Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins

The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs.     

Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma

Background

At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA).

Methods

Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium.

Results

The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44^high, CD24^low, and AC133⁺. These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α₂β₁, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α₂β₁, and CD146 surface marker expression than the epithelial type cells.

Conclusion

The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.     

Characterization of immortalized rabbit lacrimal gland epithelial cells

Summary To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm² plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 μM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.     

Gland formation from human endometrial epithelial cells in vitro

Summary We have developed methods for the culture of human endometrial glandular epithelia in vitro. The culture medium is serum-free and is used in combination with Matrigel, an extracellular matrix material applied as a coating on cell culture plates. Cell growth begins as a monolayer, but the cells subsequently form glandular or organoid structures. The glands are composed of polar columnar cells facing a central lumen, which is enclosed by the apical surfaces of cells displaying numerous microvilli and sealed by tight junction complexes. The ability to study in vitro the complex process of glandular morphogenesis represents an important new tool in cell biology which may be used to investigate growth regulation, hormone production and dependency, and cellular recognition and interactions. Ultimately, these characteristics may be applied to study the alterations of glandular epithelia associated with neoplasia. This work was supported by NIH grants CA31733 and CA09156 and NIEHS contract ES55092     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Isolation and monolayer culture of human fallopian tube epithelial cells

Summary In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy. The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would permit effective study of co-culture with embryos.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues

Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium

Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF) was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture. The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding density of 17 cells/cm² with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a 0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for the study of cell differentiation of tracheal epithelium.     

Human pancreatic adenocarcinoma: In vitro and in vivo morphology of a new tumor line established from ascites

Summary A human pancreatic tumor cell line has been established from the ascites of a patient with histopathologically confirmed adenocarcinoma of the head of the pancreas and maintained for more than 12 months in the laboratory. Epitheloid tumor cell colonies, which resulted from primary tissue cultures of the ascitic cell component, were mechanically isolated by needle micromanipulation. Tumorigenicity was proven in athymic nude mice. Morphologically the pancreatic tumor epithelial cells grew to confluency with moderately tight adhesion to the culture plastic surface and with free-floating cells in the medium. Upon re-establishment of the tumoral xenograft in tissue culture, the epithelial cells retained their original morphology. Histologically the tumor grown in nude mice exhibited prototypic characteristics of the primary adenocarcinoma in the patient, producing abundant mucin and displaying a broad spectrum of glandular differentiation, which ranged from well to poorly differentiated adenocarcinomas with occasionally localized lymphocytic infiltrations. Furthermore, the tumor expressed carcinoembryonic antigen and human pancreas cancer associated antigen. This tumor line, designated AsPC-1, has been cultured for at least 10 passages in vitro and 3 in vivo. It represents a new model for human pancreatic cancer. This work was supported in part by Research Grant CA-18410 awarded by the National Cancer Institute through the National Pancreatic Cancer Project.     

A virus-producing cell line developed by transformation of human parapharyngeal cells with SV40

Summary Normal cultures of epithelial appearance were initiated by trypsinization of a surgically resected, histologically normal branchial cyst. Cellular morphology was consistent with derivation from the stratified squamous epithelium of the cyst or from vascular endothelium, although electron micrographs of the cultured cells failed to show any junctional complexes. Infection with SV40 produced transformants which were also epithelioid in appearance. These grew vigorously for 22 to 50 population doublings (about 23 to 32 subcultures, depending upon regimen) and then became quiescent. During this evolution, virus was detectable at all stages by both direct isolation (cell extracts) and cocultivation with permissive cells. In two sublines in which selection for rapidly growing cell types occurred, virus was detected only by cocultivation. The work confirms that of others in the finding that normal human epithelial cells are susceptible to transformation by oncogenic viruses, but are apparently less responsive than are fibroblasts to such transforming agents. It also suggests that subcultivation techniques that maintain the populations of transformed cells at low density tend to select against cell strains that are continuous producers of infectious virus. This study was supported by Grant CA 13494 from the National Cancer Institute.     

人胎儿鼻咽上皮细胞的背景氯电流

采用膜片钳和图像分析技术,研究人胎儿鼻咽上皮细胞背景电流的特性及其与容积激活性氯电流的关系。在等张溶液中,可记录到一背景电流,该电流呈微弱的外向整流性,无明显时间依赖性失活,其翻转电位为(?0.73±1.7)mV(n=21),接近氯离子平衡电位(?0.9mV)。细胞外高张刺激(440mOsmol/L)明显抑制此电流(59.6±7.1)%,而低张刺激(160mOsmol/L)则诱发细胞产生容积激活性氯电流。氯通道阻断剂tamoxifen和5-硝基-2-(3-苯丙胺基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB]显著地抑制背景电流并使细胞基础容积增大。上述结果表明,人胎儿鼻咽上皮细胞的背景Cl?电流是背景电流的重要成分,此Cl?电流与容积激活性氯电流及细胞基础容积调节有关。     

A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse

Summary A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Rat lens epithelial cells in vitro

Summary Lens epithelial cells from rats aged 5 days were grown in long-term cultures. These cells age, differentiate and transform spontaneously. Morphological observations indicate five different stages (A-E). The epithelial character is lost after the first two passages. Elongated cells appearing afterwards are considered as cells that have started differentiation to fiberlike cells. Big flattened cells are considered as senescent cells that have lost their proliferative capacity. Data from population kinetics also reflect these five stages. Chromosome analysis shows that three of the five stages are no longer diploid. Two alternative modes of spontaneous transformation are possible. The proliferative capacity of rat lens epithelial cells is higher than that of rat embryonic fibroblast systems. The investigations were supported by the Deutsche Forschungsgemeinschaft (Biology of Aging, Grants Ri 285/2 and Ri 285/3).     

Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody

Summary Two monoclonal antibodies respectively recognizing cytokeratins (CK) 18 and 19 were applied to the human thymic epithelium (in vivo and in vitro) in normal and pathological conditions, including 12 thymomas. We observed that in both normal and hyperplastic thymuses (from patients with myasthenia gravis) virtually the entire epithelial network was CK19-positive as were the majority of cells growing in culture. In four thymomas, however, the expression of cytokeratin 19 was not detected by immunofluorescence. On the other hand, CK18 was expressed by a discrete subset of medullary thymic epithelial cells in normal and in hyperplastic thymuses. Among the thymomas a large majority was either negative or contained few isolated CK18-positive cells scattered within the tumour. Conversely, in the two undifferentiated epithelial thymomas, virtually all the tumoral network was strongly labeled with the anti-CK18 monoclonal antibody. The present investigation thus not only defines the human thymic epithelial cell subset on the basis of differential cytokeratin expression but also indicates that anti-CK antibodies with single cytokeratin specificities can be regarded as useful tools to study the heterogeneity of thymomas.     

Novel type of interstitial cell (Cajal-like) in human fallopian tube

We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.