cancer Research

Cancer.gov

All cells within a tumor are not identical. In fact, only a small subset appears to be capable of actually generating the tumor. These tumor-initiating cells tend to resemble normal stem cells, which have the unique ability to give rise to differentiated cells while simultaneously producing additional undifferentiated stem cells. Most chemotherapeutics affect the bulk of a

UTF1, a Putative Marker for Spermatogonial Stem Cells in Stallions

PubMed Central

Jung, Heejun; Roser, Janet F.; Yoon, Minjung

2014-01-01

Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of spermatogonia that express UTF1. Testicular samples were collected and categorized based on the age of the horses as follows: pre-pubertal (<1 yr), pubertal (1–1.5 yr), post-pubertal (2–3 yr), and adult (4–8 yr). Western blot analysis was utilized to determine the cross-activity of the UTF1 antibody to horse testes tissues. Immunohistochemistry was conducted to investigate the UTF1 expression pattern in germ cells at different reproductive stages. Whole mount staining was applied to determine the subpopulation of UTF1-positive spermatogonia. Immunohistological analysis showed that most germ cells in the pre-pubertal and pubertal stages were immunolabeled with UTF1, whereas only a few germ cells in the basal compartment of the seminiferous tubule cross-sections of post-pubertal and adult tissues were UTF1-positive. No staining was observed in the Sertoli or Leydig cells at any reproductive stages. Whole mount staining showed that As, Apr, and chains of 4, 8, 16 Aal spermatogonia were immunolabeled with UTF1 in the post-pubertal stallion tubule. Isolated single germ cells were also immunolabeled with UTF1. In conclusion, UTF1 is expressed in undifferentiated spermatogonia, and its antibody can be used as a putative marker for SSCs in stallions. PMID:25272017

Morphological differentiation of N1E-115 neuroblastoma cells by dimethyl sulfoxide activation of lipid second messengers.

PubMed

Clejan, S; Dotson, R S; Wolf, E W; Corb, M P; Ide, C F

1996-04-10

Quantitative changes in the lipid second messenger diacylglycerol (DAG) were studied in the rat neuroblastoma N1E-115 following exposure to the differentiating agent dimethylsulfoxide (DMSO). Relatively high basal levels of DAG are present in these cells, and addition of 2% DMSO elicited a biphasic increase in DAG levels, dependent on the presence of extracellular Ca2+. Exposure to DMSO also elicited a rapid increase in inositol phosphate and a slight increase in phosphatidic acid (PA), trailing that of DAG. The molecular species (MS) of DAG were analyzed. Within 60 s of DMSO application there were transient increases of DAG representative of phosphatidylinositol (PI) hydrolysis. At longer intervals, more DAG originated from phosphatidylcholine. The MS composition of newly formed PA resembled that of PI and native DAG. Inhibition studies indicated that DAG is formed in the DMSO-treated cells by phospholipases C and that PA formed later is a result of DAG phosphorylation and not activity of phospholipase D (PLD). Undifferentiated cells exhibited an active PLD pathway. In contrast, PLD in DMSO-differentiated cells was not active. In examining the involvement of the sphingomyelin pathway, DMSO exposure was found to increase ceramide levels with a concomitant decrease in sphingomyelin. Addition of the exogenous, soluble analog C6-ceramide to undifferentiated cells resulted in dramatic reductions in DAG and PA levels and PLD activity. These results indicate that DMSO treatment inactivates PLD while activating phospholipases C and the sphingomyelin pathway, suggesting a "switch" between signal transduction pathways in the undifferentiated and differentiated states of N1E-115.

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

PubMed

Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

2005-04-01

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations of reduced NPSH in renal epithelial cells than in hepatocytes.

Potential Role for a Carbohydrate Moiety in Anti-Candida Activity of Human Oral Epithelial Cells

PubMed Central

Steele, Chad; Leigh, Janet; Swoboda, Rolf; Ozenci, Hatice; Fidel, Paul L.

2001-01-01

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose- and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety. PMID:11598085

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

PubMed

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

2016-11-15

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

PubMed Central

Grodzki, Ana Cristina G.; Giulivi, Cecilia; Lein, Pamela J.

2013-01-01

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology. PMID:23355903

Pathological modifications of plant stem cell destiny

USDA-ARS?s Scientific Manuscript database

In higher plants, the shoot apex contains undifferentiated stem cells that give rise to various tissues and organs. The fate of these stem cells determines the pattern of plant growth as well as reproduction; and such fate is genetically preprogrammed. We found that a bacterial infection can derai...

Histogenesis of the epithelial component of rat thymus: an ultrastructural and immunohistological analysis.

PubMed

Vicente, A; Varas, A; Sacedón, R; Zapata, A G

1996-04-01

Despite the assumed importance of thymic cell microenvironments for governing T-cell maturation, little is known about the ontogeny of their cell components. A few studies have analyzed previously the ontogenetical development of rat thymic epithelium (Bogojevic et al. 1990. Period. Biol., 92:126; Kampinga and Aspinall 1990 Harwood Acad. Pub., London, pp. 149-186; Micic et al., 1991 Dev. Comp. Immunol., 15:443-450) and recently we have reported the development of both interdigitating/dendritic cells and macrophages (Vicente et al., 1994 Immunology, 82:75-81, 1995 Immunology, 85:99-105). In the present work we analyze in situ ultrastructural, immunohistochemical, and histoenzymatically the appearance and development of the thymic epithelial cell component in both embryonic and neonatal Wistar rats with special emphasis on the origin of the different epithelial cell types, the occurrence or absence of a common precursor for these, and the expression of MHC molecules. The thymic primordium of 13-day-old embryos is formed by a homogeneous population of primitive epithelial cells differentiating gradually into various epithelial cell subtypes of both the cortex and the medulla. In the cortex, subcapsular and stroma-supporting epithelial cells appear at days 14-15 as two structurally different cell entities. At the same time, stroma-supporting, keratinized, and vacuolated epithelial cells occur in the thymic medulla. These last two cell types differentiate subsequently into Hassall's bodies and hypertrophied cells. Lympho-epithelial cell complexes are identified in the deep cortex around birth, when the cortical parenchyma houses a transitional erythropoiesis. mAbs (His-39, RMC-20) which recognize medullary epithelial cells in the adult thymus stain positively cells of the thymic primordium as early as day 16 of embryonic life. Cortical epithelial cell markers (His-37, RMC-17) appear, however, slightly later and the subcapsulary region is not established until postnatal life. MHC class I and class II molecules can be identified on epithelial cells in the thymus of 15-day-old embryonic rats although they reach the highest expression around birth. Our results confirm the heterogeneity of the thymic epithelial component, the persistence of primitive, non-differentiated epithelial cells morphologically similar to those occurring in the early thymic primordium in adult thymus, and the mutual relevance of epithelial cells and thymocytes for an adequate development of rat thymus gland.

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

PubMed Central

Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

2012-01-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

PubMed

Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

2012-02-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

PubMed

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

2015-11-01

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.

The world of epithelial sheets.

PubMed

Honda, Hisao

2017-06-01

An epithelium is a layer of closely connected cells covering the body or lining a body cavity. In this review, several fundamental questions are addressed regarding the epithelium. (i) While an epithelium functions as barrier against the external environment, how is barrier function maintained during its construction? (ii) What determines the apical and basal sides of epithelial layer? (iii) Is there any relationship between the apical side of the epithelium and the apical membrane of an epithelial cell? (iv) Why are hepatocytes (liver cells) called epithelial, even though they differ completely from column-like shape of typical epithelial cells? Keeping these questions in mind, multiple shapes of epithelia were considered, extracting a few of their elemental processes, and constructing a virtual world of epithelia by combining them. Epithelial cells were also classified into several types based on the number of apical domains of each cell. In addition, an intracellular organelle was introduced within epithelial cells, the vacuolar apical compartment (VAC), which is produced within epithelial cells surrounded by external cell matrix (ECM). The VAC interacts with areas of cell-cell contact of the cell surface membrane and is converted to apical membrane. The properties of VACs enable us to answer the initial questions posed above. Finally, the genetic and molecular mechanisms of epithelial morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES ...

Epstein-Barr Virus Infection of Polarized Epithelial Cells via the Basolateral Surface by Memory B Cell-Mediated Transfer Infection

PubMed Central

Shannon-Lowe, Claire; Rowe, Martin

2011-01-01

Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b–negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo. PMID:21573183

Palliative Care in Improving Quality of Life and Symptoms in Patients With Stage III-IV Pancreatic or Ovarian Cancer

ClinicalTrials.gov

2014-12-18

Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Pancreatic Cancer; Stage III Pancreatic Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Pancreatic Cancer

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

PubMed

Hu, Tao; Abbah, Sunny Akogwu; Toh, Soo Yein; Wang, Ming; Lam, Raymond Wing Moon; Naidu, Mathanapriya; Bhakta, Gajadhar; Cool, Simon M; Bhakoo, Kishore; Li, Jun; Goh, James Cho-Hong; Wong, Hee-Kit

2015-12-01

The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. A prospective study using a rodent model of posterolateral spinal fusion was carried out. Thirty-six syngeneic Fischer rats comprised the patient sample. Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct. Copyright © 2015 Elsevier Inc. All rights reserved.

Isolation, separation, and characterization of epithelial and connective cells from rat palate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terranova, Victor Paul

1979-01-01

Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means ofmore » labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.« less

Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish

PubMed Central

Nóbrega, Rafael Henrique; Greebe, Caaj Douwe; van de Kant, Henk; Bogerd, Jan; de França, Luiz Renato; Schulz, Rüdiger W.

2010-01-01

Background Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called “niche” which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate. Methodology/Principal Findings Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5′-bromo-2′-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish. Conclusions/Significance We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs. PMID:20862221

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

PubMed

Cruickshank, S M; Southgate, J; Selby, P J; Trejdosiewicz, L K

1998-10-01

Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation.

PubMed

Menon, Alessandra; Creo, Pasquale; Piccoli, Marco; Bergante, Sonia; Conforti, Erika; Banfi, Giuseppe; Randelli, Pietro; Anastasia, Luigi

2018-01-01

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the "hypoxic niches" present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

5-Azacitidine Monotherapy Followed by Related Haploidentical Hematopoietic Stem Cell Transplantation Achieves Durable Remission in a Pediatric Patient With Acute Undifferentiated Leukemia Refractory to High-Dose Chemotherapy.

PubMed

Polishchuk, Veronika; Khazal, Sajad; Berulava, Giorgi; Roth, Michael; Mahadeo, Kris M

2016-06-01

Patients with acute leukemias of undifferentiated lineage (AUL) generally have guarded prognosis. Here, we describe the first reported pediatric patient with AUL refractory to high-dose chemotherapy who achieved clinical remission with ALL maintenance therapy and 5-azacitidine. His induction remission was followed by consolidation with reduced toxicity haploidentical hematopoietic stem cell transplant (HSCT). At 9 months post-HSCT, the patient is alive and in remission. This combination therapy of remission induction with ALL maintenance therapy and 5-azacitidine and consolidation with reduced toxicity haploidentical HSCT is novel and promising for patients who lack conventional donors and are not candidates for myeloablative therapy. © 2016 Wiley Periodicals, Inc.

Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

PubMed Central

Arlt, Volker M.; Indra, Radek; Joel, Madeleine; Stiborová, Marie; Eardley, Ian; Ahmad, Niaz; Otto, Wolfgang; Burger, Maximilian; Rubenwolf, Peter; Phillips, David H.; Southgate, Jennifer

2018-01-01

Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies. PMID:29323757

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

PubMed

Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

2017-08-15

Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Mechanical stretch triggers rapid epithelial cell division through Piezo1.

PubMed

Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

2017-03-02

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells

PubMed Central

Fu, Xing; Du, Min

2018-01-01

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health. PMID:29643147

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

PubMed

Shi, Yanhong; Chichung Lie, D; Taupin, Philippe; Nakashima, Kinichi; Ray, Jasodhara; Yu, Ruth T; Gage, Fred H; Evans, Ronald M

2004-01-01

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

PubMed Central

Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

2015-01-01

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

PubMed

Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

2013-09-24

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition. Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model.

PubMed

Chang, Chih-Hung; Kuo, Tzong-Fu; Lin, Feng-Huei; Wang, Jyh-Horng; Hsu, Yuan-Ming; Huang, Huei-Ting; Loo, Shiao-Tung; Fang, Hsu-Wei; Liu, Hwa-Chang; Wang, Wen-Chih

2011-12-01

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF-β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF-β-induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF-β-induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel-only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF-β-induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects. Copyright © 2011 Orthopaedic Research Society.

The epithelial-mesenchymal transition generates cells with properties of stem cells.

PubMed

Mani, Sendurai A; Guo, Wenjun; Liao, Mai-Jing; Eaton, Elinor Ng; Ayyanan, Ayyakkannu; Zhou, Alicia Y; Brooks, Mary; Reinhard, Ferenc; Zhang, Cheng Cheng; Shipitsin, Michail; Campbell, Lauren L; Polyak, Kornelia; Brisken, Cathrin; Yang, Jing; Weinberg, Robert A

2008-05-16

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

PubMed

Smith, I M; Baker, A; Arneborg, N; Jespersen, L

2015-11-01

The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics. © 2015 The Society for Applied Microbiology.

Dedifferentiated endometrial carcinomas with neuroendocrine features: a clinicopathologic, immunohistochemical, and molecular genetic study.

PubMed

Espinosa, Iñigo; De Leo, Antonio; D'Angelo, Emanuela; Rosa-Rosa, Juan M; Corominas, Marina; Gonzalez, Alan; Palacios, José; Prat, Jaime

2018-02-01

Undifferentiated endometrial carcinoma is an aggressive type of uterine cancer, which is occasionally associated with a low-grade endometrioid carcinoma component. This combination is referred to as "dedifferentiated endometrioid endometrial carcinoma." Neuroendocrine expression may occur in undifferentiated endometrial carcinoma, but its significance in dedifferentiated endometrial carcinomas is unknown. To gain insight into the pathogenesis of these tumors we have analyzed the immunophenotype (ARID1A, MLH1, PMS2, MSH2, MSH6, p53, β-catenin, SMARCB1, synaptophysin, chromogranin A, and CD56) and mutational status (PTEN, KRAS, PIK3CA, TP53 and POLE) of 4 dedifferentiated endometrial carcinomas with strong and diffuse neuroendocrine expression. All tumors demonstrated neuroendocrine expression in ≥70% of the cells in the undifferentiated carcinoma areas. Loss of expression of at least 1 DNA mismatch repair protein was observed in 2 cases, and p53 immunoreaction was aberrant (mutated/inactivated) in one case. All carcinomas were negative for β-catenin and maintained nuclear SMARCB1 (INI1) and ARID1A expression. Three tumors shared identical endometrioid molecular profile (PTEN and/or PIK3CA mutations) in both components. One tumor had POLE exonuclease domain mutation in the undifferentiated component. In one case, TP53 mutation was found exclusively in the undifferentiated component. Two patients died with peritoneal carcinomatosis and abdominal metastases, respectively; one patient died of a renal failure without evidence of disease, and the last patient is alive and free of disease at 3.3 years. Dedifferentiated endometrial carcinomas with neuroendocrine features are clinically and molecularly heterogeneous tumors. Probably, these carcinomas might acquire undifferentiated phenotype through mutations in TP53 and POLE. Copyright © 2017 Elsevier Inc. All rights reserved.

Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

PubMed

Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

2015-01-01

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

PubMed

Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

2015-05-01

Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. © 2014 AlphaMed Press.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

PubMed Central

Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

2017-01-01

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

Cystic fibrosis epithelial cells are primed for apoptosis as a result of increased Fas (CD95).

PubMed

Chen, Qiwei; Pandi, Sudha Priya Soundara; Kerrigan, Lauren; McElvaney, Noel G; Greene, Catherine M; Elborn, J Stuart; Taggart, Clifford C; Weldon, Sinéad

2018-02-24

Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells. Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines. Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR. Copyright © 2018 European Cystic Fibrosis Society. All rights reserved.

Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing.

PubMed

Lin, Louis M; Huang, George T-J; Rosenberg, Paul A

2007-08-01

There is continuing controversy regarding the potential for inflammatory apical cysts to heal after nonsurgical endodontic therapy. Molecular cell biology may provide answers to a series of related questions. How are the epithelial cell rests of Malassez stimulated to proliferate? How are the apical cysts formed? How does the lining epithelium of apical cysts regress after endodontic therapy? Epithelial cell rests are induced to divide and proliferate by inflammatory mediators, proinflammatory cytokines, and growth factors released from host cells during periradicular inflammation. Quiescent epithelial cell rests can behave like restricted-potential stem cells if stimulated to proliferate. Formation of apical cysts is most likely caused by the merging of proliferating epithelial strands from all directions to form a three-dimensional ball mass. After endodontic therapy, epithelial cells in epithelial strands of periapical granulomas and the lining epithelium of apical cysts may stop proliferating because of a reduction in inflammatory mediators, proinflammatory cytokines, and growth factors. Epithelial cells will also regress because of activation of apoptosis or programmed cell death through deprivation of survival factors or by receiving death signals during periapical wound healing.

Vibrational mapping of sinonasal lesions by Fourier transform infrared imaging spectroscopy

NASA Astrophysics Data System (ADS)

Giorgini, Elisabetta; Sabbatini, Simona; Conti, Carla; Rubini, Corrado; Rocchetti, Romina; Re, Massimo; Vaccari, Lisa; Mitri, Elisa; Librando, Vito

2015-12-01

Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes in tumoral tissues. The head and neck region is characterized by a great variety of lesions, with different degrees of malignancy, which are often difficult to diagnose. Schneiderian papillomas are sinonasal benign neoplasms arising from the Schneiderian mucosa; they can evolve into malignant tumoral lesions (squamous cell carcinoma). In addition, they can sometimes be confused with the more common inflammatory polyps. Therefore, an early and definitive diagnosis of this pathology is mandatory. Progressing in our research on the study of oral cavity lesions, 15 sections consisting of inflammatory sinonasal polyps, benign Schneiderian papillomas, and sinonasal undifferentiated carcinomas were analyzed using FTIRI. To allow a rigorous description of these pathologies and to gain objective diagnosis, the epithelial layer and the adjacent connective tissue of each section were separately investigated by following a multivariate analysis approach. According to the nature of the lesion, interesting modifications were detected in the average spectra of the different tissue components, above all in the lipid and protein patterns. Specific band-area ratios acting as spectral markers of the different pathologies were also highlighted.

In vivo retroviral gene transfer into human bronchial epithelia of xenografts.

PubMed

Engelhardt, J F; Yankaskas, J R; Wilson, J M

1992-12-01

Cystic fibrosis (CF) is the most common lethal inherited disease in the Caucasian population with an incidence of approximately 1 in 2,500 live births. Pulmonary complications of CF, which are the most morbid aspects of the disease, are caused by primary abnormalities in epithelial cells that lead to impaired mucociliary clearance. One potential therapeutic strategy is to reconstitute expression of the CF gene in airway epithelia by somatic gene transfer. To this end, we have developed an animal model of the human airway using bronchial xenografts and have tested the efficiency of in vivo retroviral gene transfer. Using the LacZ reporter gene, we find the efficiency of in vivo retroviral gene transfer to be dramatically dependent on the regenerative and mitotic state of the epithelium. Within an undifferentiated regenerating epithelium in which 40% of nuclei labeled with BrdU, 5-10% retroviral gene transfer was obtained. In contrast, no gene transfer was noted in a fully differentiated epithelium in which 1% of nuclei labeled with BrdU. These findings suggest that retroviral mediated gene transfer to the airway in vivo may be feasible if the proper regenerative state can be induced.

Production Methods for a Mesenchymal Stem Cell Therapeutic as a Medical Defense Countermeasure

DTIC Science & Technology

2012-02-01

differentiation of murine embryonic stem cells into vascular progenitors. BMC Cell Biol. 2008;9:2. 56. Johnson EA, Dao TL, Kan RK. Status epilepticus ...their undifferentiated and multipotent status . BMC Cell Biol. 2011;12:12. 52. Sun Y, Chen L, Hou XG, Hou WK, Dong JJ, Sun L, et al. Differentiation of

A Stochastic Polygons Model for Glandular Structures in Colon Histology Images.

PubMed

Sirinukunwattana, Korsuk; Snead, David R J; Rajpoot, Nasir M

2015-11-01

In this paper, we present a stochastic model for glandular structures in histology images of tissue slides stained with Hematoxylin and Eosin, choosing colon tissue as an example. The proposed Random Polygons Model (RPM) treats each glandular structure in an image as a polygon made of a random number of vertices, where the vertices represent approximate locations of epithelial nuclei. We formulate the RPM as a Bayesian inference problem by defining a prior for spatial connectivity and arrangement of neighboring epithelial nuclei and a likelihood for the presence of a glandular structure. The inference is made via a Reversible-Jump Markov chain Monte Carlo simulation. To the best of our knowledge, all existing published algorithms for gland segmentation are designed to mainly work on healthy samples, adenomas, and low grade adenocarcinomas. One of them has been demonstrated to work on intermediate grade adenocarcinomas at its best. Our experimental results show that the RPM yields favorable results, both quantitatively and qualitatively, for extraction of glandular structures in histology images of normal human colon tissues as well as benign and cancerous tissues, excluding undifferentiated carcinomas.

Endoscopic craniofacial resection. Indications and technical aspects.

PubMed

Llorente, José Luis; López, Fernando; Suárez, Vanessa; Costales, María; Moreno, Carla; Suárez, Carlos

2012-01-01

Anterior craniofacial resection (CFR) is a standardised procedure for the treatment of tumours involving the anterior skull base. We present our experience in the endoscopic treatment of these tumours. A retrospective analysis was performed of patients treated by endoscopic anterior CFR in our Department from 2004 until 2011. Thirty-two patients were analysed. Mean follow-up was 28 months (range: 6-84 months). The most frequent pathological entity was adenocarcinoma (60%), followed by undifferentiated carcinoma (13%). According to TNM classification, malignant epithelial tumour staging was T3 in 9%, T4a in 53% and T4b in 19% of the malignant epithelial tumours. The complication rate was 6% and the resection was complete in 91% of cases. During follow-up, 9% of patients developed recurrence. The 5-year overall survival rate was 70% and the 5-year disease-free survival rate was 85% These results seem to indicate that properly planned endoscopic CFR may be a valid alternative to traditional open approaches for the management of malignancies of the anterior skull base. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Role of Alpha-Smooth Muscle Actin and Fibroblast Activation Protein Alpha in Ovarian Neoplasms.

PubMed

da Silva, Ana Carolinne; Jammal, Millena Prata; Etchebehere, Renata Margarida; Murta, Eddie Fernando Candido; Nomelini, Rosekeila Simões

2018-04-05

Studies show that tumor growth is not just determined by the presence of malignant cells, since interactions between cancer cells and stromal microenvironment have important impacts on the cancer growth and progression. Cancer-associated fibroblasts play a prominent role in this process. The aims of the study were to investigate 2 cancer-associated fibroblasts markers, alpha-smooth muscle actin (α-SMA), and fibroblast activation protein alpha (FAP) in the stromal microenvironment of benign and malignant ovarian epithelial neoplasms, and to relate their tissue expression with prognostic factors in ovarian cancer. α-SMA and FAP were evaluated by immunohistochemistry in malignant (n = 28) and benign (n = 28) ovarian neoplasms. Fisher's exact test was used with a significance level lower than 0.05. FAP immunostaining was stronger in ovarian cancer when compared to benign neoplasms (p = 0.0366). There was no significant difference in relation to α-SMA expression between malignant and benign ovarian neoplasms as well as prognostic factors. In ovarian cancer, FAP stainings 2/3 was significantly related to histological grades 2 and 3 (p = 0.0183). FAP immunostaining is more intense in malignant neoplasms than in benign ovarian neoplasms, as well as in moderately differentiated and undifferentiated ovarian carcinomas compared to well-differentiated neoplasms, thus indicating that it can be used as a marker of worse prognosis. © 2018 S. Karger AG, Basel.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

PubMed Central

2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

Selection and dynamics of embryonic stem cell integration into early mouse embryos

PubMed Central

Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

2016-01-01

The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Documentation of angiotensin II receptors in glomerular epithelial cells

NASA Technical Reports Server (NTRS)

Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

1998-01-01

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells

PubMed Central

Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J.

2012-01-01

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here. PMID:23150208

Langerhans cells from human oral epithelium are more effective at stimulating allogeneic T cells in vitro than Langerhans cells from skin.

PubMed

Hasséus, B; Jontell, M; Bergenholtz, G; Dahlgren, U I

2004-06-01

This report is focused on the functional capacity of Langerhans cells (LC) in the epithelium of skin and oral mucosa, which both meet different antigenic challenges. The capacity of LC from human oral and skin epithelium to provide co-stimulatory signals to T cells in vitro was compared. LC in a crude suspension of oral epithelial cells had a significantly enhanced T cell co-stimulatory capacity compared to skin epithelial cells. This applied both to cultures with concanavalin A (con-A)-stimulated syngeneic T cells and to a mixed epithelial cell lymphocyte reaction involving allogeneic T cells. The co-stimulatory capacity of oral and skin epithelial cells was reduced by >70% if monoclonal antibodies against HLA-DR, -DP and -DQ were added to the cultures with allogeneic T cells, indicating the involvement of HLA class II expressing LC. Immunohistochemistry revealed that 6% of the epithelial cells were CD1a + LC in sections from both oral and skin epithelium. Interleukin (IL)-8 production was higher in cultures of oral epithelial cells and con-A stimulated T cells than in corresponding cultures with skin epithelial cells as accessory cells. The results suggest that LC in human oral epithelium are more efficient at stimulating T cells than those of skin.

CD4+ acute undifferentiated leukaemia, probably early monoblastic type, developing in a patient with myelodysplastic syndrome and bone marrow fibrosis.

PubMed

Guenova, M; Taskov, H; Zechev, J

1997-07-01

We report here a patient who presented with pancytopenia, hypercellular bone marrow and three-lineage dysplasia associated with an increase of reticulin fibres. After a 5-month period, anaemia and thrombocytopenia progressed very rapidly and the white blood count increased showing 45% blasts with monocytoid morphology, but cytochemically undifferentiated in nature. The immunophenotype revealed an unusual expression of CD4, CD36 and HLA-DR in the absence of any other myeloid or lymphoid lineage-associated markers. The patient died unexpectedly during the course of chemotherapy. The occurrence of CD4, CD36 and HLA-DR on the blast cells cannot determine the lineage of differentiation with certainty but provides some evidence that the leukaemic cells were probably derived from a very early monocytic progenitor with maturation arrest. These cells had apparently complex interactions with pathologic megakaryocytes and cytokine production.

Characterization of newly established bovine intestinal epithelial cell line.

PubMed

Miyazawa, Kohtaro; Hondo, Tetsuya; Kanaya, Takashi; Tanaka, Sachi; Takakura, Ikuro; Itani, Wataru; Rose, Michael T; Kitazawa, Haruki; Yamaguchi, Takahiro; Aso, Hisashi

2010-01-01

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.

Epithelial-to-mesenchymal transition in penile squamous cell carcinoma.

PubMed

Masferrer, Emili; Ferrándiz-Pulido, Carla; Masferrer-Niubò, Magalí; Rodríguez-Rodríguez, Alfredo; Gil, Inmaculada; Pont, Antoni; Servitje, Octavi; García de Herreros, Antonio; Lloveras, Belen; García-Patos, Vicenç; Pujol, Ramon M; Toll, Agustí; Hernández-Muñoz, Inmaculada

2015-02-01

Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma. We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification. Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition. Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Warthin adenocarcinoma: analysis of 2 cases of a distinct salivary neoplasm.

PubMed

Bell, Diana; Luna, Mario A

2009-06-01

Carcinomas arising in or from the epithelial component of preexisting parotid Warthin tumors (WTs) are rare; the other histologic types of carcinoma found to arise from WTs are adenocarcinoma not otherwise specified, undifferentiated, mucoepidermoid, squamous cell, and oncocytic. The aim of this study is to describe the clinicopathologic features of a distinct salivary gland neoplasm, previously undescribed, with a striated duct phenotype arising from WT. We have designated this neoplasm "Warthin adenocarcinoma" (WA). In this retrospective study, we searched the surgical pathology files of the Department of Pathology at The University of Texas M.D. Anderson Cancer Center for cases of malignant WT and salivary adenocarcinoma not otherwise specified diagnosed from January 1, 1985, through December 31, 2006, and evaluated patients' medical records and pathologic material. We obtained tissue sections and immunohistochemically stained them with antibodies against p63; Bcl-2; cytokeratin (CK)903, CK7, CK14, and CK18; antimitochondrial antibody (AMA); smooth muscle actin; calponin; S-100; and Ki-67. We identified 2 cases of WA; both patients were women, 44 and 60 years of age, with 4.0- and 4.5-cm tumors in the left parotid gland. Histologically, the tumors were composed of bilayered duct-like structures: The inner layer was formed by a single row of columnar oxyphilic cells expressing CK7, CK14, CK18, and AMA. The outer layer was composed of multiple layers of small round dark cells with scanty cytoplasm that expressed p63, Bcl-2, and CK903 and were focally positive for AMA and negative for myoepithelial markers. The Ki-67 proliferative indices were 20%; and 25%. A residual WT with transition to carcinoma was identified in both cases. Treatment had consisted of total parotidectomy with postoperative irradiation. Patients were free of disease 1 and 3 years after treatment. Warthin adenocarcinoma is a unique salivary gland carcinoma representing the malignant epithelial counterpart of WT. The identification of additional cases would help to better elucidate the line of differentiation of the tumor and further define its natural history.

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

PubMed

Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

2018-04-01

Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Twite, Nicolas; Andrei, Graciela; Kummert, Caroline

2014-07-15

Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMVmore » by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.« less

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

PubMed

Pan, J-L; Yuan, D-Z; Zhao, Y-B; Nie, L; Lei, Y; Liu, M; Long, Y; Zhang, J-H; Blok, L J; Burger, C W; Yue, L-M

2017-03-01

This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G 1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

Epithelial cells as alternative human biomatrices for comet assay

PubMed Central

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells

PubMed Central

Li, Hui; Dai, Lu; Frank, Jacqueline A.; Peng, Shaojun; Wang, Siying; Chen, Gang

2017-01-01

The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages. PMID:28423485

Interaction of chitin/chitosan with salivary and other epithelial cells-An overview.

PubMed

Patil, Sharvari Vijaykumar; Nanduri, Lalitha S Y

2017-11-01

Chitin and its deacetylated form, chitosan, have been widely used for tissue engineering of both epithelial and mesenchymal tissues. Epithelial cells characterised by their sheet-like tight cellular arrangement and polarised nature, constitute a major component in various organs and play a variety of roles including protection, secretion and maintenance of tissue homeostasis. Regeneration of damaged epithelial tissues has been studied using biomaterials such as chitin, chitosan, hyaluronan, gelatin and alginate. Chitin and chitosan are known to promote proliferation of various embryonic and adult epithelial cells. However it is not clearly understood how this activity is achieved or what are the mechanisms involved in the chitin/chitosan driven proliferation of epithelial cells. Mechanistic understanding of influence of chitin/chitosan on epithelial cells will guide us to develop more targeted regenerative scaffold/hydrogel systems. Therefore, current review attempts to elicit a mechanistic insight into how chitin and chitosan interact with salivary, mammary, skin, nasal, lung, intestinal and bladder epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

In situ electrochemical detection of embryonic stem cell differentiation.

PubMed

Yea, Cheol-Heon; An, Jeung Hee; Kim, Jungho; Choi, Jeong-Woo

2013-06-20

Stem cell sensors have emerged as a promising technique to electrochemically monitor the functional status and viability of stem cells. However, efficient electrochemical analysis techniques are required for the development of effective electrochemical stem cell sensors. In the current study, we report a newly developed electrochemical cyclic voltammetry (CV) system to determine the status of mouse embryonic stem (ES) cells. 1-Naphthly phosphate (1-NP), which was dephosphorylated by alkaline phosphatase into a 1-naphthol on an undifferentiated mouse ES cell, was used as a substrate to electrochemically monitor the differentiation status of mouse ES cells. The peak current in the cyclic voltammetry of 1-NP increased linearly with the concentration of pure 1-NP (R(2)=0.9623). On the other hand, the peak current in the electrochemical responses of 1-NP decreased as the number of undifferentiated ES cells increased. The increased dephosphorylation of 1-NP to 1-naphthol made a decreased electrochemical signal. Non-toxicity of 1-NP was confirmed. In conclusion, the proposed electrochemical analysis system can be applied to an electrical stem cell chip for diagnosis, drug detection and on-site monitoring. Copyright © 2013 Elsevier B.V. All rights reserved.

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells.

PubMed

Amatngalim, Gimano D; Broekman, Winifred; Daniel, Nadia M; van der Vlugt, Luciën E P M; van Schadewijk, Annemarie; Taube, Christian; Hiemstra, Pieter S

2016-01-01

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.

Soluble Proteins Produced by Probiotic Bacteria Regulate Intestinal Epithelial Cell Survival and Growth

PubMed Central

YAN, FANG; CAO, HANWEI; COVER, TIMOTHY L.; WHITEHEAD, ROBERT; WASHINGTON, M. KAY; POLK, D. BRENT

2011-01-01

Background & Aims Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases. PMID:17258729

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.

PubMed

Kicic, A; Stevens, P T; Sutanto, E N; Kicic-Starcevich, E; Ling, K-M; Looi, K; Martinovich, K M; Garratt, L W; Iosifidis, T; Shaw, N C; Buckley, A G; Rigby, P J; Lannigan, F J; Knight, D A; Stick, S M

2016-11-01

The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-β production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-β production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-β restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-β. © 2016 John Wiley & Sons Ltd.

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues.

PubMed

Roy, M J

1987-06-01

Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.

Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris.

PubMed

Sweat JMDunigan, D D; Wright, S D

2001-06-01

The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.

High levels of telomere dysfunction bestow a selective disadvantage during the progression of human oral squamous cell carcinoma.

PubMed

Gordon, Katrina E; Ireland, Hazel; Roberts, Meryl; Steeghs, Karen; McCaul, James A; MacDonald, D Gordon; Parkinson, E Kenneth

2003-01-15

Human epithelial cells experience multiple barriers to cellular immortality in culture (mortality mechanisms 0, 1, and 2). Mortality mechanism 2 (M2) is termed crisis and involves telomere dysfunction due to lack of telomerase. However, proliferating normal keratinocytes in vivo can express telomerase, so it is unclear whether human squamous cell carcinomas (SCCs), which usually have high telomerase levels, develop from preexisting telomerase-positive precursors or by the activation of telomerase in telomerase-deficient somatic cells. We show that 6 of 29 oral SCCs show characteristics of M2 crisis in vivo, as indicated by a high anaphase bridge index (ABI), which is a good correlate of telomere dysfunction, and that 25 of 29 tumors possess some anaphase bridges. ABIs in excess of 0.2 in the primary tumor showed a decrease in the corresponding lymph node metastases. This suggests that high levels of telomere dysfunction (>0.2) and, by inference, M2 crisis bestow a selective disadvantage on SCCs during progression stages of the disease. Supporting this, SCCs with high levels of telomere dysfunction grow poorly in culture, and the ectopic expression of telomerase corrects this, together with other features of M2 crisis. Our data suggest that a substantial proportion of oral SCCs in vivo ultimately arise from telomerase-deficient keratinocytes rather than putative telomerase-proficient cells in the undifferentiated parts of the epithelium. Furthermore, the presence of significant levels of telomere dysfunction in a high proportion of SCCs at diagnosis but not in the normal epithelium implies that the therapeutic inhibition of telomerase should selectively compromise the growth of such tumors.

[BIOCOMPATIBILITY OF POLY-LACTIDE-CO-GLYCOLIDE/COLLAGEN TYPE I SCAFFOLD WITH RAT VAGINAL EPITHELIAL CELLS].

PubMed

Li, Yachai; Huang, Xianghua; Zhang, Mingle; Li, Yanan; Chen, Yexing; Jia, Jingfei

2015-09-01

To explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. PLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. The epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane-like layer on the scaffold. The keratin expression of the epithelium was positive. At 3 months after transplantation in situ, the vaginal mucosa showed pink and lustrous epithelialization, and the majority of scaffold degraded. After 6 months, the neovagina length was 1.2 cm, without obvious stenosis; the vaginal mucosa had similar appearance and epithelial layer to normal vagina, but it had less duplicature; there were nail-like processes in the basal layer, but the number was less than that of normal vagina. The immunohistochemistry staining for keratin was positive. The PLGA/collagen type I scaffolds have good cytocompatibility with the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

PubMed

Davidson, E J; Morris, L S; Scott, I S; Rushbrook, S M; Bird, K; Laskey, R A; Wilson, G E; Kitchener, H C; Coleman, N; Stern, P L

2003-01-27

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Physiology and pathophysiology of apoptosis in epithelial cells of the liver, pancreas, and intestine.

PubMed

Jones, B A; Gores, G J

1997-12-01

Cell death of gastrointestinal epithelial cells occurs by a process referred to as apoptosis. In this review, we succinctly define apoptosis and summarize the role of apoptosis in the physiology and pathophysiology of epithelial cells in the liver, pancreas, and small and large intestine. The physiological mediators regulating apoptosis in gastrointestinal epithelial cells, when known, are discussed. Selected pathophysiological consequences of excessive apoptosis and inhibition of apoptosis are used to illustrate the significance of apoptosis in disease processes. These examples demonstrate that excessive apoptosis may result in epithelial cell atrophy, injury, and dysfunction, whereas inhibition of apoptosis results in hyperplasia and promotes malignant transformation. The specific cellular mechanisms responsible for dysregulation of epithelial cell apoptosis during pathophysiological disturbances are emphasized. Potential future areas of physiological research regarding apoptosis in gastrointestinal epithelia are highlighted when appropriate.

CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

USDA-ARS?s Scientific Manuscript database

After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

PubMed Central

Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

2013-01-01

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiangjun; Yao, Qisheng, E-mail: yymcyqs@126.com; Sun, Xinbo

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treatedmore » with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates fibrosis after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates inflammation and fibrosis after hypoxia-and LPS-induced renal epithelial cells injury via TLR4/NF-κB.« less

A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

PubMed

Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

2014-01-01

The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

Changes in the regulation of heat shock gene expression in neuronal cell differentiation.

PubMed

Oza, Jay; Yang, Jingxian; Chen, Kuang Yu; Liu, Alice Y-C

2008-01-01

Neuronal differentiation of the NG108-15 neuroblastoma-glioma hybrid cells is accompanied by a marked attenuation in the heat shock induction of the Hsp70-firefly luciferase reporter gene activity. Analysis of the amount and activation of heat shock factor 1, induction of mRNA(hsp), and the synthesis and accumulation of heat shock proteins (HSPs) in the undifferentiated and differentiated cells suggest a transcriptional mechanism for this attenuation. Concomitant with a decreased induction of the 72-kDa Hsp70 protein in the differentiated cells, there is an increased abundance of the constitutive 73-kDa Hsc70, a protein known to function in vesicle trafficking. Assessment of sensitivity of the undifferentiated and differentiated cells against stress-induced cell death reveals a significantly greater vulnerability of the differentiated cells toward the cytotoxic effects of arsenite and glutamate/glycine. This study shows that changes in regulation of the HSP and HSC proteins are components of the neuronal cell differentiation program and that the attenuated induction of HSPs likely contributes to neuronal vulnerability whereas the increased expression of Hsc70 likely has a role in neural-specific functions.

Constitutive Proteasomal Degradation of TWIST-1 in Epithelial Ovarian Cancer Stem Cells Impacts Differentiation and Metastatic Potential

PubMed Central

Yin, Gang; Alvero, Ayesha B.; Craveiro, Vinicius; Holmberg, Jennie C.; Fu, Han-Hsuan; Montagna, Michele K.; Yang, Yang; Chefetz-Menaker, Ilana; Nuti, Sudhakar; Rossi, Michael; Silasi, Dan-Arin; Rutherford, Thomas; Mor, Gil

2013-01-01

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that Epithelial Ovarian Cancer Stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and Mesenchymal-Epithelial Transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected i.p in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to “home” to the ovaries and establish tumors. Most interestingly, we found that TWIST1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells. PMID:22349827

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

PubMed

Morita, Maresuke; Fujita, Naoki; Takahashi, Ayaka; Nam, Eun Ryel; Yui, Sho; Chung, Cheng Shu; Kawahara, Naoya; Lin, Hsing Yi; Tsuzuki, Keiko; Nakagawa, Takayuki; Nishimura, Ryohei

2015-01-01

To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively. © 2014 American College of Veterinary Ophthalmologists.

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

NASA Astrophysics Data System (ADS)

Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

2006-07-01

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1α-dependent pathway.

PubMed

Kang, Min-Jung; Song, Eun-Jung; Kim, Bo-Yeon; Kim, Dong-Jae; Park, Jong-Hwan

2014-12-01

Although Helicobacter pylori have been known to induce vascular endothelial growth factor (VEGF) production in gastric epithelial cells, the precise mechanism for cellular signaling is incompletely understood. In this study, we investigated the role of bacterial virulence factor and host cellular signaling in VEGF production of H. pylori-infected gastric epithelial cells. We evaluated production of VEGF, activation of nuclear factor nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) and hypoxia-inducible factor-1α (HIF-1α) stabilization in gastric epithelial cells infected with H. pylori WT or isogenic mutants deficient in type IV secretion system (T4SS). H. pylori induced VEGF production in gastric epithelial cells via both T4SS-dependent and T4SS-independent pathways, although T4SS-independent pathway seems to be the dominant signaling. The inhibitor assay implicated that activation of NF-κB and MAPKs is dispensable for H. pylori-induced VEGF production in gastric epithelial cells. H. pylori led to HIF-1α stabilization in gastric epithelial cells independently of T4SS, NF-κB, and MAPKs, which was essential for VEGF production in these cells. N-acetyl-cysteine (NAC), a reactive oxygen species (ROS) inhibitor, treatment impaired H. pylori-induced HIF-1α stabilization and VEGF production in gastric epithelial cells. We defined the important role of ROS-HIF-1α axis in VEGF production of H. pylori-infected gastric epithelial cells, and bacterial T4SS has a minor role in H. pylori-induced VEGF production of gastric epithelial cells. © 2014 John Wiley & Sons Ltd.

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

PubMed

Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

2017-08-01

Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R (epithelial-like)-derived xenograft tumors but not TE-8 (mesenchymal-like)-derived xenograft tumors. The factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.

Low- and high-LET radiation drives clonal expansion of lung progenitor cells in vivo

PubMed Central

Farin, Alicia M.; Manzo, Nicholas D.; Kirsch, David G.; Stripp, Barry R.

2015-01-01

Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X-ray or 600 MeV/nucleon 56Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X-rays and 56Fe resulted in a dose dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X-rays and 56Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior. PMID:25564721

Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells

PubMed Central

Loureiro, Renata Ruoco; Cristovam, Priscila Cardoso; Martins, Caio Marques; Covre, Joyce Luciana; Sobrinho, Juliana Aparecida; Ricardo, José Reinaldo da Silva; Hazarbassanov, Rossen Myhailov; Höfling-Lima, Ana Luisa; Belfort, Rubens; Nishi, Mauro

2013-01-01

Purpose To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. Methods Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT–PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. Results Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. Conclusions Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM. PMID:23378720

Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

PubMed

Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

2015-08-01

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model

PubMed Central

Gao, Yang; Li, Shu; Li, Qinglei

2014-01-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. PMID:24770950

ALTERATION OF CATECHOLAMINES IN PHOECHROMOCYTOMA (PC12) CELLS IN VITRO BY THE METABOLITES OF CHLOROTRIAZINE HERBICIDE

EPA Science Inventory

The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified following treatme...

EphA2 and Src regulate equatorial cell morphogenesis during lens development

PubMed Central

Cheng, Catherine; Ansari, Moham M.; Cooper, Jonathan A.; Gong, Xiaohua

2013-01-01

High refractive index and transparency of the eye lens require uniformly shaped and precisely aligned lens fiber cells. During lens development, equatorial epithelial cells undergo cell-to-cell alignment to form meridional rows of hexagonal cells. The mechanism that controls this morphogenesis from randomly packed cuboidal epithelial cells to highly organized hexagonal fiber cells remains unknown. In Epha2-/- mouse lenses, equatorial epithelial cells fail to form precisely aligned meridional rows; moreover, the lens fulcrum, where the apical tips of elongating epithelial cells constrict to form an anchor point before fiber cell differentiation and elongation at the equator, is disrupted. Phosphorylated Src-Y424 and cortactin-Y466, actin and EphA2 cluster at the vertices of wild-type hexagonal epithelial cells in organized meridional rows. However, phosphorylated Src and phosphorylated cortactin are not detected in disorganized Epha2-/- cells with altered F-actin distribution. E-cadherin junctions, which are normally located at the basal-lateral ends of equatorial epithelial cells and are diminished in newly differentiating fiber cells, become widely distributed in the apical, lateral and basal sides of epithelial cells and persist in differentiating fiber cells in Epha2-/- lenses. Src-/- equatorial epithelial cells also fail to form precisely aligned meridional rows and lens fulcrum. These results indicate that EphA2/Src signaling is essential for the formation of the lens fulcrum. EphA2 also regulates Src/cortactin/F-actin complexes at the vertices of hexagonal equatorial cells for cell-to-cell alignment. This mechanistic information explains how EphA2 mutations lead to disorganized lens cells that subsequently contribute to altered refractive index and cataracts in humans and mice. PMID:24026120

Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

PubMed

Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

2016-01-01

This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

Fluorescent in situ hybridization (FISH) on corneal impression cytology specimens (CICS): study of epithelial cell survival after keratoplasty.

PubMed

Catanese, Muriel; Popovici, Cornel; Proust, Hélène; Hoffart, Louis; Matonti, Frédéric; Cochereau, Isabelle; Conrath, John; Gabison, Eric E

2011-02-22

To assess corneal epithelial cell survival after keratoplasty. Corneal impression cytology (CIC) was performed on sex-mismatched corneal transplants. Fluorescent in situ hybridization (FISH) with sex chromosome-specific probes was performed to identify epithelial cell mosaicism and therefore allocate the donor or recipient origin of the cells. Twenty-four samples of corneal epithelial cells derived from 21 transplanted patients were analyzed. All patients received post-operative treatment using dexamethasone eye drops, with progressive tapering over 18 months, and nine patients also received 2% cyclosporine eye drops. Out of the 24 samples reaching quality criteria, sex mosaicism was found in 13, demonstrating the presence of donor-derived cells at the center of the graft for at least 211 days post keratoplasty. Kaplan-Meier analysis established a median survival of donor corneal epithelial cells of 385 days. Although not statistically significant, the disappearance of donor cells seemed to be delayed and the average number of persistent cells appeared to be greater when 2% cyclosporine was used topically as an additional immunosuppressive therapy. The combination of corneal impressions and FISH analysis is a valuable tool with negligible side effects to investigate the presence of epithelial cell mosaicism in sex-mismatched donor transplants. Epithelial cells survived at the center of the graft with a median survival of more than one year, suggesting slower epithelial turnover than previously described.

GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

PubMed Central

Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

2012-01-01

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Wenyao; Li, Xuezhong; Xu, Tong

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferationmore » and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.« less

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4[S

PubMed Central

Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena

2016-01-01

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4. In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4. Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

Coreceptors and Their Ligands in Epithelial γδ T Cell Biology

PubMed Central

Witherden, Deborah A.; Johnson, Margarete D.; Havran, Wendy L.

2018-01-01

Epithelial tissues line the body providing a protective barrier from the external environment. Maintenance of these epithelial barrier tissues critically relies on the presence of a functional resident T cell population. In some tissues, the resident T cell population is exclusively comprised of γδ T cells, while in others γδ T cells are found together with αβ T cells and other lymphocyte populations. Epithelial-resident γδ T cells function not only in the maintenance of the epithelium, but are also central to the repair process following damage from environmental and pathogenic insults. Key to their function is the crosstalk between γδ T cells and neighboring epithelial cells. This crosstalk relies on multiple receptor–ligand interactions through both the T cell receptor and accessory molecules leading to temporal and spatial regulation of cytokine, chemokine, growth factor, and extracellular matrix protein production. As antigens that activate epithelial γδ T cells are largely unknown and many classical costimulatory molecules and coreceptors are not used by these cells, efforts have focused on identification of novel coreceptors and ligands that mediate pivotal interactions between γδ T cells and their neighbors. In this review, we discuss recent advances in the understanding of functions for these coreceptors and their ligands in epithelial maintenance and repair processes. PMID:29686687

Design and characterization of hybrid peptide sol-gel materials for the solid state induction of neuronal differentiation

NASA Astrophysics Data System (ADS)

Jedlicka, Sabrina S.

2007-12-01

Cell-based therapeutics are a rapidly growing area of research, with considerable promise in the treatment of neurological diseases. One of the primary limitations to neuronal cell-based devices is the necessity to maintain cells in an immature or undifferentiated state in culture prior to transplantation. In many cases, the undifferentiated cell does not express the desired characteristics for implantation. Biologically functional nanomaterials provide the ability to manipulate the direct extracellular environment surrounding cells; influencing their fate and differentiation path. The ability to engineer the interface between the cells and culture materials provides a repeatable, stable means of directing cells down a specific growth path determined by endogenous signaling pathways. This materials approach to cellular engineering can limit the need for added exogenous growth factors, "feeder" layers, or animal sera, in addition to creating a homogenous cell population for transplantation. In this work, hybrid peptide ormosil materials were developed; designed to mimic the developing mammalian brain during corticogenesis. These materials have been developed to enhance the GABAergic phenotype of P19 embryonic carcinoma cells and immature immortalized neurons. The ability to develop a homogenous, directed cell population has implications in stem cell research, regenerative medicine, cell-based devices and biosensing technology.

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation

PubMed Central

Creo, Pasquale; Bergante, Sonia; Conforti, Erika; Banfi, Giuseppe

2018-01-01

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the “hypoxic niches” present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue. PMID:29713352

Basal cell adenocarcinoma of minor salivary and seromucous glands of the head and neck region.

PubMed

Fonseca, I; Soares, J

1996-05-01

Basal cell adenocarcinoma of salivary glands is an uncommon and recently described entity occurring almost exclusively at the major salivary glands. This report provides an overview of the clinicopathologic profile of this neoplasm by including the personal experience on the clinical features, microscopic and ultrastructural characteristics, proliferation activity, and DNA tumor patterns of 12 lesions occurring at the minor salivary glands of the head and neck region, where basal cell adenocarcinoma is probably an underecognized entity, previously reported under different designations. Basal cell adenocarcinoma predominates at the seventh decade without sex preference. The tumors affecting the minor salivary glands occur most frequently at the oral cavity (jugal mucosa, palate) and the upper respiratory tract. The prevalent histologic tumor pattern is represented by solid neoplastic aggregates with a peripheral cell palisading arrangement frequently delineated by basement membrane-like material. The neoplastic clusters are formed by two cell populations: the small dark cell type (that predominates) and a large cell type. Necrosis, either of the comedo or the apoptotic type, is a frequent finding. Perineural growth occurs in 50% of the cases and vascular permeation in 25%. Immunohistochemistry identifies a dual differentiation with a reactivity pattern indicative of ductal epithelial and myoepithelial differentiation, which can be confirmed by electron microscopy. The differential diagnosis of the neoplasm includes its benign counterpart, the basal cell adenoma, solid variant of adenoid cystic carcinoma, undifferentiated carcinoma, and basaloid squamous carcinoma. The tumors recur more frequently than lesions originating in major salivary glands. Mortality is associated with the anatomic site of the lesion, advanced stage, residual neoplasia at surgery, and tumor recurrence. The importance of recognizing basal cell adenocarcinoma outside major salivary glands is related to the clinical behavior of the neoplasm that seems to be less indolent than that of tumors arising in major salivary glands.

Cytokine Expression and Production by Purified Helicobacter pylori Urease in Human Gastric Epithelial Cells

PubMed Central

Tanahashi, Toshihito; Kita, Masakazu; Kodama, Tadashi; Yamaoka, Yoshio; Sawai, Naoki; Ohno, Tomoyuki; Mitsufuji, Shoji; Wei, Ya-Ping; Kashima, Kei; Imanishi, Jiro

2000-01-01

Cytokines have been proposed to play an important role in Helicobacter pylori-associated gastroduodenal diseases, but the exact mechanism of the cytokine induction remains unclear. H. pylori urease, a major component of the soluble proteins extracted from bacterial cells, is considered to be one of the virulence factors for the inflammation in the gastric mucosa that is produced in H. pylori infection. However, the response of human gastric epithelial cells to the stimulation of urease has not been investigated. In the present study, we used human gastric epithelial cells in a primary culture system and examined whether H. pylori urease stimulates the gastric epithelial cells to induce proinflammatory cytokines by reverse transcription-PCR and enzyme-linked immunosorbent assay. First, by using peripheral blood mononuclear cells (PBMC) and a gastric cancer cell line (MKN-45 cells), we confirmed the ability of purified H. pylori urease to induce the production of proinflammatory cytokines. Furthermore, we demonstrated that the human gastric epithelial cells produced interleukin-6 (IL-6) and tumor necrosis factor alpha, but not IL-8, following stimulation with purified urease. The patterns of cytokine induction differed among human PBMC, MKN-45 cells, and human gastric epithelial cells. These results suggest that the human gastric epithelial cells contribute to the induction of proinflammatory cytokines by the stimulation of H. pylori urease, indicating that the epithelial cells were involved in the mucosal inflammation that accompanied H. pylori infection. PMID:10639431

Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

PubMed Central

Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

2016-01-01

The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Histone Deacetylase Inhibitor Induces the Expression of Select Epithelial Genes in Mouse Utricle Sensory Epithelia-Derived Progenitor Cells

PubMed Central

Wang, Jue

2014-01-01

Abstract Mouse utricle sensory epithelial cell–derived progenitor cells (MUCs), which have hair cell progenitor and mesenchymal features via epithelial-to-mesenchymal transition (EMT) as previously described, provide a potential approach for hair cell regeneration via cell transplantation. In this study, we treated MUCs with trichostatin A (TSA) to determine whether histone deacetylase inhibitor is able to stimulate the expression of epithelial genes in MUCs, an essential step for guiding mesenchymal-like MUCs to become sensory epithelial cells. After 72 h of TSA treatment, MUCs acquired epithelial-like features, which were indicated by increased expression of epithelial markers such as Cdh1, Krt18, and Dsp. Additionally, TSA decreased the expression of mesenchymal markers, including Zeb1, Zeb2, Snai1, and Snai2, and prosensory genes Lfng, Six1, and Dlx5. Moreover, the expression of the hair cell genes Atoh1 and Myo6 was increased in TSA-treated MUCs. We also observed significantly decreased expression of Hdac2 and Hdac3 in TSA-treated MUCs. However, no remarkable change was detected in protein expression using immunofluorescence, indicating that TSA-induced HDAC inhibition may contribute to the initial stage of the mesenchymal-to-epithelial phenotypic change. In the future, more work is needed to induce hair cell regeneration using inner ear tissue–derived progenitors to achieve an entire mesenchymal-to-epithelial transition. PMID:24945595

The Contribution of the Airway Epithelial Cell to Host Defense.

PubMed

Stanke, Frauke

2015-01-01

In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

Targeting the (Un)differentiated State of Cancer.

PubMed

Kemeny, Lajos V; Fisher, David E

2018-05-14

Dedifferentation in cancer is associated with intrinsic and acquired resistance to therapies. In this issue of Cancer Cell, Tsoi et al. identify four differentiation states in melanoma and provide evidence that melanoma cells develop drug resistance through a stepwise dedifferentiation process, making them vulnerable to ferroptotic cell death-inducing compounds. Copyright © 2018 Elsevier Inc. All rights reserved.

Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine.

PubMed

Neal, Rebekah A; Lenz, Steven M; Wang, Tiffany; Abebayehu, Daniel; Brooks, Benjamin P C; Ogle, Roy C; Botchwey, Edward A

2014-09-01

Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.

Zap70 functions to maintain stemness of mouse embryonic stem cells by negatively regulating Jak1/Stat3/c-Myc signaling

PubMed Central

Cha, Young; Moon, Bo-Hyun; Lee, Mi-Ok; Ahn, Hee-Jin; Lee, Hye-Jin; Lee, Kyung-Ah; Fornace, Albert J.; Kim, Kwang-Soo; Cha, Hyuk-Jin; Park, Kyung-Soon

2011-01-01

Zeta-chain associated protein kinase-70 (Zap70), a Syk family tyrosine kinase, has been reported to be present exclusively in normal T cells, Natural Killer (NK) cells, and B cells, serving as a pivotal regulator of antigen-mediated receptor signaling and development. In this study, we report that Zap70 is expressed in undifferentiated mouse embryonic stem cells (mESCs) and may critically regulate self-renewal and pluripotency in mESCs. We found that Zap70 knocked-down mESCs (Zap70KD) show sustained self-renewal and defective differentiation. In addition, we present evidence that the sustained self-renewal in Zap70KD is associated with enhanced Jak/Stat3 signaling and c-Myc induction. These altered signaling appears to result from up-regulated LIFR and down-regulated SHP-1 phosphatase activity. Based on these results, we propose that, in undifferentiated mESCs, Zap70 plays important roles in modulating the balance between self-renewal capacity and pluripotent differentiation ability as a key regulator of the Jak/Stat3/c-Myc signaling pathway. PMID:20641039

Role of medullary progenitor cells in epithelial cell migration and proliferation

PubMed Central

Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

2014-01-01

This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity.

PubMed

Acharya, Aviseka; Brungs, Sonja; Henry, Margit; Rotshteyn, Tamara; Singh Yaduvanshi, Nirmala; Wegener, Lucia; Jentzsch, Simon; Hescheler, Jürgen; Hemmersbach, Ruth; Boeuf, Helene; Sachinidis, Agapios

2018-06-15

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-β signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

PubMed

Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

2009-04-01

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

PubMed

Mesa, Kailin R; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Y; Brown, Samara; Gonzalez, David G; Blagoev, Krastan B; Haberman, Ann M; Greco, Valentina

2015-06-04

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

PubMed

Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

2015-11-01

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions.

Generation of Epithelial Cell Populations from Human Pluripotent Stem Cells Using a Small-Molecule Inhibitor of Src Family Kinases.

PubMed

Selekman, Joshua A; Lian, Xiaojun; Palecek, Sean P

2016-01-01

Human pluripotent stem cells (hPSCs), under the right conditions, can be engineered to generate populations of any somatic cell type. Knowledge of what mechanisms govern differentiation towards a particular lineage is often quite useful for efficiently producing somatic cell populations from hPSCs. Here, we have outlined a strategy for deriving populations of simple epithelial cells, as well as more mature epidermal keratinocyte progenitors, from hPSCs by exploiting a mechanism previously shown to direct epithelial differentiation of hPSCs. Specifically, we describe how to direct epithelial differentiation of hPSCs using an Src family kinase inhibitor, SU6656, which has been shown to modulate β-catenin translocation to the cell membrane and thus promote epithelial differentiation. The differentiation platform outlined here produces cells with the ability to terminally differentiate to epidermal keratinocytes in culture through a stable simple epithelial cell intermediate that can be expanded in culture for numerous (>10) passages.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues.

PubMed

Xiong, Jimin; Gronthos, Stan; Bartold, P Mark

2013-10-01

Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth-supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal-regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem-cell populations that demonstrate similar properties to mesenchymal stromal/stem cells, both functionally and phenotypically. Therefore, the epithelial cell rests of Malassez, rather than being 'cell rests', as indicated by their name, are an important source of stem cells that might play a pivotal role in periodontal regeneration. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Epithelial stem cells are formed by small-particles released from particle-producing cells

PubMed Central

Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

2017-01-01

Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

[Pathological and immunohistochemical analysis of giant cells of pancreas].

PubMed

Miyake, T; Suda, K; Yamamura, A; Tada, Y

1997-10-01

Multinucleated giant cells in the pancreas (five giant cell carcinomas, a mucinous cystadenocarcinoma attended with many osteoclast-like giant cells, 42 invasive ductal carcinomas and 29 chronic pancreatitises) were examined. Three types of multinucleated giant cell were identified: epithelial type, coexpressive type, mesenchymal type. Epithelial type expressed epithelial markers, such as keratin and EMA in 23 ductal carcinomas. Coexpressive type expressed both epithelial markers and mesenchymal marker vimentin was in four ductal carcinomas. Mesenchymal type expressed mesenchymal markers, vimentin and CD68 in four osteoclastoid type giant cell carcinomas, the mucinous cystadenocarcinoma, six ductal carcinomas and ten chronic pancreatitises. Epithelial and coexpressive type were considered to be epithelial neoplastic origin, those had bizarre appearance and transitional area from definite adenocarcinoma area. Vimentin expression is associated with sarcomatous proliferation. Mesenchymal type was considered to be nonneoplastic and a certain type of macrophage polykaryons.

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

PubMed Central

Gibson, P; Birchall, I; Rosella, O; Albert, V; Finch, C; Barkla, D; Young, G

1998-01-01

Background—The functions of urokinase in intestinal epithelia are unknown. 
Aims—To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. 
Methods—Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. 
Results—From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. 
Conclusions—Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine. 

 Keywords: urokinase; intestinal epithelium; colon; epithelial proliferation PMID:9824347

Epithelial junctions, cytoskeleton, and polarity.

PubMed

Pásti, Gabriella; Labouesse, Michel

2014-11-04

A distinctive feature of polarized epithelial cells is their specialized junctions, which contribute to cell integrity and provide platforms to orchestrate cell shape changes. This chapter discusses the composition, assembly and remodeling of C. elegans cell-cell (CeAJ) and hemidesmosome-like cell-extracellular matrix junctions (CeHD), proteins that anchor the cytoskeleton, and mechanisms involved in establishing epithelial polarity. Major recent progress in this area has come from the analysis of mechanisms that maintain cell polarity, which involve lipids and trafficking, and on the impact of mechanical forces on junction remodeling. This chapter focuses on cellular, rather than developmental, aspects of epithelial cells.

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

PubMed

Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2008-09-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

PubMed

Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.

NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

USDA-ARS?s Scientific Manuscript database

Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells.

PubMed

Curto, Pedro; Simões, Isaura; Riley, Sean P; Martinez, Juan J

2016-01-01

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells

PubMed Central

Curto, Pedro; Simões, Isaura; Riley, Sean P.; Martinez, Juan J.

2016-01-01

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals. PMID:27525249

Epithelial Cell Rests of Malassez Contain Unique Stem Cell Populations Capable of Undergoing Epithelial–Mesenchymal Transition

PubMed Central

Xiong, Jimin; Mrozik, Krzysztof; Gronthos, Stan

2012-01-01

The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial–mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α6/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial–mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament. PMID:22122577

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

PubMed

Tong, Hui-Li; Li, Qing-Zhang; Gao, Xue-Jun; Yin, De-Yun

2012-03-01

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and β-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

PubMed Central

Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G.; Kuemmerle, John F.; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I.

2014-01-01

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA–depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. PMID:25143399

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

PubMed Central

Huang, G T; Eckmann, L; Savidge, T C; Kagnoff, M F

1996-01-01

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens. PMID:8755670

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

PubMed

Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

2016-01-01

Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

PubMed Central

Lange, Alexander W.; Sridharan, Anusha; Xu, Yan; Stripp, Barry R.; Perl, Anne-Karina; Whitsett, Jeffrey A.

2015-01-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. PMID:25480985

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

PubMed

Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

2018-04-24

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

PubMed Central

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-01-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. PMID:26602832

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

NASA Astrophysics Data System (ADS)

Sorce, Barbara; Escobedo, Carlos; Toyoda, Yusuke; Stewart, Martin P.; Cattin, Cedric J.; Newton, Richard; Banerjee, Indranil; Stettler, Alexander; Roska, Botond; Eaton, Suzanne; Hyman, Anthony A.; Hierlemann, Andreas; Müller, Daniel J.

2015-11-01

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

5-Oxo-ETE from Nasal Epithelial Cells Upregulates Eosinophil Cation Protein by Eosinophils in Nasal Polyps in vitro.

PubMed

Lin, Lin; Chen, Zheng; Tang, Xinyue; Dai, Fei; Wei, Jinjin; Sun, Guangbin

2018-06-13

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant and activator that is synthesized not only in inflammatory cells but also in bronchial epithelial cells. The purpose of this study is to clarify whether 5-oxo-ETE can promote the production of eosinophil cation protein (ECP) by eosinophils in nasal polyps (NP) in vitro, and whether normal nasal epithelial cells can produce this lipid mediator in response to oxidative stress. Nasal biopsy samples were obtained from normal subjects or subjects with chronic rhinosinusitis with NP. The infiltration of eosinophil in NP was detected and cultured. After that, concentrations of ECP in eosinophil and NP cultures were evaluated after the treatment of 5-oxo-ETE or 5-oxo-ETE + its receptor (OXER) antagonist, pertussis toxin (PT). Then we studied the synthesis of 5-oxo-ETE after H2O2 stimulation by normal nasal epithelial cells and by epithelial cells of NP alone in the cultures, and also determined the OXER expression in NP. The number of infiltrative eosinophils in NP was increased. The ECP levels in eosinophil and NP cultures were enhanced after the administration of 5-oxo-ETE, and decreased by the PT treatment. 5-Oxo-ETE was upregulated in the cultures of nasal epithelial cells in the presence of H2O2 and of NP epithelial cells alone. The OXER was expressed in inflammatory cells, and not in epithelial cells. 5-Oxo-ETE produced by nasal epithelial cells may play a role in the formation and development of NP. © 2018 S. Karger AG, Basel.

Multi-functionality and plasticity characterize epithelial cells in Hydra

PubMed Central

Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

2015-01-01

Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

Control of stomach smooth muscle development and intestinal rotation by transcription factor BARX1

PubMed Central

Jayewickreme, Chenura D.; Shivdasani, Ramesh A.

2015-01-01

Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1−/− embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1+ intestinal mesenchyme and reduced in Barx1−/− stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors. PMID:26057579

Control of stomach smooth muscle development and intestinal rotation by transcription factor BARX1.

PubMed

Jayewickreme, Chenura D; Shivdasani, Ramesh A

2015-09-01

Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1(-/)(-) embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1(+) intestinal mesenchyme and reduced in Barx1(-/-) stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcription Factor-Mediated Control of Anthocyanin Biosynthesis in Vegetative Tissues1[OPEN

PubMed Central

Outchkourov, Nikolay S.; Schrama, Xandra; Blilou, Ikram; Jongedijk, Esmer; Simon, Carmen Diez; Bosch, Dirk; Hall, Robert D.

2018-01-01

Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology. PMID:29192027

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

PubMed

Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

2008-04-01

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

Bioactive interleukin-1alpha is cytolytically released from Candida albicans-infected oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H; Villar, C Cunha

2004-12-01

Oral epithelial cells are primary targets of Candida albicans in the oropharynx and may regulate the inflammatory host response to this pathogen. This investigation studied the mechanisms underlying interleukin-1alpha (IL-1alpha) release by oral epithelial cells and the role of IL-1alpha in regulating the mucosal inflammatory response to C. albicans. Infected oral epithelial cells released processed IL-1alpha protein in culture supernatants. The IL-1alpha generated was stored intracellularly and was released upon cell lysis. This was further supported by the fact that different C. albicans strains induced variable IL-1alpha release, depending on their cytolytic activity. IL-1alpha from C. albicans-infected oral epithelial cells upregulated proinflammatory cytokine secretion (IL-8 and GM-CSF) in uninfected oral epithelial or stromal cells. Our studies suggest that production of IL-1alpha, IL-8 and GM-CSF may take place in the oral mucosa in response to lytic infection of epithelial cells with C. albicans. This process can act as an early innate immune surveillance system and may contribute to the clinicopathologic signs of infection in the oral mucosa.

Phenotypic characterization of collagen gel embedded primary human breast epithelial cells in athymic nude mice.

PubMed

Yang, J; Guzman, R C; Popnikolov, N; Bandyopadhyay, G K; Christov, K; Collins, G; Nandi, S

1994-06-30

We have developed a method to characterize the phenotypes and tumorigenicity of dissociated human breast epithelial cells. The dissociated cells were first embedded in collagen gels and subsequently transplanted subcutaneously in vivo in athymic nude mice. The transplantation of dissociated epithelial cells from reduction mammoplasties, presumed to be normal, always resulted in normal histomorphology. Epithelial cells were arranged as short tubular structures consisting of lumina surrounded by epithelial cells with an occasional more complex branching structure. These outgrowths were surrounded by intact basement membrane and were embedded in collagen gel that, at termination, contained collagenous stroma with fibroblasts and blood vessels. In contrast, transplantation of dissociated breast epithelial cells from breast cancer specimens resulted in outgrowths with an invasive pattern infiltrating the collagen gel as well as frank invasion into vascular space, nerves and muscles. These observations were made long before the subsequent palpable stage which resulted if left in the mouse for a long enough time. The dissociated human breast epithelial cells thus retained their intrinsic property to undergo morphogenesis to reflect their original phenotype when placed in a suitable environment, the collagen gel.

Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

PubMed Central

Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

2017-01-01

Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

Role of contact inhibition of locomotion and junctional mechanics in epithelial collective responses to injury

NASA Astrophysics Data System (ADS)

Coburn, Luke; Lopez, Hender; Schouwenaar, Irin-Maya; Yap, Alpha S.; Lobaskin, Vladimir; Gomez, Guillermo A.

2018-03-01

Epithelial tissues form physically integrated barriers against the external environment protecting organs from infection and invasion. Within each tissue, epithelial cells respond to different challenges that can potentially compromise tissue integrity. In particular, cells collectively respond to injuries by reorganizing their cell-cell junctions and migrating directionally towards the sites of damage. Notwithstanding, the mechanisms that drive collective responses in epithelial aggregates remain poorly understood. In this work, we develop a minimal mechanistic model that is able to capture the essential features of epithelial collective responses to injuries. We show that a model that integrates the mechanics of cells at the cell-cell and cell-substrate interfaces as well as contact inhibition of locomotion (CIL) correctly predicts two key properties of epithelial response to injury as: (1) local relaxation of the tissue and (2) collective reorganization involving the extension of cryptic lamellipodia that extend, on average, up to 3 cell diameters from the site of injury and morphometric changes in the basal regions. Our model also suggests that active responses (like the actomyosin purse string and softening of cell-cell junctions) are needed to drive morphometric changes in the apical region. Therefore, our results highlight the importance of the crosstalk between junctional biomechanics, cell substrate adhesion, and CIL, as well as active responses, in guiding the collective rearrangements that are required to preserve the epithelial barrier in response to injury.

Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells

PubMed Central

Chen, Zhuo; Evans, W. Howard; Pflugfelder, Stephen C.; Li, De-Quan

2010-01-01

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells. PMID:16424398

Contraction and elongation: Mechanics underlying cell boundary deformations in epithelial tissue.

PubMed

Hara, Yusuke

2017-06-01

The cell-cell boundaries of epithelial cells form cellular frameworks at the apical side of tissues. Deformations in these boundaries, for example, boundary contraction and elongation, and the associated forces form the mechanical basis of epithelial tissue morphogenesis. In this review, using data from recent Drosophila studies on cell boundary contraction and elongation, I provide an overview of the mechanism underlying the bi-directional deformations in the epithelial cell boundary, that are sustained by biased accumulations of junctional and apico-medial non-muscle myosin II. Moreover, how the junctional tensions exist on cell boundaries in different boundary dynamics and morphologies are discussed. Finally, some future perspectives on how recent knowledge about single cell boundary-level mechanics will contribute to our understanding of epithelial tissue morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

Continual exposure to cigarette smoke extracts induces tumor-like transformation of human nontumor bronchial epithelial cells in a microfluidic chip.

PubMed

Li, Encheng; Xu, Zhiyun; Liu, Fen; Wang, Huiling; Wen, Jiabin; Shao, Shujuan; Zhang, Lichuan; Wang, Lei; Liu, Chong; Lu, Jianxin; Wang, Wenxin; Gao, Zhancheng; Wang, Qi

2014-08-01

Heavy cigarette smoking-related chronic obstructive pulmonary disease is an independent risk factor for lung squamous carcinoma. However, the mechanisms underlying the malignant transformation of bronchial epithelial cells are unclear. In our study, human tumor-adjacent bronchial epithelial cells were obtained from 10 cases with smoking-related chronic obstructive pulmonary disease and lung squamous carcinoma and cultured in an established microfluidic chip for continual exposure to cigarette smoke extracts (CSE) to investigate the potential tumor-like transformation and mechanisms. The integrated microfluidic chip included upstream concentration gradient generator and downstream cell culture chambers supplied by flowing medium containing different concentrations of CSE. Our results showed that continual exposure to low doses of CSE promoted cell proliferation whereas to high doses of CSE triggered cell apoptosis. Continual exposure to CSE promoted reactive oxygen species production in human epithelial cells in a dose-dependent manner. More importantly, continual exposure to low dose of CSE promoted the epithelial-to-mesenchymal transition process and anchorage-independent growth, and increased chromosome instability in bronchial epithelial cells, accompanied by activating the GRP78, NF-κB, and PI3K pathways. The established microfluidic chip is suitable for primary culture of human tumor-adjacent bronchial epithelial cells to investigate the malignant transformation. Continual exposure to low doses of CSE promoted tumor-like transformation of human nontumor bronchial epithelial cells by inducing reactive oxygen species production and activating the relevant signaling.

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

PubMed Central

Ryan, Aideen E; Lohan, Paul; O'Flynn, Lisa; Treacy, Oliver; Chen, Xizhe; Coleman, Cynthia; Shaw, Georgina; Murphy, Mary; Barry, Frank; Griffin, Matthew D; Ritter, Thomas

2014-01-01

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3+ and CD68+ infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use. PMID:24184966

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction.

PubMed

Cohen, Daniel J; Gloerich, Martijn; Nelson, W James

2016-12-20

Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

PubMed Central

Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

2012-01-01

Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

PubMed Central

Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N.; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C.

2013-01-01

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling. PMID:23700468

Niche induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

PubMed Central

Mesa, Kailin R.; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Yang; Brown, Samara; Gonzalez, David; Blagoev, Krastan B.; Haberman, Ann M.; Greco, Valentina

2015-01-01

Summary Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression)1,2. Contrary to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration3. Here we show by intravital microscopy in live mice4–6 that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbors. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through TGFβ activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviors and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis. PMID:25849774

Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, J.A.; Reynolds-Kohler, C.; Smith, B.A.

1987-11-01

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells.more » However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three-to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negatively modulates expression in nonpermissive cells but positively influences expression in permissive cells.« less

Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer.

PubMed

Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hirose, Michiko; Noda, Shinichi; Kim, Jin-Moon; Aoki, Fugaku; Miyoshi, Hiroyuki; Ogura, Atsuo

2006-05-15

In general, cloning undifferentiated preimplantation embryos (blastomeres) or embryonic stem cells is more efficient than cloning differentiated somatic cells. Therefore, there has been an assumption that tissue-specific stem cells might serve as efficient donors for nuclear transfer because of the undifferentiated state of their genome. Here, we show that this is not the case with adult hematopoietic stem cells (HSCs). Although we have demonstrated for the first time that mouse HSCs can be cloned to generate offspring, the birth rates (0-0.7%) were lowest among the clones tested (cumulus, immature Sertoli and fibroblast cells). Only 6% of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus clones; P < 5 x 10(-10)). Transcription and gene expression analyses of HSC clone embryos revealed that they initiated zygotic gene activation (ZGA) at the appropriate timing, but failed to activate five out of six important embryonic genes examined, including Hdac1 (encoding histone deacetylase 1), a key regulator of subsequent ZGA. These results suggest that the HSC genome has less plasticity than we imagined, at least in terms of reprogrammability in the ooplasm after nuclear transfer.

Cellular network entropy as the energy potential in Waddington's differentiation landscape

PubMed Central

Banerji, Christopher R. S.; Miranda-Saavedra, Diego; Severini, Simone; Widschwendter, Martin; Enver, Tariq; Zhou, Joseph X.; Teschendorff, Andrew E.

2013-01-01

Differentiation is a key cellular process in normal tissue development that is significantly altered in cancer. Although molecular signatures characterising pluripotency and multipotency exist, there is, as yet, no single quantitative mark of a cellular sample's position in the global differentiation hierarchy. Here we adopt a systems view and consider the sample's network entropy, a measure of signaling pathway promiscuity, computable from a sample's genome-wide expression profile. We demonstrate that network entropy provides a quantitative, in-silico, readout of the average undifferentiated state of the profiled cells, recapitulating the known hierarchy of pluripotent, multipotent and differentiated cell types. Network entropy further exhibits dynamic changes in time course differentiation data, and in line with a sample's differentiation stage. In disease, network entropy predicts a higher level of cellular plasticity in cancer stem cell populations compared to ordinary cancer cells. Importantly, network entropy also allows identification of key differentiation pathways. Our results are consistent with the view that pluripotency is a statistical property defined at the cellular population level, correlating with intra-sample heterogeneity, and driven by the degree of signaling promiscuity in cells. In summary, network entropy provides a quantitative measure of a cell's undifferentiated state, defining its elevation in Waddington's landscape. PMID:24154593

Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

PubMed

Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

2014-01-01

Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

PubMed Central

Oslund, Karen L.; Hyde, Dallas M.; Putney, Leialoha F.; Alfaro, Mario F.; Walby, William F.; Tyler, Nancy K.; Schelegle, Edward S.

2008-01-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2′-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress–positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways. PMID:18390473

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

PubMed

Sessa, F; Bonato, M; Frigerio, B; Capella, C; Solcia, E; Prat, M; Bara, J; Samloff, I M

1990-06-01

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

Pathological changes of thymic epithelial cells and autoimmune disease in NZB, NZW and (NZB × NZW)F1 mice

PubMed Central

Vries, M. J. De; Hijmans, W.

1967-01-01

An extensive histological study was carried out of NZB, NZW and (NZB × NZW)F1, (BWF1), mice of all ages between birth and 18 months. The thymuses of these mice were compared to those of three normal mouse strains. The study of the NZW mice showed that these mice, although they only occasionally have weakly positive Coombs' tests, may develop a renal disease probably of an autoimmune nature, similar to that of the NZB and the BWF1 mice. Mice of all the three NZ strains developed lesions of the skin, liver, intestines, lymphatic tissues and kidneys much resembling those occurring in human systemic lupus erythematosus (SLE), neonatally thymectomized mice and, with the exception of the renal changes, the lesions of graft versus host disease. The comparative study of the thymus in autoimmune and normal strains, revealed that important changes of the large medullary epithelial cells, involved in the formation of Hassall's corpuscles, occur very early in the three autoimmune strains. In the NZB mice the large epithelial cells are severely decreased in number in the first weeks following birth. The depletion of epithelial cells could be ascribed to a secondary degeneration of these cells soon after birth. In contrast with the NZB mice, an extensive hyperplasia of the large epithelial cells and Hassall's corpuscles was observed in the NZW and BWF1 mice, and was already apparent in the newborn animal. Many of the epithelial aggregates seemed to have been invaded by lymphoid cells. Both epithelial cells and the lymphoid cells engaged in this process showed a variety of degenerative changes. As in the NZB, a depletion of epithelial cells occurred in a later phase, at the age of 8 months in the BWF1 and at 1 year in the NZW. In the majority of young mice of the normal strains invasion of islands of epithelial cells by lymphoid cells may also be observed, although this process is far less extensive than in the autoimmune strains and does not result in either epithelial hyperplasia or depletion of epithelial cells. The described phenomenon of invasion of epithelial structures in the thymus by subsequently disintegrating lymphoid cells seems to support Burnet's concept, that so-called `forbidden clones' of lymphoid cells are eliminated in the thymus. ImagesFIG. 18FIG. 19FIG. 20FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7FIG. 8FIG. 9FIG. 10FIG. 11FIG. 12FIG. 13FIG. 14FIG. 15FIG. 16FIG. 17 PMID:6020121

Moderate plasma activated media suppresses proliferation and migration of MDCK epithelial cells

NASA Astrophysics Data System (ADS)

Mohades, Soheila; Laroussi, Mounir; Maruthamuthu, Venkat

2017-05-01

Low-temperature plasma has been shown to have diverse biomedical uses, including its applications in cancer and wound healing. One recent approach in treating mammalian cells with plasma is through the use of plasma activated media (PAM), which is produced by exposing cell culture media to plasma. While the adverse effects of PAM treatment on cancerous epithelial cell lines have been recently studied, much less is known about the interaction of PAM with normal epithelial cells. In this paper, non-cancerous canine kidney MDCK (Madin-Darby Canine Kidney) epithelial cells were treated by PAM and time-lapse microscopy was used to directly monitor their proliferation and random migration upon treatment. While longer durations of PAM treatment led to cell death, we found that moderate levels of PAM treatment inhibited proliferation in these epithelial cells. We also found that PAM treatment reduced random cell migration within epithelial islands. Immunofluorescence staining showed that while there were no major changes in the actin/adhesion apparatus, there was a significant change in the nuclear localization of proliferation marker Ki-67, consistent with our time-lapse results.

Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances.

PubMed

Broaders, Kyle E; Cerchiari, Alec E; Gartner, Zev J

2015-12-01

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.

Application of Stem Cells in Oral Disease Therapy: Progresses and Perspectives

PubMed Central

Yang, Bo; Qiu, Yi; Zhou, Niu; Ouyang, Hong; Ding, Junjun; Cheng, Bin; Sun, Jianbo

2017-01-01

Stem cells are undifferentiated and pluripotent cells that can differentiate into specialized cells with a more specific function. Stem cell therapies become preferred methods for the treatment of multiple diseases. Oral and maxillofacial defect is one kind of the diseases that could be most possibly cured by stem cell therapies. Here we discussed oral diseases, oral adult stem cells, iPS cells, and the progresses/challenges/perspectives of application of stem cells for oral disease treatment. PMID:28421002

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

PubMed

Gao, Yang; Li, Shu; Li, Qinglei

2014-08-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

PubMed

Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

2017-03-21

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung.

PubMed

Lange, Alexander W; Sridharan, Anusha; Xu, Yan; Stripp, Barry R; Perl, Anne-Karina; Whitsett, Jeffrey A

2015-02-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Characterization of cultivated murine lacrimal gland epithelial cells

PubMed Central

Kobayashi, Shinya; Kawashima, Motoko; Okada, Naoko; Mishima, Kenji; Saito, Ichiro; Ito, Masataka; Shimmura, Shigeto; Tsubota, Kazuo

2012-01-01

Purpose To date, mouse lacrimal gland epithelial cells have been cultured successfully but only in cases involving newborn mouse lacrimal glands. In this work, we attempted to cultivate and characterize adult mouse lacrimal gland epithelial cells. Methods Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland epithelial cells were seeded onto tissue culture treated or low adherent culture dishes in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were characterized by immunostaining with pan-cytokeratin, α-smooth muscle actin, and lactoferrin antibodies. Lacrimal gland cells from 7-week-old green fluorescent protein (GFP) and non-GFP (C57B/6) mice were mixed and seeded onto uncoated dishes to assess sphere-forming efficiency. Cells were also seeded onto 3T3 cell feeder layers to assess colony forming efficiency. Results Lacrimal gland epithelial cells were selectively cultured with cholera toxin, and cell type was verified by pan-cytokeratin and α-smooth muscle actin immunostaining. Sphere formation from single cells of adult mice was observed using specific medium and low adherent culture dishes. These cells could also undergo colony formation on 3T3 feeder cells. Conclusions Adult mouse lacrimal gland epithelial cells were successfully cultivated in cholera toxin-containing medium, and were observed to form spheres from single cells. PMID:22665974

Mesenchymal Stem Cells Promote Diabetic Corneal Epithelial Wound Healing Through TSG-6-Dependent Stem Cell Activation and Macrophage Switch.

PubMed

Di, Guohu; Du, Xianli; Qi, Xia; Zhao, Xiaowen; Duan, Haoyun; Li, Suxia; Xie, Lixin; Zhou, Qingjun

2017-08-01

To explore the role and mechanism of bone marrow-derived mesenchymal stem cells (BM-MSCs) in corneal epithelial wound healing in type 1 diabetic mice. Diabetic mice were treated with subconjunctival injections of BM-MSCs or recombinant tumor necrosis factor-α-stimulated gene/protein-6 (TSG-6). The corneal epithelial wound healing rate was examined by fluorescein staining. The mRNA and protein expression levels of TSG-6 were measured by quantitative RT-PCR and Western blot. The infiltrations of leukocytes and macrophages were analyzed by flow cytometry and immunofluoresence staining. The effect of TSG-6 was further evaluated in cultured limbal epithelial stem/progenitor cells, macrophages, and diabetic mice by short hairpin RNA (shRNA) knockdown. Local MSC transplantation significantly promoted diabetic corneal epithelial wound healing, accompanied by elevated corneal TSG-6 expression, increased corneal epithelial cell proliferation, and attenuated inflammatory response. Moreover, in cultured human limbal epithelial stem/progenitor cells, TSG-6 enhanced the colony-forming efficiency, stimulated mitogenic proliferation, and upregulated the expression level of ΔNp63. Furthermore, in diabetic mouse cornea and in vitro macrophage culture, TSG-6 alleviated leukocyte infiltration and promoted the polarization of recruited macrophages to anti-inflammatory M2 phenotypes with increased phagocytotic capacity. In addition, the promotion of epithelial stem/progenitor cell activation and macrophage polarization by MSC transplantation was largely abrogated by shRNA knockdown of TSG-6. This study provided the first evidence of TSG-6 secreted by MSCs promoting corneal epithelial wound healing in diabetic mice through activating corneal epithelial stem/progenitor cells and accelerating M2 macrophage polarization.

Undifferentiated Connective Tissue Disease

MedlinePlus

... Home Conditions Undifferentiated Connective Tissue Disease (UCTD) Undifferentiated Connective Tissue Disease (UCTD) Make an Appointment Find a Doctor ... by Barbara Goldstein, MD (February 01, 2016) Undifferentiated connective tissue disease (UCTD) is a systemic autoimmune disease. This ...

Nitric Oxide Promotes Airway Epithelial Wound Repair through Enhanced Activation of MMP-9

PubMed Central

Bove, Peter F.; Wesley, Umadevi V.; Greul, Anne-Katrin; Hristova, Milena; Dostmann, Wolfgang R.; van der Vliet, Albert

2007-01-01

The airway epithelium provides a protective barrier against inhaled environmental toxins and microorganisms, and epithelial injury initiates a number of processes to restore its barrier integrity, including activation of matrix metalloproteinases such as MMP-9 (92-kD gelatinase B). Airway epithelial cells continuously produce nitric oxide (NO), which has been linked to cell migration and MMP-9 regulation in several cell types, but the importance of epithelial NO in mediating airway epithelial repair or MMP-9 activation is unknown. Using primary or immortalized human bronchial epithelial cells, we demonstrate that low concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which was associated with increased localized expression and activation of MMP-9. In addition, in HBE1 cells that were stably transfected with inducible NOS (NOS2), to mimic constitutive epithelial NOS2 expression in vivo, NOS inhibition decreased epithelial wound repair and MMP-9 expression. The stimulatory effects of NO on epithelial wound repair and MMP-9 expression were dependent on cGMP-mediated pathways and were inhibited by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase. Inhibition of cGMP-dependent protein kinase (PKG) attenuated NO-mediated epithelial wound closure, but did not affect MMP-9 expression. However, pharmacologic MMP inhibition and siRNA knockdown of MMP-9 expression demonstrated the contribution of MMP-9 to NO-mediated wound closure. Overall, our results demonstrate that NOS2-derived NO contributes to airway epithelial repair by both PKG-dependent and -independent mechanisms, and involves NO-dependent expression and activation of MMP-9. PMID:16980554

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis

PubMed Central

Bosveld, Floris; Markova, Olga; Guirao, Boris; Martin, Charlotte; Wang, Zhimin; Pierre, Anaëlle; Balakireva, Maria; Gaugue, Isabelle; Ainslie, Anna; Christophorou, Nicolas; Lubensky, David K.; Minc, Nicolas; Bellaïche, Yohanns

2017-01-01

The orientation of cell division along the interphase cell long-axis, the century old Hertwig’s rule, has profound roles in tissue proliferation, morphogenesis, architecture and mechanics1,2. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways3–12. At mitosis, epithelial cells usually round up to ensure faithful chromosome segregation and to promote morphogenesis1. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. We found that in Drosophila epithelia, tricellular junctions (TCJ) localize microtubule force generators, orienting cell division via the Dynein associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJ emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues. PMID:26886796

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

PubMed Central

2012-01-01

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. PMID:22857383

Isolation of Mouse Primary Gastric Epithelial Cells to Investigate the Mechanisms of Helicobacter pylori Associated Disease.

PubMed

Tran, Le Son; Ferrero, Richard L

2018-01-01

The gastrointestinal epithelium provides the first line of defense against invading pathogens, among which Helicobacter pylori is linked to numerous gastric pathologies, including chronic gastritis and cancer. Primary gastric epithelial cells represent a useful model for the investigation of the underlying molecular and cellular mechanisms involved in these H. pylori associated diseases. In this chapter, we describe a method for the isolation of primary gastric epithelial cells from mice and detection of epithelial cell adhesion molecule (EpCAM) expression in the isolated cells.

CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.

2014-09-10

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells.more » Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.« less

Sex cord-gonadal stromal tumor of the rete testis.

PubMed

Sajadi, Kamran P; Dalton, Rory R; Brown, James A

2009-01-01

A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

Accumulation of high OPDA level correlates with reduced ROS and elevated GSH benefiting white cell survival in variegated leaves

USDA-ARS?s Scientific Manuscript database

Variegated Epipremnum aureum ‘Marble Queen’ plant has white (VMW) and green (VMG) sectors within the same leaf. The white sector cells containing undifferentiated chloroplasts are viable, but the underlying mechanism for their survival is not clear. Because phytohormones are important for plant grow...

Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

1995-12-31

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2},more » and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.« less

Acute leukemia coexpressing myeloid, B- and T-lineage associated markers: multiparameter analysis of criteria defining lineage commitment and maturational stage in a case of undifferentiated leukemia.

PubMed

Meckenstock, G; Heyll, A; Schneider, E M; Hildebrandt, B; Runde, V; Aul, C; Bartram, C R; Ludwig, W D; Schneider, W

1995-02-01

Coexpression of myeloid, B-, and T-lineage associated markers was found in a patient with morphologically and cytochemically undifferentiated acute leukemia. Surface marker analysis using two-color immunofluorescence staining characterized blast cells to express CD34, CD38, CD117, and class II antigens, coexpressing TdT, CD4, CD7, CD13, CD19, and CD33. Cytoplasmic expression of myeloperoxidase, CD3, and CD22 could not be demonstrated. Monosomy for chromosome 7 was found by cytogenetic analysis. The absence of clonal rearrangements of immunoglobulin or T-cell receptor genes was shown by Southern blot analysis. Using a 3H-thymidine incorporation assay, DNA synthesis of leukemic blasts could be stimulated by IL-3, IL-6 and G-CSF in vitro. The present case did not offer specific criteria of lineage commitment. Corresponding to an equivalent counterpart in normal hematopoiesis, the involved cell population may reflect an early, most immature developmental stage within a multipotent progenitor cell compartment.

Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

PubMed Central

Wang, Xiaocheng; Tan, Bie; Li, Tiejun; Yin, Yulong

2016-01-01

The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets. PMID:27022727

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Jung Ar; Chung, Jin Sil; Cho, Sang-Ho

Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain.more » Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H{sub 2}O{sub 2}) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H{sub 2}O{sub 2} treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.« less

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

PubMed Central

Tron, Victor A.; Trotter, Martin J.; Tang, Liren; Krajewska, Maryla; Reed, John C.; Ho, Vincent C.; Li, Gang

1998-01-01

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction. PMID:9708817

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

PubMed

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Cell death at the intestinal epithelial front line.

PubMed

Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

2016-07-01

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

PubMed Central

Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

2012-01-01

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

PubMed

Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

2015-01-01

One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

PubMed Central

2010-01-01

Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues. PMID:20964822

Genetics and epithelial cell dysfunction in cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riordan, J.R.; Buchwald, M.

1987-01-01

This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

Cripto-1 Ablation Disrupts Alveolar Development in the Mouse Mammary Gland through a Progesterone Receptor–Mediated Pathway

PubMed Central

Klauzinska, Malgorzata; McCurdy, David; Rangel, Maria Cristina; Vaidyanath, Arun; Castro, Nadia P.; Shen, Michael M.; Gonzales, Monica; Bertolette, Daniel; Bianco, Caterina; Callahan, Robert; Salomon, David S.; Raafat, Ahmed

2016-01-01

Cripto-1, a member of the epidermal growth factor–Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways. PMID:26429739

Adherence of Lactobacillus crispatus to vaginal epithelial cells from women with or without a history of recurrent urinary tract infection.

PubMed

Kwok, Louisa; Stapleton, Ann E; Stamm, Walter E; Hillier, Sharon L; Wobbe, Cheryl L; Gupta, Kalpana

2006-11-01

Lactobacillus crispatus strain CTV-05 is a vaginal probiotic proposed for use in women with recurrent urinary tract infection to reduce vaginal colonization with Escherichia coli and the risk of urinary tract infection. However, the ability of this probiotic strain to adhere to the target mucosa, vaginal epithelial cells, has not been assessed in women with recurrent urinary tract infection. We measured the adherence of L. crispatus strain CTV-05 to vaginal epithelial cells collected from more than 100 premenopausal women with (cases) and without (controls) a history of recurrent urinary tract infection. We also examined the effects of relevant host factors on bacterial adherence. Bacterial adherence assays were performed by combining L. crispatus CTV-05 with exfoliated vaginal epithelial cells collected from 51 case women and 51 controls. L. crispatus CTV-05 adhered in high numbers to vaginal epithelial cells from women with recurrent urinary tract infection (mean adherence of 50.5 lactobacilli per vaginal epithelial cell) and controls (mean adherence of 39.4 lactobacilli per vaginal epithelial cell). Adherence was significantly higher using vaginal epithelial cells from women with a maternal history of urinary tract infection (p = 0.036) and a nonsecretor phenotype (p < 0.001), but was not significantly affected by recent spermicide use, oral contraceptive use, menstrual cycle phase or sexual activity. L. crispatus strain CTV-05 is highly adherent to vaginal epithelial cells collected from a large sample of premenopausal women with or without a history of recent recurrent urinary tract infection. These data strongly support further evaluation of this probiotic in clinical trials of women with recurrent urinary tract infection.

TRPA1 channels: expression in non-neuronal murine lung tissues and dispensability for hyperoxia-induced alveolar epithelial hyperplasia.

PubMed

Kannler, Martina; Lüling, Robin; Yildirim, Ali Önder; Gudermann, Thomas; Steinritz, Dirk; Dietrich, Alexander

2018-05-12

Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca 2+ ([Ca 2+ ] i ) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1-/-) mouse lungs. In all cell types, we detected hyperoxia-induced rises in [Ca 2+ ] i levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1-/- controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1-/- mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in [Ca 2+ ] i levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.

Psychosexual Intervention in Patients With Stage I-III Gynecologic or Breast Cancer

ClinicalTrials.gov

2018-05-25

Ovarian Sarcoma; Ovarian Stromal Cancer; Stage I Uterine Sarcoma; Stage I Vaginal Cancer; Stage I Vulvar Cancer; Stage IA Cervical Cancer; Stage IA Endometrial Carcinoma; Stage IA Fallopian Tube Cancer; Stage IA Ovarian Epithelial Cancer; Stage IA Ovarian Germ Cell Tumor; Stage IA Primary Peritoneal Cavity Cancer; Stage IB Cervical Cancer; Stage IB Endometrial Carcinoma; Stage IB Fallopian Tube Cancer; Stage IB Ovarian Epithelial Cancer; Stage IB Ovarian Germ Cell Tumor; Stage IB Primary Peritoneal Cavity Cancer; Stage IC Fallopian Tube Cancer; Stage IC Ovarian Epithelial Cancer; Stage IC Ovarian Germ Cell Tumor; Stage IC Primary Peritoneal Cavity Cancer; Stage II Endometrial Carcinoma; Stage II Gestational Trophoblastic Tumor; Stage II Uterine Sarcoma; Stage II Vaginal Cancer; Stage II Vulvar Cancer; Stage IIA Cervical Cancer; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Epithelial Cancer; Stage IIA Ovarian Germ Cell Tumor; Stage IIA Primary Peritoneal Cavity Cancer; Stage IIB Cervical Cancer; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Epithelial Cancer; Stage IIB Ovarian Germ Cell Tumor; Stage IIB Primary Peritoneal Cavity Cancer; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Epithelial Cancer; Stage IIC Ovarian Germ Cell Tumor; Stage IIC Primary Peritoneal Cavity Cancer; Stage III Gestational Trophoblastic Tumor; Stage III Uterine Sarcoma; Stage III Vaginal Cancer; Stage III Vulvar Cancer; Stage IIIA Cervical Cancer; Stage IIIA Endometrial Carcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cavity Cancer; Stage IIIB Cervical Cancer; Stage IIIB Endometrial Carcinoma; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cavity Cancer; Stage IIIC Endometrial Carcinoma; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cavity Cancer; Breast Cancer

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

PubMed

Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

2017-04-01

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

Tracking and Functional Characterization of Epithelial-Mesenchymal Transition and Mesenchymal Tumor Cells During Prostate Cancer Metastasis

PubMed Central

Ruscetti, Marcus; Quach, Bill; Dadashian, Eman L.; Mulholland, David J.; Wu, Hong

2015-01-01

The epithelial-mesenchymal transition (EMT) has been postulated as a mechanism by which cancer cells acquire the invasive and stem-like traits necessary for distant metastasis. However, direct in vivo evidence for the role of EMT in the formation of cancer stem-like cells (CSC) and the metastatic cascade remains lacking. Here we report the first isolation and characterization of mesenchymal and EMT tumor cells, which harbor both epithelial and mesenchymal characteristics, in an autochthonous murine model of prostate cancer. By crossing the established Pb-Cre+/−;PtenL/L;KrasG12D/+ prostate cancer model with a vimentin-GFP reporter strain, generating CPKV mice, we were able to isolate epithelial, EMT and mesenchymal cancer cells based on expression of vimentin and EpCAM. CPKV mice (but not mice with Pten deletion alone) exhibited expansion of cells with EMT (EpCAM+/Vim-GFP+) and mesenchymal (EpCAM−/Vim-GFP+) characteristics at the primary tumor site and in circulation. These EMT and mesenchymal tumor cells displayed enhanced stemness and invasive character compared to epithelial tumor cells. Moreover, they displayed an enriched tumor-initiating capacity and could regenerate epithelial glandular structures in vivo, indicative of epithelia-mesenchyme plasticity. Interestingly, while mesenchymal tumor cells could persist in circulation and survive in the lung following intravenous injection, only epithelial and EMT tumor cells could form macrometastases. Our work extends the evidence that mesenchymal and epithelial states in cancer cells contribute differentially to their capacities for tumor initiation and metastatic seeding, respectively, and that EMT tumor cells exist with plasticity that can contribute to multiple stages of the metastatic cascade. PMID:25948589

Candida albicans triggers interleukin-8 secretion by oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-04-01

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha. Copyright 2003 Elsevier Science Ltd.

Pattern of SMARCB1 (INI1) and SMARCA4 (BRG1) in poorly differentiated endometrioid adenocarcinoma of the uterus: analysis of a series with emphasis on a novel SMARCA4-deficient dedifferentiated rhabdoid variant.

PubMed

Strehl, Johanna D; Wachter, David L; Fiedler, Jutta; Heimerl, Engelbert; Beckmann, Matthias W; Hartmann, Arndt; Agaimy, Abbas

2015-08-01

The role of the switch/sucrose nonfermenting chromatin remodeling complex in the initiation and progression of cancer is emerging. In the female genital tract, only ovarian small cell carcinoma, hypercalcemic type harbors recurrent inactivating SMARCA4 mutations. Otherwise, only rare case reports documented SMARCB1 involvement in endometrial cancer. We analyzed 24 grade 3 uterine endometrioid adenocarcinomas and 2 undifferentiated carcinomas for immunohistochemical expression of SMARCB1 and SMARCA4. All tumors showed high-grade nuclear features with a predominance of solid growth pattern. All cases showed intact nuclear SMARCB1 expression in all tumor cells. However, 1 case of a 78-year-old woman showed complete loss of SMARCA4 in 90% of the tumor with retained expression in 10% of the tumor. The SMARCA4-intact component was a moderate-to-poorly differentiated endometrioid adenocarcinoma. The SMARCA4-deficient dominating component showed solid growth of highly anaplastic undifferentiated large cells with prominent rhabdoid features. None of the 25 SMARCA4-intact cases showed rhabdoid cell morphology. To our knowledge, this is the first systematic study of SMARCB1 and SMARCA4 expression in endometrioid adenocarcinoma of uterus and the first description of a novel SMARCA4-deficient variant of dedifferentiated/undifferentiated endometrial carcinoma. The presence of a differentiated SMARCA4-intact endometrioid component points to a novel pathway of dedifferentiation in endometrioid adenocarcinoma as a consequence of a "second hit." This case further underlines the close link between the "rhabdoid phenotype" and the SWI/SNF pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

PubMed

Ho, Hoang-Yen; Moffat, Ryan C; Patel, Rupal V; Awah, Franklin N; Baloue, Kaitrin; Crowe, David L

2010-09-01

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Claudin-7-positive synchronous spontaneous intrahepatic cholangiocarcinoma, adenocarcinoma and adenomas of the gallbladder in a Bearded dragon (Pogona vitticeps).

PubMed

Jakab, Csaba; Rusvai, Miklós; Szabó, Zoltán; Gálfi, Péter; Marosán, Miklós; Kulka, Janina; Gál, János

2011-03-01

In this study, synchronous spontaneous, independent liver and gallbladder tumours were detected in a Bearded dragon (Pogona vitticeps). The multiple tumours consisted of intrahepatic cholangiocarcinoma as well as in situ adenocarcinoma and two adenomas of the gallbladder. The biliary epithelial cells and the cholangiocarcinoma showed membranous cross-immunoreactivity for claudin-7. The gallbladder epithelial cells, its adenoma and adenocarcinoma showed basolateral cross-reactivity for claudin-7. We think that the humanised anti-claudin-7 antibody is a good marker for the detection of different primary cholangiocellular and gallbladder tumours in Bearded dragons. The cholangiocytes, the cholangiocarcinoma, the endothelial cells of the liver and the epithelial cells and gallbladder tumours all showed claudin-5 cross-reactivity. The humanised anti-cytokeratin AE1-AE3 antibody showed cross-reactivity in the biliary epithelial cells, cholangiocarcinoma cells, epithelial cells and tumour cells of the gallbladder. It seems that this humanised antibody is a useful epithelial marker for the different neoplastic lesions of epithelial cells in reptiles. The humanised anti-α-smooth muscle actin (α-SMA) antibody showed intense cross-reactivity in the smooth muscle cells of the hepatic vessels and in the muscle layer of the gallbladder. The portal myofibroblasts, the endothelial cells of the sinusoids and the stromal cells of the cholangiocarcinoma and gallbladder tumours were positive for α-SMA. The antibovine anti-vimentin and humanised anti-Ki-67 antibodies did not show crossreactivity in the different samples from the Bearded dragon.

A LGG-derived protein promotes IgA production through up-regulation of APRIL expression in intestinal epithelial cells

PubMed Central

Wang, Yang; Liu, Liping; Moore, Daniel J; Shen, Xi; Peek, Richard M.; Acra, Sari A; Li, Hui; Ren, Xiubao; Polk, D Brent; Yan, Fang

2016-01-01

p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting IgA production. p40 up-regulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl , but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice. Thus, p40 up-regulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production. PMID:27353252

Assessment of cytologic evaluation of preputial epithelial cells as a diagnostic test for detection of adrenocortical disease in castrated ferrets.

PubMed

Protain, Holly J; Kutzler, Michelle A; Valentine, Beth A

2009-05-01

To determine whether results of cytologic evaluation of preputial epithelial cells correspond to results of a serum endocrine hormone assay and clinical signs associated with adrenocortical disease in castrated ferrets. 13 clinically normal ferrets and 8 ferrets with signs of adrenocortical disease. Blood and preputial lavage samples were collected from each ferret. Serum samples were submitted to the University of Tennessee Veterinary Diagnostic Laboratory for performance of an endocrine hormone assay. Differential epithelial cell counts were performed on preputial lavage samples to determine the percentage of cornified cells. Results of cytologic evaluation were compared with results of the endocrine hormone assay and clinical status of ferrets. The percentage of cornified preputial epithelial cells was not significantly correlated with serum 17B-estradiol or androstenedione concentration but was significantly correlated with serum 17-hydroxyprogesterone concentration (r = 0.60). The percentage of cornified preputial epithelial cells was higher in ferrets with clinical signs of adrenocortical disease (mean +/- SD, 71.3 +/- 16.9%) than in clinically normal ferrets (55.5 +/- 19.0%). Cornification of preputial epithelial cells was correlated with an increase in serum 17-hydroxyprogesterone concentration as well as clinical signs of adrenocortical disease in castrated ferrets. Additional investigation is needed to elucidate the mechanism of preputial epithelial cell cornification in castrated ferrets.

Review with novel markers facilitates precise categorization of 41 cases of diagnostically challenging, "undifferentiated small round cell tumors". A clinicopathologic, immunophenotypic and molecular analysis.

PubMed

Machado, Isidro; Yoshida, Akihiko; Morales, María Gema Nieto; Abrahão-Machado, Lucas Faria; Navarro, Samuel; Cruz, Julia; Lavernia, Javier; Parafioriti, Antonina; Picci, Piero; Llombart-Bosch, Antonio

2017-11-29

Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. Almost all the tumors (n=40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n=16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n=1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n=7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n=3), neuroblastoma (n=2), unclassifiable neoplasm with neuroblastic differentiation (n=1), malignant rhabdoid tumor (n=2), lymphoblastic lymphoma (n=1), clear cell sarcoma of the gastrointestinal tract (n=1), small cell carcinoma (n=1), sclerosing rhabdomyosarcoma (n=1), desmoplastic small round cell tumor (n=1), malignant peripheral sheath nerve tumor (n=1), poorly-differentiated synovial sarcoma (n=1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n=1) and possible SMARCA4-deficient-sarcoma (n=1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context. Copyright © 2017 Elsevier Inc. All rights reserved.

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

PubMed

Nguyen, Quy H; Pervolarakis, Nicholas; Blake, Kerrigan; Ma, Dennis; Davis, Ryan Tevia; James, Nathan; Phung, Anh T; Willey, Elizabeth; Kumar, Raj; Jabart, Eric; Driver, Ian; Rock, Jason; Goga, Andrei; Khan, Seema A; Lawson, Devon A; Werb, Zena; Kessenbrock, Kai

2018-05-23

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos

PubMed Central

Katow, Hideki

2015-01-01

Sea urchin mesenchyme is composed of the large micromere-derived spiculogenetic primary mesenchyme cells (PMC), veg2-tier macromere-derived non-spiculogenetic mesenchyme cells, the small micromere-derived germ cells, and the macro- and mesomere-derived neuronal mesenchyme cells. They are formed through the epithelial-to-mesenchymal transition (EMT) and possess multipotency, except PMCs that solely differentiate larval spicules. The process of EMT is associated with modification of epithelial cell surface property that includes loss of affinity to the apical and basal extracellular matrices, inter-epithelial cell adherens junctions and epithelial cell surface-specific proteins. These cell surface structures and molecules are endocytosed during EMT and utilized as initiators of cytoplasmic signaling pathways that often initiate protein phosphorylation to activate the gene regulatory networks. Acquisition of cell motility after EMT in these mesenchyme cells is associated with the expression of proteins such as Lefty, Snail and Seawi. Structural simplicity and genomic database of this model will further promote detailed EMT research. PMID:26716069

A Computational Study of the Development of Epithelial Acini: II. Necessary Conditions for Structure and Lumen Stability

PubMed Central

Rejniak, Katarzyna A.; Anderson, Alexander R.A.

2013-01-01

Simple epithelial tissues are organized as single layers of tightly packed cells that surround hollow lumens and form selective barriers separating different internal compartments of the body. The maintenance of epithelial structure and its function requires tight coordination and control of all the life processes of epithelial cells via cell-to-cell communication and signaling. These well-balanced cellular systems are, however, quite often disturbed by genetic or environmental cues that may lead to the formation of epithelial tumors (carcinomas). In fact, more than a half of all diagnosed tumors are initiated from epithelial cells. It is, therefore, important to gain a greater understanding of the factors that form and maintain the epithelial structure, as well as the features of the acinar structure that are modified during cancer development as observable in experimental and clinical research. We address these questions using the bio-mechanical model of the developing hollow epithelial acini introduced in Rejniak and Anderson (Bull. Math. Biol. 70:677–712, 2008). Here, we propose several scenarios involving various bio-mechanical interactions between neighboring cells that result in abnormal acinar development. Whenever possible, we compare our computational results with known experimental cases of mutant acini. PMID:18401665

The Role of Epithelial-Mesenchymal Transition in the Formation of Normal and Neoplastic Mammary Epithelial Stem Cells

DTIC Science & Technology

2011-09-01

separating stem cell and non- stem cell populations of normal and breast cancer cells and identified EMT transcription factors most likely involved in... stem cell biology. Preliminary results directly demonstrate that transient induction of EMT increases the number of mammary epithelial stem cells...EMT and entrance into a stem - cell state. The outcome of these experiments holds important implications for the mechanisms controlling the formation of

Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor.

PubMed

Chandrasekher, Gudiseva; Ma, Xiang; Lallier, Thomas E; Bazan, Haydee E P

2002-05-01

To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

PubMed Central

Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F.; Vörös, Andrea; Hegedűs, Zoltán; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

2015-01-01

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

A hybrid computational model to explore the topological characteristics of epithelial tissues.

PubMed

González-Valverde, Ismael; García-Aznar, José Manuel

2017-11-01

Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells.

PubMed

Eshkiki, Zahra Shokati; Ghahremani, Mohammad Hossein; Shabani, Parisa; Firuzjaee, Sattar Gorgani; Sadeghi, Asie; Ghanbarian, Hossein; Meshkani, Reza

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.

Radiation damage to the microvasculature in the rabbit ear chamber. An electron microscope study. [X radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, V.V.; Stearner, S.P.; Dimitrievich, G.S.

1977-04-01

Cell aggregates in increased numbers appear along blood vessel walls within a few days after local x irradiation of the tissue within rabbit ear chambers. At 7 days after irradiation with 400 or 700 rad of 250 kVp of x rays, electron microscopic studies of the microvasculature were carried out to determine the morphological characteristics of the cell types involved in the aggregates and the relation of these cells to vascular repair. The cell aggregates usually occur in the interstitial region subjacent to the endothelium. The cells that make up the aggregates show morphological characteristics of relatively undifferentiated mesenchymal cells;more » they have an irregularly rounded shape and contain large amounts of rough endoplasmic reticulum, Golgi vesicles, and mitochondria. In a few instances, cells of similar morphology also occur as part of the lining of the blood vessels. The perivascular cell aggregates may originate from the pericyte population or from undifferentiated mesenchymal cells that occur in the interstitial region surrounding blood vessels; it is improbable that they are dedifferentiated smooth muscle cells. It is suggested that the cells that make up these aggregates contribute to the repair of the microvasculation after radiation injury. The radiosensitivity of vascular endothelium reported by previous investigators seems to preclude endothelial proliferation as the principal repair mechanism at higher radiation doses.« less

Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

PubMed Central

Osherov, Nir

2012-01-01

Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia) into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just “innocent bystanders” or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides, and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome. PMID:23055997

Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

PubMed

Shariff, Sami; Shelfoon, Christopher; Holden, Neil S; Traves, Suzanne L; Wiehler, Shahina; Kooi, Cora; Proud, David; Leigh, Richard

2017-06-01

Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

Neuroglian stabilizes epithelial structure during Drosophila oogenesis.

PubMed

Wei, Jun; Hortsch, Michael; Goode, Scott

2004-08-01

The vertebrate L1 family of cell adhesion molecules (CAMs) and their fly homolog, Neuroglian, are members of the immunoglobulin (Ig) superfamily of CAMs. In general, Ig CAMs have been found to play critical roles in mediating axon guidance. One Ig CAM, NCAM, has also been implicated in maintaining epithelial integrity and suppressing metastatic dissemination of tumor cells. Other Ig CAMs, such as Nrg, are also expressed in epithelia. We thus tested the hypothesis that, like NCAM, Nrg might also be required for maintaining epithelial integrity and for inhibiting tumor invasion. We used the Drosophila follicular epithelium to determine the function of Nrg in vivo in maintaining epithelial structure, and in regulating the motility of migrating border cells and invasive tumorous follicle cells. Nrg(167) is expressed on the lateral membrane of follicle cells. Loss of Nrg(167) causes border cells to delay delamination and causes other follicle cells to delaminate inappropriately. The delaminated cells have aberrant epithelial polarity manifested as severe mislocalization of apical and basal membrane proteins, and uniform localization of lateral membrane proteins. Furthermore, loss of Nrg(167) dramatically enhances the invasive phenotype associated with loss of Discs Large, a neoplastic tumor suppressor. These results indicate that Nrg(167) stabilizes epithelial polarity by regulating junctional adhesion and function in normal and tumorous epithelia. Our data also suggest that Ig superfamily members have significant functional redundancy in maintaining epithelial polarity, with individual members playing subtle, unique roles during epithelial morphogenesis. Copyright 2004 Wiley-Liss, Inc.