Development and characterization of a cell line WAF from freshwater shark Wallago attu.

PubMed

Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Sharma, Bhagwati S

2014-02-01

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus.

PubMed

Ganassin, R C; Sanders, S M; Kennedy, C J; Joyce, E M; Bols, N C

1999-01-01

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.

High metastaticgastric and breast cancer cells consume oleic acid in an AMPK dependent manner.

PubMed

Li, Shuai; Zhou, Ti; Li, Cen; Dai, Zhiyu; Che, Di; Yao, Yachao; Li, Lei; Ma, Jianxing; Yang, Xia; Gao, Guoquan

2014-01-01

Gastric cancer and breast cancer have a clear tendency toward metastasis and invasion to the microenvironment predominantly composed of adipocytes. Oleic acid is an abundant monounsaturated fatty acid that releases from adipocytes and impinges on different energy metabolism responses. The effect and underlying mechanisms of oleic acid on highly metastatic cancer cells are not completely understood. We reported that AMP-activated protein kinase (AMPK) was obviously activated in highly aggressive carcinoma cell lines treated by oleic acid, including gastric carcinoma HGC-27 and breast carcinoma MDA-MB-231 cell lines. AMPK enhanced the rates of fatty acid oxidation and ATP production and thus significantly promoted cancer growth and migration under serum deprivation. Inactivation of AMPK attenuated these activities of oleic acid. Oleic acid inhibited cancer cell growth and survival in low metastatic carcinoma cells, such as gastric carcinoma SGC7901 and breast carcinoma MCF-7 cell lines. Pharmacological activation of AMPK rescued the cell viability by maintained ATP levels by increasing fatty acid β-oxidation. These results indicate that highly metastatic carcinoma cells could consume oleic acid to maintain malignancy in an AMPK-dependent manner. Our findings demonstrate the important contribution of fatty acid oxidation to cancer cell function.

[Role of let-7 in maintaining characteristics of breast cancer stem cells].

PubMed

Sun, Xin; Fan, Chong; Hu, Li-juan; Du, Ning; Xu, Chong-wen; Ren, Hong

2012-08-01

To observe the expression of let-7 in breast cancer stem cells and explore the role of let-7 in maintaining the characteristics of breast cancer stem cells. We separated breast cancer stem cells (SP and NSP) from MCF-7 cell line using SP sorting, and observed the expression of let-7a/b/c on SP and NSP cells using quantitative real-time PCR and the expressions of Ras and ERK using Western blotting to study the mechanism by which let-7 maintains the characteristics of breast cancer stem cells. The SP cells accounted for 3.3% in MCF-7 cells, however, the rate dropped to 0.4% when verapamil was added into the process of seperation. The level of Let-7a/b/c in SP cells were lower than that in NSP cells, and among let-7 miRNAs, let-7b/c showed the most obvious difference. The expressions of t-Ras and t-ERK showed no difference between SP and NSP cells, nevertheless, the expressions of p-Ras, p-ERK were higher in SP cells than in NSP cells. SP sorting is an effective method to separate cancer stem cells. There do exist cancer stem cells in MCF-7 breast cancer cell line. Let-7 is down-regulated in SP cells, and the down-regulation makes let-7 lose the opportunity to restrain Ras mRNA, finally, p-Ras and p-ERK are activated. They play an important role in maintaining the characteristics of breast cancer stem cells.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro.

PubMed

Paolinelli, Roberta; Corada, Monica; Ferrarini, Luca; Devraj, Kavi; Artus, Cédric; Czupalla, Cathrin J; Rudini, Noemi; Maddaluno, Luigi; Papa, Eleanna; Engelhardt, Britta; Couraud, Pierre Olivier; Liebner, Stefan; Dejana, Elisabetta

2013-01-01

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

History of leukemia-lymphoma cell lines.

PubMed

Drexler, Hans G; Macleod, Roderick A F

2010-08-01

We outline the near 50-year history of leukemia-lymphoma (LL) cell lines - a key model system in biomedicine. Due to the detailed documentation of their oncogenomic and transcriptional alterations via recent advances in molecular medicine, LL cell lines may be fitted to parent tumors with a degree of precision unattainable in other cancers. We have surveyed the corpus of published LL cell lines and found 637 examples that meet minimum standards of authentication and characterization. Alarmingly, the rate of establishment of new LL cell lines has plummeted over the last decade. Although the main hematopoietic developmental cell types are represented by cell lines, some LL categories stubbornly resist establishment in vitro. The advent of engineering techniques for immortalizing primary human cells that maintain differentiation means the time is ripe for renewed search for in vitro models from un(der)represented hematologic entities. Given their manifold applications in biomedicine, there is little doubt that LL-derived cell lines will continue to play a vital part well into the next half-century as well. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

PubMed

Ju, Jin Young; Park, Chun Young; Gupta, Mukesh Kumar; Uhm, Sang Jun; Paik, Eun Chan; Ryoo, Zae Young; Cho, Youl Hee; Chung, Kil Saeng; Lee, Hoon Taek

2008-05-01

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Day-night cycles and the sleep-promoting factor, Sleepless, affect stem cell activity in the Drosophila testis.

PubMed

Tulina, Natalia M; Chen, Wen-Feng; Chen, Jung Hsuan; Sowcik, Mallory; Sehgal, Amita

2014-02-25

Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. Here, we explore a role of environmental day-night cycles in modulating cell cycle progression in populations of adult stem cells. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, we reveal daily rhythms in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. We also examine whether behavioral sleep-wake cycles, which are driven by the environmental day-night cycles, regulate stem cell function. We find that flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. We suggest that alterations in sleep can influence the daily dynamics of GSC divisions.

Human squamous cell carcinoma from skin: establishment and characterization of a new cell line (HSC-5).

PubMed

Hozumi, Y; Kondo, S; Shimoura, T; Aso, K

1990-03-01

A new cell line, designated as HSC-5 and derived from human skin squamous cell carcinoma (SCC), has been established in vitro and maintained proliferative in continuous tissue culture for over two years. The cells grow in a monolayer in vitro and have anaplastic epithelioid features. The doubling time was about 35 hr at the 30th passage. Chromosome analysis showed hypotetraploidy with a modal number of 76. A trial of transplantation of the cultured cells into nude mice was not successful. Analysis of cytokeratins from HSC-5 by two-dimensional gel electrophoresis revealed polypeptides No. 5, 8, 13, 18 and 19. The cell line is available to other investigators.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

PubMed Central

Wang, Su; Gao, Yuan; Song, Xiaoqing; Ma, Xing; Zhu, Xiujuan; Mao, Ying; Yang, Zhihao; Ni, Jianquan; Li, Hua; Malanowski, Kathryn E; Anoja, Perera; Park, Jungeun; Haug, Jeff; Xie, Ting

2015-01-01

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001 PMID:26452202

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

[The concept of cellular immortality, a myth or a reality. Example of "immortalized" articular chondrocytes].

PubMed

Adolphe, M; Thenet, S

1990-01-01

The concept of cellular immortality, which arose from the historical studies of A. Carrel, is getting a new start with the progress of virology. However, the definition of cell immortalization is still ambiguous. Although scientists agree that cells regarded as immortal have acquired an infinite growth capacity, the relationship of this change with the first stages of transformation is difficult to clearly define. Immortalized cell lines have already been obtained from numerous cell types by using viral infection or transfection with viral and cellular genes. Immortalization of cells is interesting for three main reasons: it permits study of the steps in progression to transformation, allows establishment of cell lines for producing biological products, and permits various cell types to maintain a part of their differentiated functions. For example, hypothalamic neurosecretory cells, macrophages, astrocytes and intestinal epithelial cells have been immortalized and these lines can be used for understanding the balance between division and differentiation, and also for pharmacotoxicological studies. In our laboratory, we immortalized rabbit articular chondrocytes by transfection with SV40 large T and little t encoding genes. At the 9th subculture, when the control culture was senescent, clones of polygonal cells appeared in the transfected cell cultures. Three clones have been selected and have been maintained in culture for two years. Growth curves of normal and SV40-transfected chondrocytes were compared and displayed similar doubling times (approximately 20 hours). The exponential phase of growth was longer for immortalized cells resulting in a 2-fold higher saturation density. These cells appear to be not fully transformed and maintain some properties of differentiated chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

PubMed Central

O’Meally, Denis; Quinn, Alexander E.; Sarre, Stephen D.; Georges, Arthur; Marshall Graves, Jennifer A.

2009-01-01

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies. PMID:19199067

Phytochelatin accumulation and cadmium tolerance in selected tomato cell lines.

PubMed

Gupta, S C; Goldsbrough, P B

1991-09-01

Four cell lines of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, were selected for their ability to grow in the presence of up to 6 millimolar CdCl(2). The intracellular Cd concentration in these cells was at least 2.3 times higher than in the medium. Growth in media containing higher concentrations of Cd was accompanied by increased production of Cd-binding phytochelatins and a trend toward accumulation of higher molecular weight phytochelatins. At least 90% of the Cd in the most tolerant cells was associated with Cd-phytochelatin complexes. Cell lines maintained an increased tolerance of Cd in the absence of continuous selection pressure.

Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models

PubMed Central

González, Raquel; Dobrinski, Ina

2015-01-01

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals. PMID:25991701

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells.

PubMed

Sada, Aiko; Hasegawa, Kazuteru; Pin, Pui Han; Saga, Yumiko

2012-02-01

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. Copyright © 2011 AlphaMed Press.

Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

PubMed Central

Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

2016-01-01

Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

Generation of a KLF15 homozygous knockout human embryonic stem cell line using paired CRISPR/Cas9n, and human cardiomyocytes derivation.

PubMed

Noack, Claudia; Haupt, Luis Peter; Zimmermann, Wolfram-Hubertus; Streckfuss-Bömeke, Katrin; Zelarayán, Laura Cecilia

2017-08-01

Krueppel-like factor 15 (KLF15) is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO) line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Attempt to develop taste bud models in three-dimensional culture.

PubMed

Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

2011-09-01

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

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

Wang, Meng-Yu; Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no; Rekdal, Øystein

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cellmore » marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.« less

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas

USGS Publications Warehouse

Lu, Y.; Nerurkar, V.R.; Aguirre, A.A.; Work, Thierry M.; Balazs, G.H.; Yanagihara, R.

1999-01-01

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30A? C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Establishment and characterization of five immortalized human scalp dermal papilla cell lines.

PubMed

Kwack, Mi Hee; Yang, Jung Min; Won, Gong Hee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2018-02-05

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research. Copyright © 2018 Elsevier Inc. All rights reserved.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

PubMed

Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

PubMed

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Development and characterization of two new cell lines from milkfish (Chanos chanos) and grouper (Epinephelus coioides) for virus isolation.

PubMed

Parameswaran, V; Ishaq Ahmed, V P; Shukla, Ravi; Bhonde, R R; Sahul Hameed, A S

2007-01-01

Two new cell lines, SIMH and SIGE, were derived from the heart of milkfish (Chanos chanos), a euryhaline teleost, and from the eye of grouper (Epinephelus coioides), respectively. These cell lines were maintained in Leibovitz's L-15 supplemented with 20% fetal bovine serum (FBS). The SIMH cell line was subcultured more than 50 times over a period of 210 days and SIGE cell line has been subcultured 100 times over a period of 1 1/2 years. The SIMH cell line consists predominantly of fibroblastic-like cells. The SIGE cell line consists predominantly of epithelial cells. Both the cell lines were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 28 degrees C with optimum growth at the concentrations of 15% or 20% FBS. Seven marine fish viruses were tested to determine the susceptibility of these cell lines. The SIGE cell line was found to be susceptible to nodavirus, MABV NC-1 and Y6, and the infection was confirmed by cytopathic effect (CPE) and reverse transcriptase-polymerase chain reaction. When these cells were transfected with pEGFP-N1 vector DNA, significant fluorescent signals were observed, suggesting that these cell lines can be a useful tool for transgenic and genetic manipulation studies. Further, these cell lines are characterized by immunocytochemistry using confocal laser scanning microscopy (CFLSM).

Engineered Herpes Simplex Viruses for the Treatment of Malignant Peripheral Nerve Sheath Tumors

DTIC Science & Technology

2015-11-01

lines). This is an entry receptor usually limited to lymphoid cells has not been previously identified in neuroectodermal tissue. Year 3: As a... innate and adaptive immune 327 response. However, resistance is common in vitro and therefore, to the extent that tumor cell lines 328 maintain the...Handgretinger R, et al. Innate immune 461 defense defines susceptibility of sarcoma cells to measles vaccine virus-based oncolysis. J Virol. 462 2013;87

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

PubMed

Xie, Yuan; Bergström, Tobias; Jiang, Yiwen; Johansson, Patrik; Marinescu, Voichita Dana; Lindberg, Nanna; Segerman, Anna; Wicher, Grzegorz; Niklasson, Mia; Baskaran, Sathishkumar; Sreedharan, Smitha; Everlien, Isabelle; Kastemar, Marianne; Hermansson, Annika; Elfineh, Lioudmila; Libard, Sylwia; Holland, Eric Charles; Hesselager, Göran; Alafuzoff, Irina; Westermark, Bengt; Nelander, Sven; Forsberg-Nilsson, Karin; Uhrbom, Lene

2015-10-01

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease

DTIC Science & Technology

2013-08-01

Immortalization of these selected clones using Epstein - Barr viral transformation provides a method to maintain these antibody producing cell lines as a...2013 Please see next page. None provided. Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease Kevin Claffey University of

Active microwave negative-index metamaterial transmission line with gain.

PubMed

Jiang, Tao; Chang, Kihun; Si, Li-Ming; Ran, Lixin; Xin, Hao

2011-11-11

We studied the active metamaterial transmission line at microwave frequency. The active composite right-handed or left-handed transmission line was designed to incorporate a germanium tunnel diode with a negative differential resistance property as the gain device at the unit cell level. Measurements of the fabricated planar transmission line structures with one-, two-, and three-unit cells showed that the addition of the dc pumped tunnel diodes not only provided gain but also maintained the left handedness of the transmission line metamaterial. Simulation results agree well with experimental observation. This work demonstrated that negative index material can be obtained with a net gain when an external source is incorporated.

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

PubMed Central

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-01-01

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

PubMed

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-06-06

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

PubMed

Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian

2014-12-01

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

PubMed

Heiden, Katherine B; Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Wang, Yimin; Liu, Dingxie; Xing, Mingzhao; Prinz, Richard A; Xu, Xiulong

2014-11-01

Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.

A Telomerase Immortalized Human Proximal Tubule Cell Line with a Truncation Mutation (Q4004X) in Polycystin-1

PubMed Central

Herbert, Brittney-Shea; Grimes, Brenda R.; Xu, Wei Min; Werner, Michael; Ward, Christopher; Rossetti, Sandro; Harris, Peter; Bello-Reuss, Elsa; Ward, Heather H.; Miller, Caroline; Gattone, Vincent H.; Phillips, Carrie L.; Wandinger-Ness, Angela; Bacallao, Robert L.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions [1], [2]. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X) in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells. PMID:23383103

Establishment and characterization of a telomerase immortalized porcine luteal cells.

PubMed

Zhang, Liang; Huang, Yong; Wang, Zhenyu; Luo, Xiaomao; Zhang, Hongling; Du, Qian; Chang, Lingling; Zhao, Xiaomin; Tong, Dewen

2017-05-01

Luteal cells play a crucial role in pregnancy through secreting progesterone to maintain pregnancy and support of fetus. However, low cellular yields and inability to passage primary porcine luteal cells (PLCs) in vitro limit the luteal cell study. Therefore, developing an immortalized porcine luteal cell line is necessary for studying luteal cells activity and function in different diseases. In this study, primary PLCs were obtained from gilts at day 30 to day 50 of gestation and immortalized by human telomerase reverse transcriptase (hTERT). The porcine corpus luteal cell line (hTERT-PLCs) expressed hTERT gene steady, maintained high hTERT activity and normal karyotype. The phase contrast microscope and transmission electron microscope observation showed primary PLCs and hTERT-PLCs were polygonal and exhibited abundant mitochondria, smooth endoplasmic reticulum and lipid droplets. 3β hydroxysteroid dehydrogenase (3βHSD) and Oil-Red-O staining showed that hTERT-PLCs at passage 30 and 50 were similar to primary PLCs. The hTERT-PLCs expressed steroidogenesis-related proteins, enzymes and receptors, such as steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, 3βHSD, 20αHSD, luteinizing hormone receptor, progesterone receptor, prolactin receptor, estrogen receptorα/β, as well as primary PLCs. Consequently, hTERT-PLCs could secret progesterone and exhibited similar responses to luteinizing hormone and prostaglandin F2α as primary PLCs. In addition, the hTERT-PLCs did not show neoplastic transformation or anchorage independent growth. In summary, we developed an immortalized porcine luteal cell line which maintained its originally morphological, biological and functional characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Modulation of Induced Cytotoxicity of Doxorubicin by Using Apoferritin and Liposomal Cages

PubMed Central

Gumulec, Jaromir; Fojtu, Michaela; Raudenska, Martina; Sztalmachova, Marketa; Skotakova, Anna; Vlachova, Jana; Skalickova, Sylvie; Nejdl, Lukas; Kopel, Pavel; Knopfova, Lucia; Adam, Vojtech; Kizek, Rene; Stiborova, Marie; Babula, Petr; Masarik, Michal

2014-01-01

Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized novel apoferritin- and liposome-encapsulated forms of doxorubicin (“Apodox” and “lip-8-dox”) and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP. PMID:25514405

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

PubMed Central

Agris, P F

1975-01-01

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

USDA-ARS?s Scientific Manuscript database

To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

Development and characterization of a cell line TTCF from endangered mahseer Tor tor (Ham.).

PubMed

Yadav, K; Lakra, W S; Sharma, J; Goswami, M; Singh, Akhilesh

2012-08-01

Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H₂O₂ in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.

Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line.

PubMed

Guillaud-Bataille, M; Brison, O; Danglot, G; Lavialle, C; Raynal, B; Lazar, V; Dessen, P; Bernheim, A

2009-01-01

High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs. Copyright 2009 S. Karger AG, Basel.

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

PubMed

Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

1989-07-01

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.

An intact centrosome is required for the maintenance of polarization during directional cell migration.

PubMed

Wakida, Nicole M; Botvinick, Elliot L; Lin, Justin; Berns, Michael W

2010-12-23

Establishing and maintaining polarization is critical during cell migration. It is known that the centrosome contains numerous proteins whose roles of organizing the microtubule network range include nucleation, stabilization and severing. It is not known whether the centrosome is necessary to maintain polarization. Due to its role as the microtubule organizing center, we hypothesize that the centrosome is necessary to maintain polarization in a migrating cell. Although there have been implications of its role in cell migration, there is no direct study of the centrosome's role in maintaining polarization. In this study we ablate the centrosome by intracellular laser irradiation to understand the role of the centrosome in two vastly different cell types, human osteosarcoma (U2OS) and rat kangaroo kidney epithelial cells (PtK). The PtK cell line has been extensively used as a model for cytoskeletal dynamics during cell migration. The U2OS cell line serves as a model for a complex, single migrating cell. In this study we use femtosecond near-infrared laser irradiation to remove the centrosome in migrating U2OS and PtK2 cells. Immunofluorescence staining for centrosomal markers verified successful irradiation with 94% success. A loss of cell polarization is observed between 30 and 90 minutes following removal of the centrosome. Changes in cell shape are correlated with modifications in microtubule and actin organization. Changes in cell morphology and microtubule organization were quantified revealing significant depolarization resulting from centrosome irradiation. This study demonstrates that the centrosome is necessary for the maintenance of polarization during directed cell migration in two widely different cell types. Removal of the centrosome from a polarized cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. These results demonstrate that the centrosome plays a critical role in the maintenance of cytoskeletal asymmetry during cell migration.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

PubMed

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Stem cell maintenance by manipulating signaling pathways: past, current and future

PubMed Central

Chen, Xi; Ye, Shoudong; Ying, Qi-Long

2015-01-01

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676] PMID:26497581

Development and characterization of a new marine fish cell line from turbot (Scophthalmus maximus).

PubMed

Wang, N; Wang, X L; Sha, Z X; Tian, Y S; Chen, S L

2010-12-01

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n=44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.

Variability of human pluripotent stem cell lines.

PubMed

Ortmann, Daniel; Vallier, Ludovic

2017-10-01

Human pluripotent stem cells derived from embryos (human Embryonic Stem Cells or hESCs) or generated by direct reprogramming of somatic cells (human Induced Pluripotent Stem Cells or hiPSCs) can proliferate almost indefinitely in vitro while maintaining the capacity to differentiate into a broad diversity of cell types. These two properties (self-renewal and pluripotency) confers human pluripotent stem cells a unique interest for clinical applications since they could allow the production of infinite quantities of cells for disease modelling, drug screening and cell based therapy. However, recent studies have clearly established that human pluripotent stem cell lines can display variable capacity to differentiate into specific lineages. Consequently, the development of universal protocols of differentiation which could work efficiently with any human pluripotent cell line is complicated substantially. As a consequence, each protocol needs to be adapted to every cell line thereby limiting large scale applications and precluding personalised therapies. Here, we summarise our knowledge concerning the origin of this variability and describe potential solutions currently available to bypass this major challenge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

PubMed

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

PubMed

Catalina, Puri; Montes, Rosa; Ligero, Gertru; Sanchez, Laura; de la Cueva, Teresa; Bueno, Clara; Leone, Paola E; Menendez, Pablo

2008-10-03

The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

PubMed

Kumar, Amit; Singh, Neha; Goswami, Mukunda; Srivastava, J K; Mishra, Akhilesh K; Lakra, W S

2016-01-01

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

PubMed Central

Atwell, Kathryn; Qin, Zhao; Gavaghan, David; Kugler, Hillel; Hubbard, E. Jane Albert; Osborne, James M.

2015-01-01

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems. PMID:26428008

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

PubMed

Gómez, M C; Serrano, M A; Pope, C Earle; Jenkins, J A; Biancardi, M N; López, M; Dumas, C; Galiguis, J; Dresser, B L

2010-09-01

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines. Copyright 2010 Elsevier Inc. All rights reserved.

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.

FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

NASA Astrophysics Data System (ADS)

Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

2012-06-01

The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

Effects of temperature on Veronaea botryosa infections in white sturgeon Acipenser transmontanus and fungal induced cytotoxicity of fish cell lines.

PubMed

Coleman, Denver J; Camus, Alvin C; Martínez-López, Beatriz; Yun, Susan; Stevens, Brittany; Soto, Esteban

2018-02-01

Veronaea botryosa is a melanized mold and cause of systemic fungal infections in cultured sturgeon (Acipenser spp.). Mortality in adult female sturgeon caused by this emergent pathogen results in significant economic losses for the caviar industry. Little is known regarding environmental conditions conducive to V. botryosa infection. This study evaluated the effect of temperature on V. botryosa infectivity and dissemination following intramuscular injection challenge of white sturgeon maintained at 13 or 18 °C for 40 days. Daily mortality was recorded and persistence of the fungus in the livers of moribund and surviving fish was investigated using culture and histopathological analysis. Fish maintained at 18 °C developed systemic phaeohyphomycosis and had significantly greater mortality than controls and fish maintained at 13 °C (p < 0.05). Challenged fish, regardless of temperature, exhibited lesions in multiple organs. However, muscle lesions, angioinvasion, and systemic dissemination were more severe and widespread in fish challenged at the higher temperature. In vitro cytotoxicity of V. botryosa was evaluated in white sturgeon skin (WSSK-1) and epithelioma papulosum cyprini (EPC) cell lines inoculated at spore:cell ratios of 1:10, 1:1 and 10:1, then incubated 15, 20 and 25 °C. Cytotoxicity, as indicated by quantifying the release of lactate dehydrogenase into culture supernatants, increased with increasing spore dose and incubation temperature in both fish cell lines. Findings suggest that temperature significantly influences the development of systemic V. botryosa infection in white sturgeon and that WSSK-1 and EPC cells are suitable in vitro models for the study of host-pathogen interactions between V. botryosa and fish epithelial cells.

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Establishment and characterization of SUIT-58 pancreas cancer cell line and its subline S58-SF adapted to serum-free condition derived from metastatic liver tumor.

PubMed

Takahashi, Nobuyasu; Aoyama, Fumiyo; Ohuchida, Jiro; Sameshima, Naoki; Asada, Yujiro; Sawaguchi, Akira

2015-10-01

A new pancreas cancer cell line, SUIT-58, was established from metastatic liver tumor. The cultured cells exhibited polygonal shape, and proliferated in a form of sheet-structure showing prominent nucleoli and frequent mitotic features. Chromosome count ranged from 54 to 73 with modal chromosome numbers 72 and 73. It was noteworthy that this cell line grew in the serum-free media and maintained in this condition for 30 passages (designated as S58-SF). Both SUIT-58 and S58-SF cell lines were successfully transplanted into nude mice, and their tumor doubling times in xenografts were calculated as 5.4 and 2.8 days, respectively. Histopathologically, the xenografts formed glandular structure that resembled the original tumor. In culture media, the doubling time of SUIT-58 and S58-SF cell lines was calculated as 32 and 35.7 h, respectively. Although the cellular arrangements of SUIT-58 and S58-SF cell lines are different to some extent, their subcellular structures under electron microscope were similar with a large number of lysosomes and distinct desmosomes at cell-cell adhesion sites. The present SUIT-58 and its derivative cell line S58-SF will be applicable for biological studies to develop a new clinical treatment of refractory pancreatic cancer.

A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

PubMed Central

Hammiller, Brianna O.; El-Abaseri, Taghrid Bahig; Dlugosz, Andrzej A.; Hansen, Laura A.

2015-01-01

Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation. PMID:26284198

Establishment and culture optimization of a new type of pituitary immortalized cell line

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

Kokubu, Yuko; Asashima, Makoto; Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells undermore » sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.« less

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM).

PubMed

Ishiwata, Isamu; Ishiwata, Chieko; Ishiwata, Emiko; Sato, Yoshiro; Kiguchi, Kazushige; Tachibana, Toshiaki; Ishikawa, Hiroshi

2004-12-01

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model.

PubMed

Risal, Prabodh; Cho, Baik Hwan; Sylvester, Karl G; Kim, Jae-Chun; Kim, Hyoung Tae; Jeong, Yeon Jun

2011-09-01

Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.

Biphasic adaptation to osmotic stress in the C. elegans germ line.

PubMed

Davis, Michael; Montalbano, Andrea; Wood, Megan P; Schisa, Jennifer A

2017-06-01

Cells respond to environmental stress in multiple ways. In the germ line, heat shock and nutritive stress trigger the assembly of large ribonucleoprotein (RNP) granules via liquid-liquid phase separation (LLPS). The RNP granules are hypothesized to maintain the quality of oocytes during stress. The goal of this study was to investigate the cellular response to glucose in the germ line and determine if it is an osmotic stress response. We found that exposure to 500 mM glucose induces the assembly of RNP granules in the germ line within 1 h. Interestingly, the RNP granules are maintained for up to 3 h; however, they dissociate after longer periods of stress. The RNP granules include processing body and stress granule proteins, suggesting shared functions. Based on several lines of evidence, the germ line response to glucose largely appears to be an osmotic stress response, thus identifying osmotic stress as a trigger of LLPS. Although RNP granules are not maintained beyond 3 h of osmotic stress, the quality of oocytes does not appear to decrease after longer periods of stress, suggesting a secondary adaptation in the germ line. We used an indirect marker of glycerol and observed high levels after 5 and 20 h of glucose exposure. Moreover, in gpdh-1;gpdh-2 germ lines, glycerol levels are reduced concomitant with RNP granules being maintained for an extended period. We speculate that increased glycerol levels may function as a secondary osmoregulatory adaptive response in the germ line, following a primary response of RNP granule assembly. Copyright © 2017 the American Physiological Society.

An in vitro monocyte culture method and establishment of a human monocytic cell line (K63).

PubMed

Kadoi, Katsuyuki

2011-01-01

A novel method of monocyte culture in vitro was developed. The fraction of monocytes was obtained by density centrifugation of heparinised human venous blood samples. Monocytes were suspended in a modified Rosewell Park Memorial Institute medium (RPMI)-1640 (mRPMI) supplemented with 10% non-inactivated autologous serum added to the feeder cells. An avian cell line was used for feeder cells. Only those monocytes that settled on feeder cells grew rapidly at 37°C-38°C into a formation of clumped masses within two to three days. The cell mass was harvested and subcultures were made without feeder cells. A stable cell line (K63) was established from subcultures using a limited dilution method and cell cloning in microplates. K63 cells were adapted for later growth in the mRPMI medium supplemented with 10% foetal calf serum. The cells were well maintained at over 50th passage levels. This method proved to be applicable for monocyte cultures of animals as well.

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

PubMed

Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of a Brazilian line of human embryonic stem cells in defined medium: implications for cell therapy in an ethnically diverse population.

PubMed

Fraga, Ana M; Sukoyan, Marina; Rajan, Prithi; Braga, Daniela Paes de Almeida Ferreira; Iaconelli, Assumpto; Franco, José Gonçalves; Borges, Edson; Pereira, Lygia V

2011-01-01

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-1, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-1 maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-1 and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Characteristics and EGFP expression of goat mammary gland epithelial cells.

PubMed

Zheng, Y-M; He, X-Y; Zhang, Y

2010-12-01

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. © 2010 Blackwell Verlag GmbH.

A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

NASA Technical Reports Server (NTRS)

Chuman, L. M.; FINE; COHEN; Saier, M. H.

1985-01-01

The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

Cloning Components of Human Telomerase.

DTIC Science & Technology

1998-07-01

absent, and the cells are unable to double further. Somatic cells have a limited replicative capacity ( Hayflick 1961), and the lack of telomerase... Hayflick limit (Bodnar et al. 1998). Immortal cells must have a method of maintaining telomeres, and indeed it has been found that immortalized cell lines...THIS PAGE Unclassified 19. SECURITY CLASSIFICATION OF ABSTRACT Unclassified 15. NUMBER OF PAGES 12 16. PRICE CODE 20. LIMITATION OF ABSTRACT

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

PubMed

Zhou, Ruoji; Xu, An; Wang, Donghui; Zhu, Dandan; Mata, Helen; Huo, Zijun; Tu, Jian; Liu, Mo; Mohamed, Alaa M T; Jewell, Brittany E; Gingold, Julian; Xia, Weiya; Rao, Pulivarthi H; Hung, Mien-Chie; Zhao, Ruiying; Lee, Dung-Fang

2018-03-01

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Establishment and proteomic characterization of a novel cell line, NCC-UPS2-C1, derived from a patient with undifferentiated pleomorphic sarcoma.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-03-01

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal malignancy requiring novel therapeutic approaches to improve clinical outcome. Patient-derived cancer cell lines are an essential tool for investigating molecular mechanisms underlying cancer initiation and development; however, there is a lack of patient-derived cell lines of UPS available for research. The objective of this study was to develop a patient-derived cell model of UPS. A cell line designated NCC-UPS2-C1 was established from the primary tumor tissue of an 84-yr-old female patient with UPS. The short tandem repeat pattern of NCC-UPS2-C1 cells was identical to that of the original tumor and distinct from that of any other cell lines deposited in public cell banks. NCC-UPS2-C1 cells were maintained as a monolayer culture for over 80 passages during 30 mo and exhibited spindle-like morphology, continuous growth, and ability for spheroid formation and invasion. Proteomic profiling using mass spectrometry and functional treemap analysis revealed that the original tumor and the derived NCC-UPS2-C1 cells had similar but distinct protein expression patterns. Our results indicate that a novel UPS cell line was successfully established and could be used to study UPS development and effects of anti-cancer drugs. However, the revealed difference between proteomes of the original tumor and NCC-UPS2-C1 cells should be further investigated to determine the appropriate applications of this cell line in UPS research.

Establishment and culture optimization of a new type of pituitary immortalized cell line.

PubMed

Kokubu, Yuko; Asashima, Makoto; Kurisaki, Akira

2015-08-07

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

Conditioned media from a renal cell carcinoma cell line demonstrates the presence of basic fibroblast growth factor.

PubMed

Mydlo, J H; Zajac, J; Macchia, R J

1993-09-01

In a previous report, we demonstrated the isolation and purification of a heparin binding growth factor from human renal carcinoma, and suggested that this growth factor may play a role in the neovascularity and growth of the tumor. In this report, we demonstrate that the growth of the renal cell carcinoma cell line RC29 is stimulated by the addition of exogenous fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha). Also, media conditioned by this cell line was able to stimulate growth of the A431 vulvar tumor cell line, known for its high concentration of EGF receptors, 3T3 fibroblasts, human umbilical vein (HUV) cells and RC29 cells. Using heparin-sepharose chromatography and then SDS polyacrylamide gel electrophoresis (PAGE), we were able to demonstrate several proteins in the conditioned media of the RC29 cell line. Using Western blot analysis, we detected that at least one of the proteins expressed in this conditioned media was FGF and that it belongs to the basic, not acidic, family of fibroblast growth factors. These findings suggest that renal tumors may express growth factors that may play a direct role in maintaining their unrestricted proliferation.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

PubMed

Fu, Mei; Chen, Yabing; Xiong, Xianrong; Lan, Daoliang; Li, Jian

2014-01-01

This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

NASA Astrophysics Data System (ADS)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the Zr-75-1 human breast cancer cell line in defined medium.

PubMed

Allegra, J C; Korat, O; Do, H M; Lippman, M

1981-01-01

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the ZR-75-1 human breast cancer cell line in defined medium is described. ZR-75-1 cells maintained in serum free hormone supplemented medium minus estradiol lack progesterone receptor activity. Readdition of estradiol to these cells leads to a marked stimulation of progesterone receptor activity (0 to greater than 100 fmols of specifically bound progesterone per million cells). Tamoxifen (10(-6)M-10(-8)M) does not stimulate progesterone receptor activity in this cell line. The presence of progesterone receptor activity is not directly related to growth. Withdrawal of insulin in the continued presence of estradiol has no effect on progesterone receptor concentration although net cell growth ceases. Conversely, withdrawal of estradiol in the continued presence of insulin induces a cessation of net cell growth accompanied by a loss of all progesterone receptor activity within 3-5 days.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells

PubMed Central

Stelcer, Ewelina; Kulcenty, Katarzyna; Rucinski, Marcin; Jopek, Karol; Trzeciak, Tomasz; Richter, Magdalena; Wroblewska, Joanna P.; Suchorska, Wiktoria M.

2018-01-01

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies. PMID:29439516

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

PubMed

Masuda, Junko; Kawamoto, Hiroshi; Strober, Warren; Takayama, Eiji; Mizutani, Akifumi; Murakami, Hiroshi; Ikawa, Tomokatsu; Kitani, Atsushi; Maeno, Narumi; Shigehiro, Tsukasa; Satoh, Ayano; Seno, Akimasa; Arun, Vaidyanath; Kasai, Tomonari; Fuss, Ivan J; Katsura, Yoshimoto; Seno, Masaharu

2016-12-01

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

Generation and characterization of a porcine endometrial endothelial cell line susceptible to porcine reproductive and respiratory syndrome virus.

PubMed

Feng, Lili; Zhang, Xinyu; Xia, Xiaoli; Li, Yangyang; He, Shan; Sun, Huaichang

2013-01-01

Previous studies on the underlying mechanism for porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure have been focused on the viral replication in the endothelial macrophages, and the susceptibility of porcine endometrial endothelial (PEE) cells to PRRSV has not yet been investigated. Therefore, in the present study we generated a PEE cell line by transfection of the primary cells with a SV40 large T antigen expression vector. The PEE cell line maintained the endothelial morphology with a significantly faster growth rate, shorter population doubling time and higher plating efficiency than the primary cells. The endothelial origination of the cell line was confirmed by detection of the endothelial cell-specific markers. The PEE cell line had been passed successively for 60 generations with an unlimited growth potential. To further characterize the PEE cell line, cells of different passages were infected with different PRRSV strains and analyzed for the viral antigen and replication. Overt cytopathic effect was observed from 36h postinfection (HPI) and the viral antigen detected as early as 12 HPI. The infectious virus was recovered from the infected PEE cells with a titer higher than that in MARC-145 cells. Since the data presented indicate a high susceptibility of PEE cells to PRRSV, we conclude that the PEE cell line generated will be useful for growth of PRRSV and further studies on the underlying mechanism for PRRSV infection of PEE cells. The finding of the susceptibility of PEE cells to PRRSV may provide an alternative explanation for PRRSV-induced reproductive failure. Copyright © 2012 Elsevier B.V. All rights reserved.

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

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

Montani, Claudia; Steimberg, Nathalie; Boniotti, Jennifer

2014-11-01

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related tomore » cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential. - Highlights: • We here characterized the first cell line derived from an estrogen reporter mouse. • In the RCCS cells express an immortalized behavior but not a transformed phenotype. • The RCCS provides a system for maintaining cells in more physiological conditions. • RCCS-cultured fibroblasts showed higher hormonal sensitivity to estradiol. • This bioreactor is a novel 3D model to be applied to pharmacotoxicological studies.« less

KCH kinesin drives nuclear transport and cytoskeletal coalescence for tip cell growth.

PubMed

Yamada, Moé; Goshima, Gohta

2018-06-07

Long-distance transport along microtubules (MTs) is critical for intracellular organisation. In animals, antagonistic motor proteins kinesin (plus end-directed) and dynein (minus end-directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain)-which can also bind actin-drives MT minus end-directed nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell centre, but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KCH KO lines, whereas actin destabilisation also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT crosslinker, reminiscent of the versatile animal dynein. © 2018 American Society of Plant Biologists. All rights reserved.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

NASA Astrophysics Data System (ADS)

Moussa, Hassan I.; Logan, Megan; Siow, Geoffrey C.; Phann, Darron L.; Rao, Zheng; Aucoin, Marc G.; Tsui, Ting Y.

2017-12-01

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only 22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips.

PubMed

Moussa, Hassan I; Logan, Megan; Siow, Geoffrey C; Phann, Darron L; Rao, Zheng; Aucoin, Marc G; Tsui, Ting Y

2017-01-01

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

PubMed Central

Moussa, Hassan I.; Logan, Megan; Siow, Geoffrey C.; Phann, Darron L.; Rao, Zheng; Aucoin, Marc G.; Tsui, Ting Y.

2017-01-01

Abstract Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability. PMID:29152017

Phenotypic and Molecular Analysis of Mes-3, a Maternal-Effect Gene Required for Proliferation and Viability of the Germ Line in C. Elegans

PubMed Central

Paulsen, J. E.; Capowski, E. E.; Strome, S.

1995-01-01

mes-3 is one of four maternal-effect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells and no gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearance of the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmed cell death. The few germ cells that appear healthy in mes-3 worms do not differentiate into gametes, even after elimination of the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterization of mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins. PMID:8601481

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

PubMed

Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

2017-10-01

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

PubMed Central

Zhao, Xiangshan; Malhotra, Gautam K.; Band, Hamid; Band, Vimla

2011-01-01

Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers. PMID:22279424

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes.

PubMed

Zhao, Xiangshan; Malhotra, Gautam K; Band, Hamid; Band, Vimla

2011-01-01

Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells. In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells. The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

USGS Publications Warehouse

Batts, William N.; Polinski, Mark P.; Drennan, John D.; Ireland, Susan C.; Cain, Kenneth D.

2010-01-01

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle’s minimum essential medium with Earle’s salts (MEM) supplemented with GlutaMAX™, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg ml–1, respectively) did not negatively influence cell replication; however, the antimycotic Fungizone™ (2.5 µg ml–1, amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23°C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

PubMed

Polinski, Mark P; Drennan, John D; Batts, William N; Ireland, Susan C; Cain, Kenneth D

2010-05-18

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle's minimum essential medium with Earle's salts (MEM) supplemented with GlutaMAX, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg m(-1), respectively) did not negatively influence cell replication; however, the antimycotic FungizoneTM (2.5 microg m(-1), amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23 degrees C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

NASA Astrophysics Data System (ADS)

Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

2016-09-01

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

PubMed

Talbot, N C; Caperna, T J; Garrett, W M

2013-01-01

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor.

PubMed

Ingram, M; Techy, G B; Saroufeem, R; Yazan, O; Narayan, K S; Goodwin, T J; Spaulding, G F

1997-06-01

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

PubMed

Dumble, Melissa L; Croager, Emma J; Yeoh, George C T; Quail, Elizabeth A

2002-03-01

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

PubMed Central

2014-01-01

Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents. PMID:24886633

Development and characterization of two cell lines from gills of Atlantic salmon

USGS Publications Warehouse

Gjessing, Mona C.; Aamelfot, Maria; Batts, William N.; Benestad, Sylvie L.; Dale, Ole B.; Thoen, Even; Weli, Simon C.; Winton, James R.

2018-01-01

Gill disease in Atlantic salmon, Salmo salar L., causes big losses in the salmon farming industry. Until now, tools to cultivate microorganisms causing gill disease and models to study the gill responses have been lacking. Here we describe the establishment and characterization of two cell lines from the gills of Atlantic salmon. Atlantic salmon gill cell ASG-10 consisted of cells staining for cytokeratin and e-cadherin and with desmosomes as seen by transmission electron microscopy suggesting the cells to be of epithelial origin. These structures were not seen in ASG-13. The cell lines have been maintained for almost 30 passages and both cell lines are fully susceptible to infection by infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), Atlantic salmon reovirus TS (TSRV) and Pacific salmon paramyxovirus (PSPV). While infectious salmon anemia virus (ISAV) did not cause visible CPE, immunofluorescent staining revealed a sub-fraction of cells in both the ASG-10 and ASG-13 lines may be permissive to infection. ASG-10 is able to proliferate and migrate to close scratches in the monolayer within seven days in vitro contrary to ASG-13, which does not appear to do have the same proliferative and migratory ability. These cell lines will be useful in studies of gill diseases in Atlantic salmon and may represent an important contribution for alternatives to experimental animals and studies of epithelial–mesenchymal cell biology.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

PubMed

Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines.

PubMed

Peel, D J; Johnson, S A; Milner, M J

1990-01-01

We have examined the ultrastructure of cellular vesicles in primary cultures of wing imaginal disc cells of Drosophila melanogaster. These cells maintain the apico-basal polarity characteristic of epithelial cells. The apical surfaces secrete extracellular material into the lumen of the vesicle from plasma membrane plaques at the tip of microvilli. During the course of one passage, cells from the established cell lines grow to confluence and then aggregate into discrete condensations joined by aligned bridges of cells. Cells in these aggregates are tightly packed, and there appears to be a loss of the epithelial polarity characteristic of the vesicle cells. Elongated cell extensions containing numerous microtubules are found in aggregates, and we suggest that these may be epithelial feet involved in the aggregation process. Virus particles are commonly found both within the nucleus and the cytoplasm of cells in the aggregates.

In vivo physiological recording from the lateral line of juvenile zebrafish

PubMed Central

Olt, Jennifer; Allen, Claire E.

2016-01-01

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. PMID:27161862

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

PubMed

Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime

2016-08-01

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

Protective effects of nicergoline against hydrogen peroxide toxicity in rat neuronal cell line.

PubMed

Iwata, E; Miyazaki, I; Asanuma, M; Iida, A; Ogawa, N

1998-07-17

We examined the effects of nicergoline on hydrogen peroxide (H2O2)-induced neurotoxicity in cultured rat neuronal cell line (B50). H2O2 induced death of B50 cells in a dose-dependent manner. The H2O2-induced neuronal cell death was significantly decreased in B50 cells maintained in the presence of nicergoline. We compared the levels of antioxidants (glutathione, catalase and superoxide dismutase) in nicergoline-treated and untreated B50 cells. Lipid peroxidation products (thiobarbituric acid reactive substances, TBARS) levels were also measured. Cultures treated with nicergoline had higher levels of catalase activity. TBARS level was significantly lower in nicergoline-treated cells than in untreated cells. Our results suggest that nicergoline may induce the up-regulation of intracellular antioxidant defences and protect the neuronal cells against oxidative stress.

76 FR 6405 - Marine Mammals; File No. 15654

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-04

... Church, PhD, Professor of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115...-02 issued to John Wise, PhD) and maintained at Harvard University for the proposed study. Cell lines...

Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.

PubMed

Huang, Edwin P; Marquis, Christopher P; Gray, Peter P

2004-11-20

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (c) 2004 Wiley Periodicals, Inc

Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

PubMed

Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

2012-12-01

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. © 2011 Blackwell Publishing Ltd.

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Establishment and characterization of fin-derived cell line from ornamental carp, Cyprinus carpio koi, for virus isolation in India.

PubMed

Swaminathan, T Raja; Basheer, V S; Kumar, Raj; Kathirvelpandian, A; Sood, Neeraj; Jena, J K

2015-08-01

Cyprinus carpio koi fin (CCKF) cell line was established and characterized from the caudal fin tissue of ornamental common carp, C. carpio koi. This cell line has been maintained in L-15 medium supplemented with 15% foetal bovine serum (FBS) and subcultured more than 52 times over a period of 24 mo. The CCKF cell line consisted of epithelial cells and was able to grow at temperatures between 22 and 35°C with an optimum temperature of 28°C. The growth rate of these cells increased as the proportion of FBS increased from 2 to 20% with optimum growth at the concentrations of 15% FBS. Karyotype analysis revealed that the modal chromosome number of CCKF cells was 2n = 100. Partial amplification and sequencing of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CCKF cell line originated from ornamental common carp. The CCKF cells showed strong reaction to the cytokeratin marker, indicating that it was epithelial in nature. The extracellular products of Vibrio cholerae MTCC 3904 and Aeromonas hydrophila were toxic to the CCKF cells and not susceptible to viral nervous necrosis virus (VNNV). These CCKF cells were confirmed for the absence of Mycoplasma sp. by polymerase chain reaction. Furthermore, 90% of viable cells could be effectively revived 4 mo after cryopreservation from CCKF cell population suggesting the possibility of long-term storage of the cells.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

PubMed Central

Degrassi, A; Hilbert, D M; Rudikoff, S; Anderson, A O; Potter, M; Coon, H G

1993-01-01

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion. Images Fig. 1 Fig. 2 PMID:8446628

Establishment and characterization of a human pancreatic cancer cell line (SUIT-2) producing carcinoembryonic antigen and carbohydrate antigen 19-9.

PubMed

Iwamura, T; Katsuki, T; Ide, K

1987-01-01

A new tumor cell line (SUIT-2) derived from a metastatic liver tumor of human pancreatic carcinoma has been established in tissue culture and in nude mice, and maintained for over five years. In tissue culture, the cells grew in a monolayered sheet with a population doubling time of about 38.2 hr, and floated or piled up to form small buds above the monolayered surface in relatively confluent cultures. Chromosome counts ranged from 34 to 176 with a modal number of 45. Subcutaneous injection of cultured cells into nude mice resulted in tumor formation, histopathologically closely resembling the original neoplasm which had been classified as moderately differentiated tubular adenocarcinoma. Electron microscopic observation of the neoplastic cells revealed a characteristic pancreatic ductal epithelium. SUIT-2 cell line produces and releases at least two tumor markers, carcinoembryonic antigen and carbohydrate antigen 19-9, propagates even in serum-free medium, and metastasizes to the regional lymph nodes in nude mice xenografts.

Establishment and characterization of a novel osteosarcoma cell line: CHOS.

PubMed

Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

2016-12-01

Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

PubMed

Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

2015-06-01

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts.

PubMed

Rungsiwiwut, Ruttachuk; Pavarajarn, Wipawee; Numchaisrika, Pranee; Virutamasen, Pramuan; Pruksananonda, Kamthorn

2016-01-01

Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Optimization of Time-Dependent Particle Tracing Using Tetrahedral Decomposition

NASA Technical Reports Server (NTRS)

Kenwright, David; Lane, David

1995-01-01

An efficient algorithm is presented for computing particle paths, streak lines and time lines in time-dependent flows with moving curvilinear grids. The integration, velocity interpolation and step-size control are all performed in physical space which avoids the need to transform the velocity field into computational space. This leads to higher accuracy because there are no Jacobian matrix approximations or expensive matrix inversions. Integration accuracy is maintained using an adaptive step-size control scheme which is regulated by the path line curvature. The problem of cell-searching, point location and interpolation in physical space is simplified by decomposing hexahedral cells into tetrahedral cells. This enables the point location to be done analytically and substantially faster than with a Newton-Raphson iterative method. Results presented show this algorithm is up to six times faster than particle tracers which operate on hexahedral cells yet produces almost identical particle trajectories.

Establishment of immortal swine kidney epithelial cells.

PubMed

Kwak, Sungwook; Jung, Ji-Eun; Jin, Xun; Kim, Sun-Myung; Kim, Tae-Kyung; Lee, Joong-Seob; Lee, Soo-Yeon; Pian, Xumin; You, Seungkwon; Kim, Hyunggee; Choi, Yun-Jaie

2006-01-01

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.

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

3-V Solid-State Flexible Supercapacitors with Ionic-Liquid-Based Polymer Gel Electrolyte for AC Line Filtering.

PubMed

Kang, Yu Jin; Yoo, Yongju; Kim, Woong

2016-06-08

State-of-the-art solid-state flexible supercapacitors with sufficiently fast response speed for AC line filtering application suffer from limited energy density. One of the main causes of the low energy density is the low cell voltage (1 V), which is limited by aqueous-solution-based gel electrolytes. In this work, we demonstrate for the first time a 3-V flexible supercapacitor for AC line filtering based on an ionic-liquid-based polymer gel electrolyte and carbon nanotube electrode material. The flexible supercapacitor exhibits an areal energy density that is more than 20 times higher than that of the previously demonstrated 1-V flexible supercapacitor (0.66 vs 0.03 μWh/cm(2)) while maintaining excellent capacitive behavior at 120 Hz. The supercapacitor shows a maximum areal power density of 1.5 W/cm(2) and a time constant of 1 ms. The improvement of the cell voltage while maintaining the fast-response capability greatly improves the potential of supercapacitors for high-frequency applications in wearable and/or portable electronics.

High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1.

PubMed

Bakos, Agnes; Banati, Ferenc; Koroknai, Anita; Takacs, Maria; Salamon, Daniel; Minarovits-Kormuta, Susanna; Schwarzmann, Fritz; Wolf, Hans; Niller, Hans Helmut; Minarovits, Janos

2007-10-01

Transcripts for the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNAs) are initiated at alternative promoters (Wp, Cp, for EBNA 1-6 transcripts and Qp, for EBNA 1 transcripts only) located in the BamHI W, C or Q fragment of the viral genome. To understand the host-cell dependent expression of EBNAs in EBV-associated tumors (lymphomas and carcinomas) and in vitro transformed cell lines, it is necessary to analyse the regulatory mechanisms governing the activity of the alternative promoters of EBNA transcripts. Such studies focused mainly on lymphoid cell lines carrying latent EBV genomes, due to the lack of EBV-associated carcinoma cell lines maintaining latent EBV genomes during cultivation in tissue culture. We took advantage of the unique nasopharyngeal carcinoma cell line, C666-1, harboring EBV genomes, and undertook a detailed analysis of CpG methylation patterns and in vivo protein-DNA interactions at the latency promoters Qp and Cp. We found that the active, unmethylated Qp was marked with strong footprints of cellular transcription factors and the viral protein EBNA 1. In contrast, we could not detect binding of relevant transcription factors to the methylated, silent Cp. We concluded that the epigenetic marks at Qp and Cp in C666-1 cells of epithelial origin resemble those of group I Burkitt's lymphoma cell lines.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

PubMed

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

Fucoidan cytotoxicity against human breast cancer T47D cell line increases with higher level of sulfate ester group

NASA Astrophysics Data System (ADS)

Saepudin, Endang; Alfita Qosthalani, Fildzah; Sinurat, Ellya

2018-01-01

The anticancer activity of different sulfate ester group content in different molecular weight was examined. The anticancer activity was achieved in vitro on human breast cancer T47D cell line. Fucoidan with lower molecular weight (5.79 kDa) tends to have lower sulfate ester group content (8.69%) and resulted in higher IC50 value (184.22 μg/mL). While fucoidan with higher molecular weight (785.12 kDa) tends to have higher sulfate level (18.63%) and achieved lower IC50 value (75.69 μg/mL). The result showed that in order to maintain fucoidan cytotoxic activity against human breast cancer T47D cell line, the sulfate content should be remain high. Keywords: fucoidan, sulfate ester group, human breast cancer

Increased expression of c-Ski as a co-repressor in transforming growth factor-beta signaling correlates with progression of esophageal squamous cell carcinoma.

PubMed

Fukuchi, Minoru; Nakajima, Masanobu; Fukai, Yasuyuki; Miyazaki, Tatsuya; Masuda, Norihiro; Sohda, Makoto; Manda, Ryokuhei; Tsukada, Katsuhiko; Kato, Hiroyuki; Kuwano, Hiroyuki

2004-03-01

Transforming growth factor-beta (TGF-beta) regulates cell growth inhibition, and inactivation of the TGF-beta signaling pathway contributes to tumor development. In our previous study, altered expression of TGF-beta, TGF-beta-specific receptors and Smad4 was shown to correlate with tumor progression in esophageal squamous cell carcinoma (SCC). These components, however, were maintained normally in some patients with esophageal SCC. In our study, the mechanism by which aggressive esophageal SCC maintains these components was investigated, with particular emphasis on the participation of c-Ski and SnoN as transcriptional co-repressors in TGF-beta signaling. Immunohistochemistry for c-Ski and SnoN was carried out on surgical specimens obtained from 80 patients with esophageal SCC. The expression of c-Ski and SnoN was also studied in 6 established cell lines derived from esophageal SCC and compared to an immortalized human esophageal cell line by Western blotting. High levels of expression of c-Ski, detected immunohistologically, were found to correlate with depth of invasion (p = 0.0080) and pathologic stage (p = 0.0447). There was, however, no significant correlation between expression of SnoN and clinicopathologic characteristics. A significant correlation between c-Ski and TGF-beta expression was observed. Moreover, in patients with TGF-beta negative expression, the survival rates of patients with c-Ski positive expression were significantly lower than those of patients with c-Ski negative expression (p = 0.0486). c-Ski was expressed at a high level in 5 of 6 cell lines derived from esophageal SCC compared to immortalized esophageal keratinocytes. Furthermore, the cyclin-dependent kinase (CDK) inhibitor, p21 that was up-regulated by TGF-beta signaling was expressed at a low level in the 5 cell lines. The expression of c-Ski protein as a transcriptional co-repressor in TGF-beta signaling seems to be correlated with tumor progression of esophageal SCC. Copyright 2003 Wiley-Liss, Inc.

CHOgenome.org 2.0: Genome resources and website updates.

PubMed

Kremkow, Benjamin G; Baik, Jong Youn; MacDonald, Madolyn L; Lee, Kelvin H

2015-07-01

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effects and mechanisms of SLC34A2 on maintaining stem cell-like phenotypes in CD147+ breast cancer stem cells.

PubMed

Lv, Yonggang; Wang, Ting; Fan, Jing; Zhang, Zhenzhen; Zhang, Juliang; Xu, Cheng; Li, Yongping; Zhao, Ge; He, Chenyang; Meng, Huimin; Yang, Hua; Wang, Zhen; Liu, Jiayun; Chen, Jianghao; Wang, Ling

2017-04-01

The cancer stem cell (CSC) hypothesis has gained significant recognition in describing tumorigenesis. Identification of the factors critical to development of breast cancer stem cells (BCSCs) may provide insight into the improvement of effective therapies against breast cancer. In this study, we aim to investigate the biological function of SLC34A2 in affecting the stem cell-like phenotypes in BCSCs and its underlying mechanisms. We demonstrated that CD147 + cells from breast cancer tissue samples and cell lines possessed BCSC-like features, including the ability of self-renewal in vitro, differentiation, and tumorigenic potential in vivo. Flow cytometry analysis showed the presence of a variable fraction of CD147 + cells in 9 of 10 tumor samples. Significantly, SLC34A2 expression in CD147 + BCSCs was enhanced compared with that in differentiated adherent progeny of CD147 + BCSCs and adherently cultured cell line cells. In breast cancer patient cohorts, SLC34A2 expression was found increased in 9 of 10 tumor samples. By using lentiviral-based approach, si-SLC34A2-transduced CD147 + BCSCs showed decreased ability of sphere formation, cell viability in vitro, and tumorigenicity in vivo, which suggested the essential role of SLC34A2 in CD147 + BCSCs. Furthermore, PI3K/AKT pathway and SOX2 were found necessary to maintain the stemness of CD147 + BCSCs by using LY294002 or lentiviral-si-SOX2. Finally, we indicated that SLC34A2 could regulate SOX2 to maintain the stem cell-like features in CD147 + BCSCs through PI3K/AKT pathway. Therefore, our report identifies a novel role of SLC34A2 in BCSCs' state regulation and establishes a rationale for targeting the SLC34A2/PI3K/AKT/SOX2 signaling pathway for breast cancer therapy.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

PubMed

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

PubMed Central

Que, Jianwen; Lian, Qizhou; El Oakley, Reida M; Lim, Bing; Lim, Sai-Kiang

2007-01-01

Background We have previously derived highly similar lineage-restricted stem cell lines, RoSH and E-RoSH cell lines from mouse embryos and CD9hi SSEA-1- differentiated mouse embryonic stem cells, respectively. These cell lines are not pluripotent and differentiate readily into endothelial cells in vitro and in vivo. Results We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines. Conclusion This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells. PMID:17892597

An Inducible, Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

PubMed Central

Bailis, Julie M.; Gordon, Marcia L.; Gurgel, Jesse L.; Komor, Alexis C.; Barton, Jacqueline K.; Kirsch, Ilan R.

2013-01-01

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells. PMID:24205301

Cell culture of the mucinous variant of human colorectal carcinoma.

PubMed

Tibbetts, L M; Chu, M Y; Vezeridis, M P; Miller, P G; Tibbetts, L L; Poisson, M H; Camara, P D; Calabresi, P

1988-07-01

Two cell lines, RW-2982 and RW-7213, have been established for the first time from the mucinous variant of human colorectal carcinoma, which is a distinctive and important subtype that has a worse prognosis than the more common nonmucogenic large bowel carcinoma. Methods of establishment and observations made during 7 and 3 years, respectively, of continuous culture are described. These cell lines required 4-9 months of adaptation to tissue culture conditions before noticeable growth occurred. Both cell lines have the following unique properties: (a) growth in vitro as delicate branching three-dimensional tumor particles within a wide gel of insoluble, often translucent mucus (proteoglycan); (b) production of large quantities of carcinoembryonic antigen; (c) ability to survive or adapt to growth in media free of serum, hormones, growth factors, and all protein; and (d) tumorigenicity in multiple sites in nude mice, including liver, with especially rapid growth in the peritoneal cavity as gelatinous material that is nonadherent and noninvasive and thus resembles pseudomyxoma peritonei. Unlike other reported colorectal cell lines, these mucus-coated particulate cell lines will not readily grow as monolayers and grow much more slowly with a doubling time of 2 weeks or more. A serially transplantable tumor from the RW-7213 surgical specimen has also been maintained in nude mice since August 8, 1984. This tumor retains properties of the original specimen. Observations made on the tumor biology of mucogenic colorectal carcinoma using these cell lines are discussed.

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone.

PubMed

Sakumoto, Marimu; Takahashi, Mami; Oyama, Rieko; Takai, Yoko; Kito, Fusako; Shiozawa, Kumiko; Qiao, Zhiwei; Yoshida, Akihiko; Endo, Makoto; Kawai, Akira; Kondo, Tadashi

2017-10-01

Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

PubMed

Zwollo, Patty; Hennessey, Erin; Moore, Catherine; Marancik, David P; Wiens, Gregory D; Epp, Lidia

2017-09-01

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM + and IgM ++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT + and/or IgT ++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT + B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT + B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM + B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT + B lineage cells may have a protective function in the immune response to Fp. Copyright © 2017 Elsevier Ltd. All rights reserved.

Avian germplasm preservation: embryonic stem cells or primordial germ cells?

PubMed

Petitte, J N

2006-02-01

Presently, avian genetic resources are best maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be destroyed because of the decline in the number of Poultry Science Departments and pressures to cut costs at land grant institutions. Cryopreservation of semen is often suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does not allow for full reconstitution of the genetics of the original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primordial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal cells, ESC, or PGC might offer a means to preserve the entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with varying degrees of efficiency. Similarly, the effectiveness of generating germ line chimeras using blastodermal cells, ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the low efficiency rate of reconstitution and the high cost associated with current technologies makes these approaches prohibitive. Significant challenges remain to be overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted.

EphA3 maintains radioresistance in head and neck cancers through epithelial mesenchymal transition.

PubMed

Kim, Song Hee; Lee, Won Hyeok; Kim, Seong Who; Je, Hyoung Uk; Lee, Jong Cheol; Chang, Hyo Won; Kim, Young Min; Kim, Kyungbin; Kim, Sang Yoon; Han, Myung Woul

2018-07-01

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases-Friedreich's Ataxia Example.

PubMed

Li, Yanjie; Polak, Urszula; Clark, Amanda D; Bhalla, Angela D; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David; Butler, Jill S; Napierala, Marek

2016-08-01

Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to <1000 and is tightly correlated with age of onset and severity of the disease symptoms. The heterogeneity of Friedreich's ataxia stresses the need for a large cohort of patient samples to conduct studies addressing the mechanism of disease pathogenesis or evaluate novel therapeutic candidates. Herein, we report the establishment and characterization of an FRDA fibroblast repository, which currently includes 50 primary cell lines derived from FRDA patients and seven lines from mutation carriers. These cells are also a source for generating induced pluripotent stem cell (iPSC) lines by reprogramming, as well as disease-relevant neuronal, cardiac, and pancreatic cells that can then be differentiated from the iPSCs. All FRDA and carrier lines are derived using a standard operating procedure and characterized to confirm mutation status, as well as expression of FXN mRNA and protein. Consideration and significance of creating disease-focused cell line and tissue repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community.

Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases—Friedreich's Ataxia Example

PubMed Central

Li, Yanjie; Polak, Urszula; Clark, Amanda D.; Bhalla, Angela D.; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David

2016-01-01

Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to <1000 and is tightly correlated with age of onset and severity of the disease symptoms. The heterogeneity of Friedreich's ataxia stresses the need for a large cohort of patient samples to conduct studies addressing the mechanism of disease pathogenesis or evaluate novel therapeutic candidates. Herein, we report the establishment and characterization of an FRDA fibroblast repository, which currently includes 50 primary cell lines derived from FRDA patients and seven lines from mutation carriers. These cells are also a source for generating induced pluripotent stem cell (iPSC) lines by reprogramming, as well as disease-relevant neuronal, cardiac, and pancreatic cells that can then be differentiated from the iPSCs. All FRDA and carrier lines are derived using a standard operating procedure and characterized to confirm mutation status, as well as expression of FXN mRNA and protein. Consideration and significance of creating disease-focused cell line and tissue repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community. PMID:27002638

Thermionic photovoltaic energy converter

NASA Technical Reports Server (NTRS)

Chubb, D. L. (Inventor)

1985-01-01

A thermionic photovoltaic energy conversion device comprises a thermionic diode mounted within a hollow tubular photovoltaic converter. The thermionic diode maintains a cesium discharge for producing excited atoms that emit line radiation in the wavelength region of 850 nm to 890 nm. The photovoltaic converter is a silicon or gallium arsenide photovoltaic cell having bandgap energies in this same wavelength region for optimum cell efficiency.

Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties.

PubMed

Yoshimoto, Nobuo; Kuroda, Shun'ichi

2014-04-01

For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

PubMed

Osada, Masako; Singh, Varan J; Wu, Kenmin; Sant'Angelo, Derek B; Pezzano, Mark

2013-01-01

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

Alternative lengthening of telomeres can be maintained by preferential elongation of lagging strands

PubMed Central

Min, Jaewon; Wright, Woodring E.

2017-01-01

Abstract Alternative lengthening of telomeres (ALT) is a telomerase independent telomere maintenance mechanism that occurs in ∼15% of cancers. The potential mechanism of ALT is homology-directed telomere synthesis, but molecular mechanisms of how ALT maintains telomere length in human cancer is poorly understood. Here, we generated TERC (telomerase RNA) gene knockouts in telomerase positive cell lines that resulted in long-term surviving clones acquiring the ALT pathway but at a very low frequency. By comparing these ALT cells with parental telomerase positive cells, we observed that ALT cells possess excessively long telomeric overhangs derived from telomere elongation processes that mostly occur during S phase. ALT cells exhibited preferential elongation of the telomeric lagging strands, whereas telomerase positive cells exhibited similar elongation between leading and lagging strands. We propose that the ALT pathway preferentially occurs at telomeric lagging strands leading to heterogeneous telomere lengths observed in most ALT cancers. PMID:28082393

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

PubMed

Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

2016-01-01

A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family

PubMed Central

Soyombo, Abigail A.; Wu, Yipin; Kolski, Lauren; Rios, Jonathan J.; Rakheja, Dinesh; Chen, Alice; Kehler, James; Hampel, Heather; Coughran, Alanna; Ross, Theodora S.

2013-01-01

Summary Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability. PMID:24319668

Telomere lengthening and other functions of telomerase.

PubMed

Rubtsova, M P; Vasilkova, D P; Malyavko, A N; Naraikina, Yu V; Zvereva, M I; Dontsova, O A

2012-04-01

Telomerase is an enzyme that maintains the length of the telomere. The telomere length specifies the number of divisions a cell can undergo before it finally dies (i.e. the proliferative potential of cells). For example, telomerase is activated in embryonic cell lines and the telomere length is maintained at a constant level; therefore, these cells have an unlimited fission potential. Stem cells are characterized by a lower telomerase activity, which enables only partial compensation for the shortening of telomeres. Somatic cells are usually characterized by the absence of telomerase activity. Telomere shortening leads to the attainment of the Hayflick limit, the transition of cells to a state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death. The surviving cells become cancer cells, which are capable both of dividing indefinitely and maintaining telomere length (usually with the aid of telomerase). Telomerase is a reverse transcriptase. It consists of two major components: telomerase RNA (TER) and reverse transcriptase (TERT). TER is a non-coding RNA, and it contains the region which serves as a template for telomere synthesis. An increasing number of articles focussing on the alternative functions of telomerase components have recently started appearing. The present review summarizes data on the structure, biogenesis, and functions of telomerase.

Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device.

PubMed

Enosawa, Shin; Miyamoto, Yoshitaka; Kubota, Hisayo; Jomura, Tomoko; Ikeya, Takeshi

2012-01-01

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-μm diameter at 100-μm intervals. Our standard protocol is to seed with feeder HH cells and then seed with primary hepatic parenchymal cells. The composite cell spheroids thus obtained are called heterospheroids. Feeder cells that were attached to the plate migrated and encompassed the spheroidal hepatocyte mass. Electron microscopy revealed Disse space-like structures characterized by hepatocyte-rooted microvilli rooted between hepatocyte and feeder epithelial HH cells. Differentiated hepatic functions such as albumin synthesis and cytochrome P450 subfamily CYP3A activities were maintained for 28 days in the heterospheroid versus monospheroid and monolayer cultures. In addition, glucuronide conjugation activity was maintained at a high level in heterospheroids. These results indicate that structurally similar hepatic lobules were formed in a microfabricated cell array coculture system and that the culture conditions are beneficial for maintaining differentiated hepatic functions.

[Formation of protodioscin and deltoside isomers in suspension cultures of Nepal yam (Dioscorea deltoidea Wall.) cells].

PubMed

Khandy, M T; Titova, M V; Konstantinova, S V; Kochkin, D V; Ivanov, I M; Nosov, A M

2016-01-01

Changes in the content of the furostanol glycosides protodioscin and deltoside, particularly that of the (25S)-isomers of the glycosides, during suspension cultivation of different lines of Nepal yam (Dioscorea deltoidea Wall.) cells of the strain IFR-DM-0.5 has been investigated. The composition of furostanol glycosides has been characterized, and the dynamics of the accumulation of individual glycosides during lengthy subcultivation of cells maintained in flasks or in a barbotage bioreactor has been analyzed. A positive correlation between the growth and accumulation of substances that belonged to the class of furostanol glycosides has been demonstrated for cultured dioscorea cells, whereas the content of some of the individual glycosides varied considerably between the lines of the strain, cultures maintained under different conditions, and even between cells in different phases of the growth cycle. The increased content of (25R)-forms of the glycosides (protodioscin and deltoside) was correlated with a decrease in the cellular growth rate, whereas an increase in culture growth intensity occurred concomitantly to an increase of the amount of (25S)-isomers. This may be indicative of the specific stimulatory effect of (25S)-glycosides, but not the (25R)-forms, on cell proliferation in vitro. Thus, the concentration of (25S)-forms may increase due to the autoselection of cells capable of intensive division during prolonged cultivation.

Establishment and characterization of a new marine fish cell line from ovary of barfin flounder ( Verasper moseri)

NASA Astrophysics Data System (ADS)

Xu, Xiaohui; Fan, Tingjun; Jiang, Guojian; Yang, Xiuxia

2015-12-01

A novel continuous ovary cell line from barfin flounder ( Verasper moseri) (BFO cell line) was established with its primitive application in transgenic expression demonstrated in this study. Primarily cultured cells grew well at 22°C in Dulbecco's modified Eagle medium/F12 medium (DMEM/F12, 1:1; pH 7.2) supplemented with 20% fetal bovine serum (FBS), carboxymethyl chitooligosaccharide, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The primary BFO cells in fibroblastic morphology proliferated into a confluent monolayer about 2 weeks later, and were able to be subcultured. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 22°C. The BFO cells can be continuously subcultured to Passage 120 steadily with a population doubling time of 32.7 h at Passage 60. Chromosome analysis revealed that 72% of BFO cells at Passage 60 maintained the normal diploid chromosome number (46) with a normal karyotype of 2st+44t. The results of gene transformation indicated that green fluorescence protein (GFP) positively expressed in these cells after being transformed with pcDNA3.1-GFP. Therefore, a continuous and transformable BFO cell line was successfully established, which may serve as a useful tool for cytotechnological manipulation and transgenic modification of this fish.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emergedmore » cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.« less

Pineal Photoreceptor Cells Are Required for Maintaining the Circadian Rhythms of Behavioral Visual Sensitivity in Zebrafish

PubMed Central

Li, Xinle; Montgomery, Jake; Cheng, Wesley; Noh, Jung Hyun; Hyde, David R.; Li, Lei

2012-01-01

In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry)] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity. PMID:22815753

Long-term cultivation of human corneal endothelial cells by telomerase expression.

PubMed

Liu, Zhiping; Zhuang, Jing; Li, Chaoyang; Wan, Pengxia; Li, Naiyang; Zhou, Qiang; Zhou, Chenjing; Huang, Zheqian; Wang, Zhichong

2012-07-01

The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vivo physiological recording from the lateral line of juvenile zebrafish.

PubMed

Olt, Jennifer; Allen, Claire E; Marcotti, Walter

2016-10-01

Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

A p53-dependent response limits the viability of mammalian haploid cells

PubMed Central

Olbrich, Teresa; Mayor-Ruiz, Cristina; Vega-Sendino, Maria; Gomez, Carmen; Ortega, Sagrario; Ruiz, Sergio; Fernandez-Capetillo, Oscar

2017-01-01

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell–sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells. PMID:28808015

Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

PubMed Central

Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

2015-01-01

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963

In vitro evidence of glucose-induced toxicity in GnRH secreting neurons: high glucose concentrations influence GnRH secretion, impair cell viability, and induce apoptosis in the GT1-1 neuronal cell line.

PubMed

Pal, Lubna; Chu, Hsiao-Pai; Shu, Jun; Topalli, Ilir; Santoro, Nanette; Karkanias, George

2007-10-01

To evaluate for direct toxic effects of high glucose concentrations on cellular physiology in GnRH secreting immortalized GT1-1 neurons. Prospective experimental design. In vitro experimental model using a cell culture system. GT1-1 cells were cultured in replicates in media with two different glucose concentrations (450 mg/dL and 100 mg/dL, respectively) for varying time intervals (24, 48, and 72 hours). Effects of glucose concentrations on GnRH secretion by the GT1-1 neurons were evaluated using a static culture model. Cell viability, cellular apoptosis, and cell cycle events in GT1-1 neurons maintained in two different glucose concentrations were assessed by flow cytometry (fluorescence-activated cell sorter) using Annexin V-PI staining. Adverse influences of high glucose concentrations on GnRH secretion and cell viability were noted in cultures maintained in high glucose concentration (450 mg/dL) culture medium for varying time intervals. A significantly higher percentage of cells maintained in high glucose concentration medium demonstrated evidence of apoptosis by a fluorescence-activated cell sorter. We provide in vitro evidence of glucose-induced cellular toxicity in GnRH secreting GT1-1 neurons. Significant alterations in GnRH secretion, reduced cell viability, and a higher percentage of apoptotic cells were observed in GT1-1 cells maintained in high (450 mg/dL) compared with low (100 mg/dL) glucose concentration culture medium.

Vinblastine 20' Amides: Synthetic Analogues That Maintain or Improve Potency and Simultaneously Overcome Pgp-Derived Efflux and Resistance.

PubMed

Lukesh, John C; Carney, Daniel W; Dong, Huijun; Cross, R Matthew; Shukla, Vyom; Duncan, Katharine K; Yang, Shouliang; Brody, Daniel M; Brütsch, Manuela M; Radakovic, Aleksandar; Boger, Dale L

2017-09-14

A series of 180 vinblastine 20' amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure-activity relationships and a structural model were developed in the studies which provided many such 20' amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogues that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity, and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

PubMed

Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

2017-10-01

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

Diminished origin licensing capacity specifically sensitises tumour cells to replication stress

PubMed Central

Zimmerman, Kristin M.; Jones, Rebecca M.; Petermann, Eva; Jeggo, Penelope A.

2013-01-01

Previous studies have shown that dormant licensed replication origins can be exploited to enhance recovery from replication stress. Since tumour cells express high levels of origin licensing proteins, we examined whether depletion of such factors might specifically sensitise tumour versus non-tumour cells. Consistent with previous findings, we observed that three tumour-derived cell lines overexpress ORC1, a licensing component, compared to four non-tumour cell lines and that a greater level of ORC1 was required to maintain viability in the tumour cells. We determined siRNA-mediated knockdown conditions for each line that maximally reduced ORC1 but did not impact upon viability, which we considered would optimally deplete dormant origins. ORC1 depletion hypersensitised the tumour-derived cells to hydroxyurea (HU) and H202 but did not affect the sensitivity of the non-tumour lines. Similar results were observed following depletion of ORC6 or CDC6. Further, co-depletion of p53 and ORC1 modestly impaired viability of 1BR3hTERT non-tumour fibroblasts and more dramatically caused hypersensitivity to HU. Finally, overexpression of the c-Myc oncogene combined with ORC1 depletion in non-tumour BJhTERT cells diminished viability. Collectively, these findings suggest that tumour cells may have a reliance on origin licensing capacity, suggesting that licensing factors could represent a target for drug-based cancer therapy. PMID:23364533

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

PubMed Central

Arsenakis, M; Hubenthal-Voss, J; Campadelli-Fiume, G; Pereira, L; Roizman, B

1986-01-01

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells. Images PMID:3022001

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

[The level of superoxide dismutase expression in primary and metastatic colorectal cancer cells in hypoxia and tissue normoxia].

PubMed

Skrzycki, Michał; Czeczot, Hanna; Chrzanowska, Alicja; Otto-Ślusarczyk, Dagmara

2015-11-01

Superoxide oxidase (SOD) is a key antioxidant enzyme protecting cells against oxidative stress, which might induce cancerogenesis. In tumor cells SOD influences the level of the reactive oxygen species (ROS) allowing for survival and proliferation. High rate of cells proliferation in tumor leads to their temporary hypoxia due to lower rate of angiogenesis. Therefore during tumor development, cancer cells function in conditions of hypoxia or tissue normoxia. The aim of study was to evaluate of SOD isoenzymes (SOD1 and SOD2) expression level in cell lines of primary (SW 480) and metastatic (SW 620) colorectal cancer, cultured in hypoxia (1% oxygen), tissue normoxia (10% oxygen), and atmospheric normoxia (21% oxygen). Cells were cultured in MEM medium in different oxygen concentrations (1%, 10%, 21%) in hypoxic chamber with oxygenation regulator. The number of living cells in lines SW 480 and 620 was determined by trypan blue method. Expression of SOD1 and SOD2 at the mRNA level was determined by RT-PCR and PCR. In both studied cell lines (SW 480 and SW 620), the number of living cells (viability) was increased in hypoxia and atmospheric normoxia. The expression level of SOD1 and SOD2 in studied cell lines was different. The lowest level of expression of both SOD isoenzymes was observed in hypoxia. In conditions of atmospheric normoxia the expression level of SOD1 in SW480 cell line was increased, and similar in SW620 cell line comparing to tissue normoxia. Whereas the SOD2 expression level in atmospheric normoxia conditions in both cell lines was significantly increased. Observed differences were statistically significant (p ≤ 0,05). The profile of expression of SOD1 and SOD2 in cell lines SW480 and SW620 indicates differentiated response of tumor cells depending on access to oxygen. Low level of SOD isoenzymes expression in SW480 and SW620 cells in hypoxia indicates decreased production of ROS. Differences of SOD isoenzymes expression level in tissue normoxia indicate their compensatory action, allowing to maintain the balance between O₂- removal and H₂O₂production in studied tumor cells. In atmospheric normoxia conditions increased expression level of SOD1 and SOD2 observed in studied cell lines points to oxidative stress. © 2015 MEDPRESS.

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression

PubMed Central

Hang, Su; Tiwari, Agnes F.Y.; Ngan, Hextan Y.S.; Yip, Yim-Ling; Cheung, Annie L.M.; Tsao, Sai Wah; Deng, Wen

2016-01-01

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer. PMID:27344169

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

PubMed Central

Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

2016-01-01

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID:27977675

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome.

PubMed

Jeyapalan, Jennie N; Varley, Helen; Foxon, Jenny L; Pollock, Raphael E; Jeffreys, Alec J; Henson, Jeremy D; Reddel, Roger R; Royle, Nicola J

2005-07-01

Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT- cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.

Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

PubMed Central

2013-01-01

Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

PubMed

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Manipulation of the sodium-potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures.

PubMed

Wang, Samantha B; Lee-Goldman, Alexandria; Ravikrishnan, Janani; Zheng, Lili; Lin, Henry

2018-04-01

Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed. © 2017 Wiley Periodicals, Inc.

Mammalian X Chromosome Dosage Compensation: Perspectives From the Germ Line.

PubMed

Sangrithi, Mahesh N; Turner, James M A

2018-06-01

Sex chromosomes are advantageous to mammals, allowing them to adopt a genetic rather than environmental sex determination system. However, sex chromosome evolution also carries a burden, because it results in an imbalance in gene dosage between females (XX) and males (XY). This imbalance is resolved by X dosage compensation, which comprises both X chromosome inactivation and X chromosome upregulation. X dosage compensation has been well characterized in the soma, but not in the germ line. Germ cells face a special challenge, because genome wide reprogramming erases epigenetic marks responsible for maintaining the X dosage compensated state. Here we explain how evolution has influenced the gene content and germ line specialization of the mammalian sex chromosomes. We discuss new research uncovering unusual X dosage compensation states in germ cells, which we postulate influence sexual dimorphisms in germ line development and cause infertility in individuals with sex chromosome aneuploidy. © 2018 The Authors. BioEssays Published by Periodicals, Inc.

Cell cloning-on-the-spot by using an attachable silicone cylinder.

PubMed

Park, Hong Bum; Son, Wonseok; Chae, Dong Han; Lee, Jisu; Kim, Il-Woung; Yang, Woomi; Sung, Jae Kyu; Lim, Kyu; Lee, Jun Hee; Kim, Kyung-Hee; Park, Jong-Il

2016-06-10

Cell cloning is a laboratory routine to isolate and keep particular properties of cultured cells. Transfected or other genetically modified cells can be selected by the traditional microbiological cloning. In addition, common laboratory cell lines are prone to genotypic drift during their continual culture, so that supplementary cloning steps are often required to maintain correct lineage phenotypes. Here, we designed a silicone-made attachable cloning cylinder, which facilitated an easy and bona fide cloning of interested cells. This silicone cylinder was easy to make, showed competent stickiness to laboratory plastics including culture dishes, and hence enabled secure isolation and culture for days of selected single cells, especially, on the spots of preceding cell-plating dishes under microscopic examination of visible cellular phenotypes. We tested the silicone cylinder in the monoclonal subcloning from a heterogeneous population of a breast cancer cell line, MDA-MB-231, and readily established independent MDA-MB-231 subclones showing different sublineage phenotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

PubMed Central

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-01-01

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

PubMed

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-07-07

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

PubMed

Rasmussen, B; Davis, R; Thomas, J; Reddy, P

1998-11-01

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.

Toward a framework linkage map of the canine genome.

PubMed

Langston, A A; Mellersh, C S; Wiegand, N A; Acland, G M; Ray, K; Aguirre, G D; Ostrander, E A

1999-01-01

Selective breeding to maintain specific physical and behavioral traits has made the modern dog one of the most physically diverse species on earth. One unfortunate consequence of the common breeding practices used to develop lines of dogs with the desired traits is amplification and propagation of genetic diseases within distinct breeds. To map disease loci we have constructed a first-generation framework map of the canine genome. We developed large numbers of highly polymorphic markers, constructed a panel of canine-rodent hybrid cell lines, and assigned those markers to chromosome groups using the hybrid cell lines. Finally, we determined the order and spacing of markers on individual canine chromosomes by linkage analysis using a reference panel of 17 outbred pedigrees. This article describes approaches and strategies to accomplish these goals.

Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

PubMed

Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

2014-07-01

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α. © 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.

Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

PubMed Central

Liu, Chieh-Lun; Watson, Aaron M.; Place, Allen R.; Jagus, Rosemary

2017-01-01

Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity. PMID:28587087

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

PubMed Central

Sansom, Stephen N.; Alsiö, Jessica M.; Kaneda, Masahiro; Smith, James; O'Carroll, Donal; Tarakhovsky, Alexander; Livesey, Frederick J.

2010-01-01

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation. PMID:20976144

Variable TERRA abundance and stability in cervical cancer cells.

PubMed

Oh, Bong-Kyeong; Keo, Ponnarath; Bae, Jaeman; Ko, Jung Hwa; Choi, Joong Sub

2017-06-01

Telomeres are transcribed into long non-coding RNA, referred to as telomeric repeat-containing RNA (TERRA), which plays important roles in maintaining telomere integrity and heterochromatin formation. TERRA has been well characterized in HeLa cells, a type of cervical cancer cell. However, TERRA abundance and stability have not been examined in other cervical cancer cells, at least to the best of our knowledge. Thus, in this study, we measured TERRA levels and stability, as well as telomere length in 6 cervical cancer cell lines, HeLa, SiHa, CaSki, HeLa S3, C-33A and SNU-17. We also examined the association between the TERRA level and its stability and telomere length. We found that the TERRA level was several fold greater in the SiHa, CaSki, HeLa S3, C-33A and SNU-17 cells, than in the HeLa cells. An RNA stability assay of actinomycin D-treated cells revealed that TERRA had a short half-life of ~4 h in HeLa cells, which was consistent with previous studies, but was more stable with a longer half-life (>8 h) in the other 5 cell lines. Telomere length varied from 4 to 9 kb in the cells and did not correlate significantly with the TERRA level. On the whole, our data indicate that TERRA abundance and stability vary between different types of cervical cancer cells. TERRA degrades rapidly in HeLa cells, but is maintained stably in other cervical cancer cells that accumulate higher levels of TERRA. TERRA abundance is associated with the stability of RNA in cervical cancer cells, but is unlikely associated with telomere length.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

PubMed

Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

PubMed

Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L

2014-01-01

The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

PubMed Central

Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.

2017-01-01

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells

PubMed Central

Luo, Lan; Kawakatsu, Miho; Guo, Chao-Wan; Urata, Yoshishige; Huang, Wen-Jing; Ali, Haytham; Doi, Hanako; Kitajima, Yuriko; Tanaka, Takayuki; Goto, Shinji; Ono, Yusuke; Xin, Hong-Bo; Hamano, Kimikazu; Li, Tao-Sheng

2014-01-01

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem (iPS) cells were investigated with two human iPS cell lines (201B7 and 253G1). Cells used in this study were expanded from a single colony of each cell line with the addition of proprietary antioxidant supplement or homemade antioxidant cocktail in medium, and maintained in parallel for 2 months. The cells grew well in all culture conditions and kept “stemness”. Although antioxidants modestly decreased the levels of intracellular reactive oxygen species, there were no differences in the expression of 53BP1 and pATM, two critical molecules related with DNA damage and repair, under various culture conditions. CGH analysis showed that the events of genetic aberrations were decreased only in the 253G1 iPS cells with the addition of homemade antioxidant cocktail. Long-term culture will be necessary to confirm whether low dose antioxidants improve the quality and genomic stability of iPS cells. PMID:24445363

Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux.

PubMed

Ahmed, Raya; Roger, Laureline; Costa Del Amo, Pedro; Miners, Kelly L; Jones, Rhiannon E; Boelen, Lies; Fali, Tinhinane; Elemans, Marjet; Zhang, Yan; Appay, Victor; Baird, Duncan M; Asquith, Becca; Price, David A; Macallan, Derek C; Ladell, Kristin

2016-12-13

Adaptive immunity requires the generation of memory T cells from naive precursors selected in the thymus. The key intermediaries in this process are stem cell-like memory T (T SCM ) cells, multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells. In theory, antigen specificity within the T SCM pool may be imprinted statically as a function of largely dormant cells and/or retained dynamically by more transitory subpopulations. To explore the origins of immunological memory, we measured the turnover of T SCM cells in vivo using stable isotope labeling with heavy water. The data indicate that T SCM cells in both young and elderly subjects are maintained by ongoing proliferation. In line with this finding, T SCM cells displayed limited telomere length erosion coupled with high expression levels of active telomerase and Ki67. Collectively, these observations show that T SCM cells exist in a state of perpetual flux throughout the human lifespan. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility.

PubMed

Knudsen, Erik S; Balaji, Uthra; Mannakee, Brian; Vail, Paris; Eslinger, Cody; Moxom, Christopher; Mansour, John; Witkiewicz, Agnieszka K

2018-03-01

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities. 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC. The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC. These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

PubMed Central

Variar, Gokul; Pant, Tarun; Singh, Apoorva; Ravichandran, Abinaya; Swami, Sushant; Kalyanaraman, Balaraman; Dhanasekaran, Anuradha

2017-01-01

Vicious cycles of mutations and reactive oxygen species (ROS) generation contribute to cancer progression. The use of antioxidants to inhibit ROS generation promotes cytostasis by affecting the mutation cycle and ROS-dependent survival signaling. However, cancer cells select mutations to elevate ROS albeit maintaining mitochondrial hyperpolarization (Δψm), even under hypoxia. From this perspective, the use of drugs that disrupt both ROS generation and Δψm is a viable anticancer strategy. Hence, we studied the effects of mitochondrially targeted carboxy proxyl nitroxide (Mito-CP) and a control ten carbon TPP moiety (Dec-TPP+) in the human Burkitt lymphoma cell line (Daudi) and normal peripheral blood mononuclear cells under hypoxia and normoxia. We found preferential localization, Δψm and adenosine triphosphate loss, and significant cytotoxicity by Mito-CP in Daudi cells alone. Interestingly, ROS levels were decreased and maintained in hypoxic and normoxic cancer cells, respectively, by Mito-CP but not Dec-TPP+, therefore preventing any adaptive signaling. Moreover, dual effects on mitochondrial bioenergetics and ROS by Mito-CP curtailed the cancer survival via Akt inhibition, AMPK-HIF-1α activation and promoted apoptosis via increased BCL2-associated X protein and poly (ADP-ribose) polymerase expression. This dual mode of action by Mito-CP provides a better explanation of the application of antioxidants with specific relevance to cancerous transformation and adaptations in the Daudi cell line. PMID:28426671

Cytokeratins in normal and malignant transitional epithelium. Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell culture lines.

PubMed Central

Moll, R.; Achtstätter, T.; Becht, E.; Balcarova-Ständer, J.; Ittensohn, M.; Franke, W. W.

1988-01-01

The pattern of cytokeratins expressed in normal urothelium has been compared with that of various forms of transitional cell carcinomas (TCCs; 21 cases) and cultured bladder carcinoma cell lines, using immunolocalization and gel electrophoretic techniques. In normal urothelium, all simple-epithelium-type cytokeratins (polypeptides 7, 8, 18, 19) were detected in all cell layers, whereas antibodies to cytokeratins typical for stratified epithelia reacted with certain basal cells only or, in the case of cytokeratin 13, with cells of the basal and intermediate layers. This pattern was essentially maintained in low-grade (G1, G1/2) TCCs but was remarkably modified in G2 TCCs. In G3 TCCs simple-epithelial cytokeratins were predominant whereas the amounts of component 13 were greatly reduced. Squamous metaplasia was accompanied generally by increased or new expression of some stratified-epithelial cytokeratins. The cytokeratin patterns of cell culture lines RT-112 and RT-4 resembled those of G1 and G2 TCCs, whereas cell line T-24 was comparable to G3 carcinomas. The cell line EJ showed a markedly different pattern. The results indicate that, in the cell layers of the urothelium, the synthesis of stratification-related cytokeratins such as component 13 is inversely oriented compared with that in other stratified epithelia where these proteins are suprabasally expressed, that TCCs retain certain intrinsic cytoskeletal features of urothelium, and that different TCCs can be distinguished by their cytokeratin patterns. The potential value of these observations in histopathologic and cytologic diagnoses is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:2456018

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

PubMed Central

Debeb, Bisrat G.; Galat, Vasiliy; Epple-Farmer, Jessica; Iannaccone, Steve; Woodward, Wendy A.; Bader, Michael; Iannaccone, Philip; Binas, Bert

2009-01-01

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation. PMID:19784378

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

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

Fang, Zhen F.; Gai, Hui; Huang, You Z.

2006-11-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ESmore » cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.« less

A Primary Xenograft Model of Small Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In-Vitro

PubMed Central

Daniel, Vincent C.; Marchionni, Luigi; Hierman, Jared S.; Rhodes, Jonathan T.; Devereux, Wendy L.; Rudin, Charles M.; Yung, Rex; Parmigani, Giovanni; Dorsch, Marion; Peacock, Craig D.; Watkins, D. Neil

2009-01-01

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then re-implanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared to normal lung, primary tumors, xenografts and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture, and that was not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development. PMID:19351829

Chemical composition and cytotoxicity of extracts of marjoram and rosemary and their activity against Sporothrix brasiliensis.

PubMed

Waller, Stefanie Bressan; Madrid, Isabel Martins; Hoffmann, Jéssica Fernanda; Picoli, Tony; Cleff, Marlete Brum; Chaves, Fábio Clasen; Faria, Renata Osório de; Meireles, Mário Carlos Araújo; Braga de Mello, João Roberto

2017-07-01

Motivated by increasing reports of antifungal resistance in human and animal sporotrichosis, this study evaluated the chemical composition, cytotoxicity and anti-Sporothrix brasiliensis activity of extracts of marjoram (Origanum majorana) and rosemary (Rosmarinus officinalis). Ten (INF10) and 60 min (INF60) infusions, a decoction and a hydroalcoholic extract (HAE, 70 %) were prepared from both plants (10 % w/v). The extract composition was analysed by liquid chromatography/mass spectrometry and the cytotoxicity was evaluated using a colorimetric assay in canine and feline kidney cells. Using a broth microdilution assay (CLSI M38-A2) adapted to the extracts, 30 Sporothrix brasiliensis isolates from dogs, cats and humans, and one Sporothrix schenckii were tested.Results/Key findings. The predominant phenolic compounds found in all extracts were 4-hydroxybenzoic acid, caffeic acid and chlorogenic acid. Luteolin was also one of the predominant compounds, but only in the HAE of marjoram. Extracts of marjoram maintained cell viability in concentrations up to 2.5 mg ml-1 for the feline cell line and up to 10 mg ml-1 for the canine cell line, whereas in rosemary, the cell viability for both kidney lines was maintained with concentrations up to 5 mg ml-1. The activity of rosemary extracts was low or absent. Among the marjoram extracts, HAE was highlighted and had fungistatic activity against Sporothrix brasiliensis (MIC5040 mg ml-1), including in all itraconazole-resistant isolates. S. schenckiisensu stricto was sensitive to marjoram extracts (MIC/MFC ≤5 mg ml-1), with the exception of INF10. These findings support the potential usefulness of the HAE of marjoram in the treatment of sporotrichosis.

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

PubMed Central

West, John D; Dorà, Natalie J; Collinson, J Martin

2015-01-01

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis. PMID:25815115

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

PubMed

Smendziuk, Christopher M; Messenberg, Anat; Vogl, A Wayne; Tanentzapf, Guy

2015-08-01

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues. © 2015. Published by The Company of Biologists Ltd.

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential.

PubMed

Green, K J; Stappenbeck, T S; Noguchi, S; Oyasu, R; Nilles, L A

1991-03-01

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Established breast cancer stem cell markers do not correlate with in vivo tumorigenicity of tumor-initiating cells

PubMed Central

LEHMANN, CHRISTIAN; JOBS, GABRIELE; THOMAS, MARKUS; BURTSCHER, HELMUT; KUBBIES, MANFRED

2012-01-01

The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24−/CD44+ and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamil-sensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells. PMID:23042145

Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy.

PubMed

Maria, Roberta M; Altei, Wanessa F; Andricopulo, Adriano D; Becceneri, Amanda B; Cominetti, Márcia R; Venâncio, Tiago; Colnago, Luiz A

2015-11-01

(1)H high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 (1)H HR-MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR-MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells. Copyright © 2015 Elsevier Inc. All rights reserved.

SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

PubMed

Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

2010-04-20

To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

Water electrolysis

NASA Technical Reports Server (NTRS)

Schubert, Franz H. (Inventor); Grigger, David J. (Inventor)

1992-01-01

This disclosure is directed to an electrolysis cell forming hydrogen and oxygen at space terminals. The anode terminal is porous and able to form oxygen within the cell and permit escape of the gaseous oxygen through the anode and out through a flow line in the presence of backpressure. Hydrogen is liberated in the cell at the opposing solid metal cathode which is permeable to hydrogen but not oxygen so that the migratory hydrogen formed in the cell is able to escape from the cell. The cell is maintained at an elevated pressure so that the oxygen liberated by the cell is delivered at elevated pressure without pumping to raise the pressure of the oxygen.

Isolation and characterization of an IGROV-1 human ovarian cancer cell line made resistant to Ecteinascidin-743 (ET-743)

PubMed Central

Erba, E; Bergamaschi, D; Bassano, L; Ronzoni, S; Liberti, G Di; Muradore, I; Vignati, S; Faircloth, G; Jimeno, J; D'Incalci, M

2000-01-01

By exposing Igrov-1 human ovarian cancer cells to increasing concentrations of Ecteinascidin-743 (ET-743), either for a short or prolonged time, we obtained sublines resistant to ET-743 which overexpress Pgp. The most resistant clone (Igrov-1/25 ET) was evaluated for biological and pharmacological characterizations. The increased Pgp levels of Igrov-1/25 ET were not due to amplification of the mdr-1 gene but to increased mRNA levels. No increase in other multidrug resistance-related proteins such as MRP or LRP was observed in Igrov-1/25 ET. The IC50values of ET-743 against Igrov-1/25 ET was approximately 50 times higher than the parental cell line. Resistance was not reversed while maintaining the cell line in drug-free medium for at least 24 months. Igrov-1/25 ET was cross-resistant to Doxorubicin and VP16 while it was equally sensitive to L-PAM, MNNG, CPT and only marginally less sensitive to Cis-DDP and Oxaliplatin compared to the parental cell line. Igrov-1/25 ET exposed to Doxorubicin retained this drug much less, mainly because of a more efficient drug efflux. The cyclosporine analogue SDZ PSC-833 reversed the resistance of Igrov-1/25 ET to ET-743, without any enhancement of the drug activity against the parental Igrov-1 cell line. Igrov-1/25 ET exhibits typical features of cell lines overexpressing the mdr-1 gene and can be a potentially useful tool in selecting ET-743 non-cross-resistant analogues as well as to investigate methods to counteract resistance to this drug. © 2000 Cancer Research Campaign PMID:10817511

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

PubMed

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

PubMed Central

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. PMID:24465853

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

PubMed Central

Pezic, Dubravka; Manakov, Sergei A.; Sachidanandam, Ravi; Aravin, Alexei A.

2014-01-01

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. PMID:24939875

Remodeling the zonula adherens in response to tension and the role of afadin in this response

PubMed Central

Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

2016-01-01

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

Generation of an immortalized mouse embryonic palatal mesenchyme cell line

PubMed Central

Soriano, Philippe

2017-01-01

Palatogenesis is a complex morphogenetic process, disruptions in which result in highly prevalent birth defects in humans. In recent decades, the use of model systems such as genetically-modified mice, mouse palatal organ cultures and primary mouse embryonic palatal mesenchyme (MEPM) cultures has provided significant insight into the molecular and cellular defects underlying cleft palate. However, drawbacks in each of these systems have prevented high-throughput, large-scale studies of palatogenesis in vitro. Here, we report the generation of an immortalized MEPM cell line that maintains the morphology, migration ability, transcript expression and responsiveness to exogenous growth factors of primary MEPM cells, with increased proliferative potential over primary cultures. The immortalization method described in this study will facilitate the generation of palatal mesenchyme cells with an unlimited capacity for expansion from a single genetically-modified mouse embryo and enable mechanistic studies of palatogenesis that have not been possible using primary culture. PMID:28582446

[Cytogenetical comparison of restorers TP-4 and D minghui63 and maintainer D46B of autotetraploid rice].

PubMed

Long, Wen-Bo; Luan, Li; Wang, Xing; Liu, Yu-Hua; Tu, Sheng-Bin; Kong, Fan-Lun; He, Tao

2007-04-01

Cytogenetical comparison was made between high seed set restorers TP-4 and D minghui63 and eminent maintainer line D46B of autotetraploid rice. The meiosis observation demonstrated the genomes of our autotetraploid materials were all 2n = 48, the same as those in mitosis observation. Low percentages of univalent and trivalent in metaphase I (MI) of restorers TP-4 and D minghui63 and in metaphase I (MI) of maintainer line D46B of autotetraploid rice were observed. And the percentages of chromosome pairing were all over 99%, showing eminent cytological character. The frequency of TP-4 and D minghui63 in metaphase I (MI) was 2.00/PMC and 2.26/PMC, respectively. However the frequency of D46B was 6.00/PMC, significantly higher than those of TP-4 and D minghui63. It indicated that the maintainer D46B has better chromosome pairing capability in metaphase I (MI). While, the frequency of lagging chromosomes of the maintainer D46B in anaphase I (AI) was 10.62%, significantly lower than that of TP-4 (19.44%) or D minghui63 (23.14%), and it was close to the level of diploid control (7.30%). In telophase I (TI), maintainer D46B exhibited a lower frequency of microkernel, and in telophase II (TII) the frequency of normal quartered microspore of maintainer D46B was not only higher than that of TP-4 or D minghui63 but also than that of diploid control. The percentage of the cell observed chromosome lagging in A1 and the percentage of abnormal cell in TI showed a greatly significant positive correlation. That may demonstrate chromo some separation in anaphase I (AI) and microkernel formation in telophase I (TI) are controlled by the same dominant single gene or the major gene of QTL.

Comparative evaluation of maintenance of cell viability of an experimental transport media “coconut water” with Hank's balanced salt solution and milk, for transportation of an avulsed tooth: An in vitro cell culture study

PubMed Central

Thomas, Toby; Gopikrishna, Velayutham; Kandaswamy, Deivanayagam

2008-01-01

The purpose of this study was to evaluate the efficiency of a new storage medium, coconut water, in comparison with other traditional storage media like Hank's balanced salt solution (HBBS) and milk, in maintaining the viability of an established cell line BHK-21/C13 (baby hamster kidney fibroblasts) using the direct suspension cell culture technique. The storage media tested in the study were divided into three major groups and two control groups - Group A: HBBS, Group B: milk, and Group C: coconut water. The positive and negative controls corresponded to 0-minute and 24-hour dry times respectively. The three groups were then divided into five subgroups, each denoting the storage time periods 15 min, 30 min, 45 min, 60 min and 120 min respectively. The cell line BHK-21/C13 was subcultured and the number of cells was standardized by making a cell suspension using Minimal Essential Medium in five culture plates. One ml of each experimental group (HBBS, milk and coconut water) was added to eight wells of each culture plate. The culture plates containing the cells and the experimental groups were incubated for the respective time periods. The cells were then counted with a Neubauer counting chamber, under light microscope. The results were statistically analyzed using One-way ANOVA and Multiple Range Test using the Tukey-HSD procedure to identify the significant groups at p ≤ 0.05. Within the parameters of this study, it appears that coconut water may be a better alternative to HBSS or milk, in terms of maintaining cell viability. Coconut water can be used as a superior transport medium for avulsed teeth. PMID:20142880

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

PubMed Central

Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

2016-01-01

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

In vitro models.

PubMed

Mather, Jennie Powell

2012-02-01

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models. Copyright © 2011 AlphaMed Press.

Construction of trypanosome artificial mini-chromosomes.

PubMed Central

Lee, M G; E, Y; Axelrod, N

1995-01-01

We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucei PARP promoter driving a chloramphenicol acetyltransferase (CAT) gene. In the 10 kb construct the CAT gene is followed by one hygromycin phosphotransferase (Hph) gene, and in the 13 kb construct the CAT gene is followed by three tandemly linked Hph genes. At each end of these linear molecules are telomere repeats and subtelomeric sequences. Electroporation of these linear DNA constructs into the procyclic form of T.brucei generated hygromycin-B resistant cell lines. In these cell lines, the input DNA remained linear and bounded by the telomere ends, but it increased in size. In the cell lines generated by the 10 kb construct, the input DNA increased in size to 20-50 kb. In the cell lines generated by the 13 kb constructs, two sizes of linear DNAs containing the input plasmid were detected: one of 40-50 kb and the other of 150 kb. The increase in size was not the result of in vivo tandem repetitions of the input plasmid, but represented the addition of new sequences. These Hph containing linear DNA molecules were maintained stably in cell lines for at least 20 generations in the absence of drug selection and were subsequently referred to as trypanosome artificial mini-chromosomes, or TACs. Images PMID:8532534

On-Line Control of Glucose Concentration in High-Yielding Mammalian Cell Cultures Enabled Through Oxygen Transfer Rate Measurements.

PubMed

Goldrick, Stephen; Lee, Kenneth; Spencer, Christopher; Holmes, William; Kuiper, Marcel; Turner, Richard; Farid, Suzanne S

2018-04-01

Glucose control is vital to ensure consistent growth and protein production in mammalian cell cultures. The typical fed-batch glucose control strategy involving bolus glucose additions based on infrequent off-line daily samples results in cells experiencing significant glucose concentration fluctuations that can influence product quality and growth. This study proposes an on-line method to control and manipulate glucose utilizing readily available process measurements. The method generates a correlation between the cumulative oxygen transfer rate and the cumulative glucose consumed. This correlation generates an on-line prediction of glucose that has been successfully incorporated into a control algorithm manipulating the glucose feed-rate. This advanced process control (APC) strategy enables the glucose concentration to be maintained at an adjustable set-point and has been found to significantly reduce the deviation in glucose concentration in comparison to conventional operation. This method has been validated to produce various therapeutic proteins across cell lines with different glucose consumption demands and is successfully demonstrated on micro (15 mL), laboratory (7 L), and pilot (50 L) scale systems. This novel APC strategy is simple to implement and offers the potential to significantly enhance the glucose control strategy for scales spanning micro-scale systems through to full scale industrial bioreactors. © 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

PubMed

Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

2017-10-02

Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

PubMed

Lawson, Bianca; Clulow, Simon; Mahony, Michael J; Clulow, John

2013-01-01

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

PubMed Central

van den Akker, Guus G. H.; Surtel, Don A. M.; Cremers, Andy; Richardson, Stephen M.; Hoyland, Judith A.; van Rhijn, Lodewijk W.

2016-01-01

Introduction Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Methods Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). Results The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. Conclusion We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair. PMID:26794306

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Voncken, Jan Willem; Welting, Tim J M

2016-01-01

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

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

Rafat, M; Bazalova, M; Palma, B

Purpose: To characterize the effect of very rapid dose delivery as compared to conventional therapeutic irradiation times on clonogenic cell survival. Methods: We used a Varian Trilogy linear accelerator to deliver doses up to 10 Gy using a 6 MV SRS photon beam. We irradiated four cancer cell lines in times ranging from 30 sec to 30 min. We also used a Varian TrueBeam linear accelerator to deliver 9 MeV electrons at 10 Gy in 10 s to 30 min to determine the effect of irradiation time on cell survival. We then evaluated the effect of using 60 and 120more » MeV electrons on cell survival using the Next Linear Collider Test Accelerator (NLCTA) beam line at the SLAC National Accelerator Laboratory. During irradiation, adherent cells were maintained at 37oC with 20%O2/5%CO2. Clonogenic assays were completed following irradiation to determine changes in cell survival due to dose delivery time and beam quality, and the survival data were fitted with the linear-quadratic model. Results: Cell lines varied in radiosensitivity, ranging from two to four logs of cell kill at 10 Gy for both conventional and very rapid irradiation. Delivering radiation in shorter times decreased survival in all cell lines. Log differences in cell kill ranged from 0.2 to 0.7 at 10 Gy for the short compared to the long irradiation time. Cell kill differences between short and long irradiations were more pronounced as doses increased for all cell lines. Conclusion: Our findings suggest that shortening delivery of therapeutic radiation doses to less than 1 minute may improve tumor cell kill. This study demonstrates the potential advantage of technologies under development to deliver stereotactic ablative radiation doses very rapidly. Bill Loo and Peter Maxim have received Honoraria from Varian and Research Support from Varian and RaySearch.« less

Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers.

PubMed

Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry

2016-08-01

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type. Copyright © 2016 Elsevier Ltd. All rights reserved.

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium. PMID:23288986

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium.

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

PubMed Central

Kaneko, Shun; Kakinuma, Sei; Asahina, Yasuhiro; Kamiya, Akihide; Miyoshi, Masato; Tsunoda, Tomoyuki; Nitta, Sayuri; Asano, Yu; Nagata, Hiroko; Otani, Satoshi; Kawai-Kitahata, Fukiko; Murakawa, Miyako; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Nakauchi, Hiromitsu; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada; Iwamoto, Masashi; Watashi, Koichi; Wakita, Takaji; Watanabe, Mamoru

2016-01-01

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies. PMID:27386799

Static feed water electrolysis subsystem development

NASA Technical Reports Server (NTRS)

Schubert, Franz H. (Inventor); Grigger, David J. (Inventor)

1991-01-01

This disclosure is directed to an electrolysis cell forming hydrogen and oxygen at spaced terminals. The anode terminal is porous and able to form oxygen within the cell and permit escape of the gaseous oxygen through the anode and out through a flow line in the presence of backpressure. Hydrogen is liberated in the cell at the opposing solid metal cathode which is permeable to hydrogen but not oxygen so that the migratory hydrogen formed in the cell is able to escape from the cell. The cell is maintained at an elevated pressure so that oxygen liberated by the cell is delivered at elevated pressure without pumping to raise the pressure of the oxygen.

The expression of cancer stem cell markers in human colorectal carcinoma cells in a microenvironment dependent manner.

PubMed

Stankevicius, Vaidotas; Kunigenas, Linas; Stankunas, Edvinas; Kuodyte, Karolina; Strainiene, Egle; Cicenas, Jonas; Samalavicius, Narimantas E; Suziedelis, Kestutis

2017-03-18

Numerous lines of evidence support the hierarchical model of cancer development and tumor initiation. According to the theory, cancer stem cells play a crucial role in the formation of the tumor and should be targeted for more effective anticancer treatment. However, cancer stem cells quickly loose their characteristics when propagated as 2D cell culture, indicating that the 2D cell culture does not provide the appropriate settings to maintain an in vivo environment. In this study we have investigated the expression of self-renewal, cancer stem cell and epithelial to mesenchymal transition markers after the transfer of human colorectal carcinoma cell DLD1 and HT29 lines from 2D cell cultures to scaffold-attached laminin rich extracellular matrix and scaffold-free multicellular spheroid 3D culture models. Based on the up-regulated expression of multipotency, CSC and EMT markers, our data suggests that human colorectal carcinoma cells grown in 3D exhibit enhanced cancer stem cell characteristics. Therefore, in order to design more efficient targeted therapies, we suggest that 3D cell culture models should be employed in cancer stem cell research. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells.

PubMed

Farnell, Yuhua Z; Ing, Nancy H

2003-03-01

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

Cisplatin-induced Casepase-3 activation in different tumor cells

NASA Astrophysics Data System (ADS)

Shi, Hua; Li, Xiao; Su, Ting; Zhang, Yu-Hai

2008-12-01

Apoptosis plays an essential role in normal organism development which is one of the main types of programmed cell death to help tissues maintain homeostasis. Defective apoptosis can result in cell accumulation and therefore effects on tumor pathogenesis, progression and therapy resistance. A family of proteins, known as caspases, is typically activated in the early stages of apoptosis. Therefore, studying the kinetics of activation of caspases induced by antitumor drugs can contribute to antitumor drug discovery and explanation of the molecular mechanisms. This paper detected the Caspase-3 activity induced by cisplatin in human adenoid cystic carcinoma cell line (ACC-M), human hepatocellular liver carcinoma cell line (HepG2) and human epithelial carcinoma cell line (Hela) with stably expressing ECFP-DEVDDsRed (CD3) probe, a fluorescent probe consisting of Enhanced Cyan Fluorescent Protein (ECFP), red fluorescent protein (DsRed) and a linker with a recognition site of Caspase-3, by using the capillary electrophoresis (CE) and fluorescence resonance energy transfer (FRET) imaging system. Under the same concentration of cisplatin, ACC-M cells responded the most rapidly, and then HepG2 cells and Hela cells, respectively, in the early 30 hours. Later, HepG2 cells represented acceleration in the Caspase-3 activation speed and reached full activation the earliest comparing to other two cell types. The results demonstrated that ACC-M cell is more sensitive than the other two cell types under the treatment of cisplatin.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

PubMed Central

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A.; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A.; Mueller, Irina A.; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M.; Gunawardane, Ruwanthi N.

2017-01-01

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. PMID:28814507

Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni.

PubMed

Rinaldi, Gabriel; Yan, Hongbin; Nacif-Pimenta, Rafael; Matchimakul, Pitchaya; Bridger, Joanna; Mann, Victoria H; Smout, Michael J; Brindley, Paul J; Knight, Matty

2015-07-01

The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transformation, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25°C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (<48 h in 5 μg/ml) facilitated use of this antibiotic, together with the cognate resistance gene (puromycin N-acetyl-transferase) for selection of Bge cells transformed with the PAC gene (puroR). Bge cells transfected with a plasmid encoding puroR were partially rescued when cultured in the presence of 5 μg/ml of puromycin. These findings pave the way for the development of functional genomic tools applied to the host-parasite interaction during schistosomiasis and neglected tropical trematodiases at large. Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Cytological characterization of a thermo-sensitive cytoplasmic male-sterile wheat line having K-type cytoplasm of Aegilops kotschyi.

PubMed

Meng, Liying; Liu, Zihan; Zhang, Lingli; Hu, Gan; Song, Xiyue

2016-12-01

Male sterility is an important tool for obtaining crop heterosis. A thermo-sensitive cytoplasmic male-sterile (TCMS) line was developed recently using a new method based on tiller regeneration. In the present study, we explored the critical growth stages required to maintain thermo-sensitive male sterility in TCMS lines and found that fertility is associated with abnormal tapetal and microspore development. We investigated the fertility and cytology of temperature-treated plant anthers at various developmental stages. TCMS line KTM3315A exhibited thermo-sensitive male sterility in Zadoks growth stages 41-49 and 58-59. Morphologically, the line exhibited thermo-sensitive male sterility at 3-9 days before heading and at 3-6 days before flowering, and it was partially restored in three locations during spring and summer. TCMS line KTM3315A plants exhibited premature tapetal programmed cell death (PCD) from the early uninucleate stage of microspore development until the tapetal cells degraded completely. Microspore development was then blocked and the pollen abortion type was stainable abortion. Thus, male fertility in the line KTM3315A is sensitive to temperature and premature tapetal PCD is the main cause of pollen abortion, where it determines the starting period and affects male fertility conversion in K-type TCMS lines at certain temperatures.

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

PubMed Central

Johnson, Laura A.; Davis, Jeremy L.; Zheng, Zhili; Woolard, Kevin D.; Reap, Elizabeth A.; Feldman, Steven A.; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A.; Rosenberg, Steven A.

2012-01-01

Abstract No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application. PMID:22780919

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

PubMed

Morgan, Richard A; Johnson, Laura A; Davis, Jeremy L; Zheng, Zhili; Woolard, Kevin D; Reap, Elizabeth A; Feldman, Steven A; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A; Rosenberg, Steven A

2012-10-01

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Evidence for idiotypic- and antiidiotypic B-B cellular interaction with the use of cloned antiidiotypic B cell line.

PubMed

Bitoh, S; Fujimoto, S; Yamamoto, H

1990-03-15

Immunization of BALB/c mice with MOPC104E myeloma protein induces antiidiotypic B lymphocytes that have Id-specific enhancing activity on antibody production. The B-B cell interaction was restricted to both Igh and class II MHC. However, anti-Thy-1 and C-treated splenic B cells were maintained for more than 1 y in a mixture of Con A-stimulated splenocyte culture supernatant and synthetic medium. In applying the long term culture method, we have established a cloned B cell line named B19-1d, B19-1d cells are specific to MOPC104E or J558 cross-reactive Id and they express surface mu, lambda but no Ly-1. B19-1d do not spontaneously secrete Ig but produce them upon stimulation with bacterial LPS. The effect of B19-1d cell line on idiotypic antibody production was tested. Addition of only 10 to 100 B19-1d cells into dextran-immune B cell culture greatly enhanced the Id+ antidextran antibody responses. On the contrary, the antidextran antibody production was suppressed by the higher doses of B19-1d cells. The effective cooperation between dextran-immune B cells and B19-1d cloned B cells was restricted to class II MHC. The role of idiotypic- and antiidiotypic B-B cell interaction in immune regulation and repertoire generation was suggested.

Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

PubMed

Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2012-06-01

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Low-temperature incubation using a water supply

USGS Publications Warehouse

Wolf, K.; Quimby, M.C.

1967-01-01

Cell and tissue culture has been concerned primarily with homiothermic vertebrate cells which require incubation at about 37 C, and there is a great variety of incubators designed to maintain temperatures which are usually above ambient. The culture of poikilothermic vertebrate cells--and invertebrate, plant, and some microbial cells--can often be carried out at ambient temperatures, but for some work cooler conditions must be provided. Variety among the so-called low-temperature incubators is somewhat restricted; there are no small units, and all require a power source to maintain temperatures below ambient. We have used a gravity-fed water supply for 5 years to provide trouble-free, constant, low-temperature incubation of stock cultures of fish and amphibian cells. Though it is but a small part of our low-temperature incubator capacity, it has no power requirements and it provides maximal protection against temperature rises which could be lethal to some of the cell lines. Though the system has limitations, there is a considerable likelihood that the domestic water supply in other laboratories can also be used to provide low-temperature incubation.

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology.

PubMed

Hu, Jianwen; Han, Jizhong; Li, Haoran; Zhang, Xian; Liu, Lan Lan; Chen, Fei; Zeng, Bin

2018-01-01

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications. © 2018 S. Karger AG, Basel.

The SH-SY5Y cell line in Parkinson's disease research: a systematic review.

PubMed

Xicoy, Helena; Wieringa, Bé; Martens, Gerard J M

2017-01-24

Parkinson's disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the molecular and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, are difficult to obtain and maintain as primary cells, current PD research is mostly performed with permanently established neuronal cell models, in particular the neuroblastoma SH-SY5Y lineage. This cell line is frequently chosen because of its human origin, catecholaminergic (though not strictly dopaminergic) neuronal properties, and ease of maintenance. However, there is no consensus on many fundamental aspects that are associated with its use, such as the effects of culture media composition and of variations in differentiation protocols. Here we present the outcome of a systematic review of scientific articles that have used SH-SY5Y cells to explore PD. We describe the cell source, culture conditions, differentiation protocols, methods/approaches used to mimic PD and the preclinical validation of the SH-SY5Y findings by employing alternative cellular and animal models. Thus, this overview may help to standardize the use of the SH-SY5Y cell line in PD research and serve as a future user's guide.

A phenotypic screening approach to identify anticancer compounds derived from marine fungi.

PubMed

Ellinger, Bernhard; Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F

2014-04-01

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

PubMed

Granja, Sara; Marchiq, Ibtissam; Le Floch, Renaud; Moura, Conceição Souto; Baltazar, Fátima; Pouysségur, Jacques

2015-03-30

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40.

PubMed Central

Niesor, E J; Wollheim, C B; Mintz, D H; Blondel, B; Renold, A E; Weil, R

1979-01-01

The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis. Images Fig. 1. PMID:222255

Genetic variability of oxalate oxidase activity and elongation in water-stressed primary roots of diverse maize and rice lines

USDA-ARS?s Scientific Manuscript database

Previous work on maize primary roots under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. In association with these responses, several proteins related to reactive oxygen species (ROS) production, part...

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Factors influencing the abundance of the side population in a human myeloma cell line.

PubMed

Mo, Sui-Lin; Li, Jia; Loh, Yen S; Brown, Ross D; Smith, Adrian L; Chen, Yuling; Joshua, Douglas; Roufogalis, Basil D; Li, George Q; Fan, Kei; Ng, Michelle C H; Sze, Daniel Man-Yuen

2011-01-01

Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research.

The Hippo Pathway Regulates Homeostatic Growth of Stem Cell Niche Precursors in the Drosophila Ovary

PubMed Central

Sarikaya, Didem P.; Extavour, Cassandra G.

2015-01-01

The Hippo pathway regulates organ size, stem cell proliferation and tumorigenesis in adult organs. Whether the Hippo pathway influences establishment of stem cell niche size to accommodate changes in organ size, however, has received little attention. Here, we ask whether Hippo signaling influences the number of stem cell niches that are established during development of the Drosophila larval ovary, and whether it interacts with the same or different effector signaling pathways in different cell types. We demonstrate that canonical Hippo signaling regulates autonomous proliferation of the soma, while a novel hippo-independent activity of Yorkie regulates autonomous proliferation of the germ line. Moreover, we demonstrate that Hippo signaling mediates non-autonomous proliferation signals between germ cells and somatic cells, and contributes to maintaining the correct proportion of these niche precursors. Finally, we show that the Hippo pathway interacts with different growth pathways in distinct somatic cell types, and interacts with EGFR and JAK/STAT pathways to regulate non-autonomous proliferation of germ cells. We thus provide evidence for novel roles of the Hippo pathway in establishing the precise balance of soma and germ line, the appropriate number of stem cell niches, and ultimately regulating adult female reproductive capacity. PMID:25643260

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

PubMed Central

Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

2009-01-01

Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. Reviewers This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter. PMID:19715575

Establishment and characterization of a new human functional cell line from a choriocarcinoma.

PubMed

Okabe, T; Sasaki, N; Matsuzaki, M; Imai, Y; Kaneko, Y; Matsuzaki, F; Takaku, F; Tsushima, T

1983-10-01

A new human functional tumor cell line, designated as T3M-3, has been established from a xenotransplanted choriocarcinoma grown in nude mice. One of the biggest problems of the in vitro culture of these tumor cells using the xenotransplanted tumors had been the dense contamination of fibroblasts of host nude mouse origin. In the present study, these fibroblasts were completely removed by incubating the cells with antiserum raised against nude mouse spleen cells. The cell line established from the remaining tumor cells has been successfully propagated in vitro for as long as 4 years. These cells show the morphology of epithelioid cells containing a prominent nucleus with one or two large nucleoli. The cells grow in a monolayered sheet with the population-doubling time of 19 hr. The cells show perfect tumor takes when they are reinoculated into nude mice. Chromosomal analysis revealed that the cell is a human aneuploid one with a hypotriploid mode. These cultured cells maintained well the function of secreting large amounts of human chorionic gonadotropin, progesterone, and estrogen. The secretion of human chorionic gonadotropin and progesterone by these cells is enhanced by stimulation with tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin B, or with epidermal growth factor in a dose-and time-dependent manner. Interestingly, however, the tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor, indicating that the receptors for these reagents in T3M-3 cells are not shared by epidermal growth factor.

Mitochondrial functionality in female reproduction.

PubMed

Gąsior, Łukasz; Daszkiewicz, Regina; Ogórek, Mateusz; Polański, Zbigniew

2017-01-04

In most animal species female germ cells are the source of mitochondrial genome for the whole body of individuals. As a source of mitochondrial DNA for future generations the mitochondria in the female germ line undergo dynamic quantitative and qualitative changes. In addition to maintaining the intact template of mitochondrial genome from one generation to another, mitochondrial role in oocytes is much more complex and pleiotropic. The quality of mitochondria determines the ability of meiotic divisions, fertilization ability, and activation after fertilization or sustaining development of a new embryo. The presence of normal number of functional mitochondria is also crucial for proper implantation and pregnancy maintaining. This article addresses issues of mitochondrial role and function in mammalian oocyte and presents new approaches in studies of mitochondrial function in female germ cells.

Device For Trapping Laser Pulses In An Optical Delay Line

DOEpatents

Yu, David U. L.; Bullock, Donald L.

1997-12-23

A device for maintaining a high-energy laser pulse within a recirculating optical delay line for a period time to optimize the interaction of the pulse with an electron beam pulse train comprising closely spaced electron micropulses. The delay line allows a single optical pulse to interact with many of the electron micropulses in a single electron beam macropulse in sequence and for the introduction of additional optical pulses to interact with the micropulses of additional electron beam macropulses. The device comprises a polarization-sensitive beam splitter for admitting an optical pulse to and ejecting it from the delay line according to its polarization state, a Pockels cell to control the polarization of the pulse within the delay line for the purpose of maintaining it within the delay line or ejecting it from the delay line, a pair of focusing mirrors positioned so that a collimated incoming optical pulse is focused by one of them to a focal point where the pulse interacts with the electron beam and then afterwards the pulse is recollimated by the second focusing mirror, and a timing device which synchronizes the introduction of the laser pulse into the optical delay line with the arrival of the electron macropulse at the delay line to ensure the interaction of the laser pulse with a prescribed number of electron micropulses in sequence. In a first embodiment of the invention, the principal optical elements are mounted with their axes collinear. In a second embodiment, all principal optical elements are mounted in the configuration of a ring.

Characterization of immortalized dairy goat male germline stem cells (mGSCs).

PubMed

Zhu, Haijing; Ma, Jing; Du, Rui; Zheng, Liming; Wu, Jiang; Song, Wencong; Niu, Zhiwei; He, Xin; Du, Enqi; Zhao, Shanting; Hua, Jinlian

2014-09-01

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs. © 2014 Wiley Periodicals, Inc.

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

PubMed

Roy, Gargi; Martin, Tom; Barnes, Arnita; Wang, Jihong; Jimenez, Rod Brian; Rice, Megan; Li, Lina; Feng, Hui; Zhang, Shu; Chaerkady, Raghothama; Wu, Herren; Marelli, Marcello; Hatton, Diane; Zhu, Jie; Bowen, Michael A

2018-04-01

The conserved glycosylation site Asn 297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn 297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

Angiocrine functions of organ-specific endothelial cells

PubMed Central

Rafii, Shahin; Butler, Jason M; Ding, Bi-Sen

2016-01-01

Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

Ethanol reduces amyloid aggregation in vitro and prevents toxicity in cell lines.

PubMed

Ormeño, David; Romero, Fernando; López-Fenner, Julio; Avila, Andres; Martínez-Torres, Ataulfo; Parodi, Jorge

2013-01-01

Alzheimer's disease (AD) alters cognitive functions. A mixture of soluble β-amyloid aggregates (Aβ) are known to act as toxic agents. It has been suggested that moderate alcohol intake reduces the development of neurodegenerative diseases, but the molecular mechanisms leading to this type of prevention have been elusive. We show the ethanol effect in the generation of complex Aβ in vitro and the impact on the viability of two cell lines. The effect of ethanol on the kinetics of β-amyloid aggregation in vitro was assessed by turbimetry. Soluble- and ethanol-treated β-amyloid were added to the cell lines HEK and PC-12 to compare their effects on metabolic activity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In addition, we used molecular modeling to assess the impact of exposure to ethanol on the structure of β-amyloid. Exposure to soluble β-amyloid was toxic to both cell lines; however, exposing the cells to β-amyloid aggregated in 10 mmol ethanol prevented the effect. In silico modeling suggested that ethanol alters the dynamics for assembling Aβ by disrupting a critical salt bridge between residues Asp 23 and Lys 28, required for amyloid dimerization. Thus, ethanol prevented the formation of complex short (∼100 nm) Aβ, which are related to higher cell toxicity. Ethanol prevents the formation of stable Aβ dimers in vitro, thus protecting the cells maintained in culture. Accordingly, in silico modelling predicts that soluble β-amyloid molecules do not form stable multimers when exposed to ethanol. Copyright © 2013 IMSS. Published by Elsevier Inc. All rights reserved.

Transcriptional profiling reveals elevated Sox2 in DNA polymerase ß null mouse embryonic fibroblasts

PubMed Central

Li, Jianfeng; Luthra, Soumya; Wang, Xiao-Hong; Chandran, Uma R; Sobol, Robert W

2012-01-01

There are over 150 human proteins that have been categorized as bona fide DNA repair proteins. These DNA repair proteins maintain the integrity of the genome, reducing the onset of cancer, disease and aging phenotypes. Variations in expression and/or function would therefore impact genome integrity as well as the cellular response to genotoxins. Global gene expression analysis is an effective approach to uncover defects in DNA repair gene expression and to discover cellular and/or organismal effects brought about by external stimuli such as environmental genotoxicants, chemotherapeutic regimens, viral infections as well as developmental and age-related stimuli. Given the significance of genome stability in cell survival and response to stimuli, we have hypothesized that cells may undergo transcriptional re-programming to accommodate defects in basal DNA repair capacity to promote survival. As a test of this hypothesis, we have compared the transcriptome in three DNA polymerase ß knockout (Polß-KO) mouse embryonic fibroblasts (MEFs) and the corresponding wild-type (WT) littermate control cell lines. Each Polß-KO cell line was found to have a range of genes up-regulated, when compared to its WT littermate control cell line. Interestingly, six (6) genes were commonly up regulated in all three Polß-KO cell lines, including Sox2, one of several genes associated with the induction of pluripotent stem cells. Herein, we present these findings and suggest that loss of DNA repair and the induction of cellular transcriptional re-programming may, in part, contribute to tumor formation and the cellular response to external stimuli. PMID:23226616

Read margin analysis of crossbar arrays using the cell-variability-aware simulation method

NASA Astrophysics Data System (ADS)

Sun, Wookyung; Choi, Sujin; Shin, Hyungsoon

2018-02-01

This paper proposes a new concept of read margin analysis of crossbar arrays using cell-variability-aware simulation. The size of the crossbar array should be considered to predict the read margin characteristic of the crossbar array because the read margin depends on the number of word lines and bit lines. However, an excessively high-CPU time is required to simulate large arrays using a commercial circuit simulator. A variability-aware MATLAB simulator that considers independent variability sources is developed to analyze the characteristics of the read margin according to the array size. The developed MATLAB simulator provides an effective method for reducing the simulation time while maintaining the accuracy of the read margin estimation in the crossbar array. The simulation is also highly efficient in analyzing the characteristic of the crossbar memory array considering the statistical variations in the cell characteristics.

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs).

PubMed

Shi, Huaiping; Shi, Hengbo; Luo, Jun; Wang, Wei; Haile, Abiel B; Xu, Huifen; Li, Jun

2014-07-01

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and 'S' sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n=60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P<0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. © 2014 Japanese Society of Animal Science.

Epigenetic Silencing of the Proapoptotic Gene BIM in Anaplastic Large Cell Lymphoma through an MeCP2/SIN3a Deacetylating Complex12

PubMed Central

Piazza, Rocco; Magistroni, Vera; Mogavero, Angela; Andreoni, Federica; Ambrogio, Chiara; Chiarle, Roberto; Mologni, Luca; Bachmann, Petra S; Lock, Richard B; Collini, Paola; Pelosi, Giuseppe; Gambacorti-Passerini, Carlo

2013-01-01

BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation. PMID:23633923

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

PubMed

Yang, William C; Lu, Jiuyi; Kwiatkowski, Chris; Yuan, Hang; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

2014-01-01

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers.

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

PubMed

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A; Mueller, Irina A; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M; Gunawardane, Ruwanthi N

2017-10-15

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. © 2017 Roberts, Haupt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Is ABA involved in tolerance responses to salinity by affecting cytoplasm ion homeostasis in rice cell lines?

PubMed

Pons, Raül; Cornejo, María Jesús; Sanz, Amparo

2013-01-01

The ability of plant cells to maintain cytoplasm ion homeostasis under saline stress is among the main mechanisms involved in salt tolerance. To cope with excess Na(+), cells extrude it from the cytoplasm, which requires expenditure of metabolic energy, provided by H(+) gradients generated by membrane-bound H(+)-pumps. ABA is well-known to be involved in physiological processes elicited or enhanced by stresses causing cell dehydration. In this work we studied the possible implication of this plant hormone in the control of salt-induced cellular mechanisms conducting to Na(+) extrusion from the cytoplasm. We used rice (Oryza sativa L.) cell lines selected for their different tolerance to salinity to measure the response to ABA of H(+)-pumps and Na(+)/H(+)-antiporters associated to the plasma membrane and the tonoplast. Our results show that ABA generally enhances H(+)-pumping under salt stress but not under control conditions. This effect occurs to a higher extent across the tonoplast in the more tolerant lines (L-T). Na(+)/H(+) antiport activity is practically undetectable in calli under control conditions, pre-treated or not with ABA, but shows a strong activation under salinity across the tonoplast, particularly in L-T lines (cv Bahia) and also across de plasma membrane in cv Bomba. In these lines, prior treatments with ABA tend to reduce the NaCl enhanced activity of both antiporters. Overall, under saline conditions ABA seems to affect synergistically H(+) pumping and antagonistically Na(+) extrusion. A complex network of positive and negative regulatory signals seems involved in restoring ion cell homeostasis under salt stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

PubMed

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

PubMed Central

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

PubMed

Ackermann, Amanda M; Zhang, Jia; Heller, Aryel; Briker, Anna; Kaestner, Klaus H

2017-03-01

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER T2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. We utilized CRISPR-Cas9 technology to insert an IRES-CreER T2 sequence into the 3' UTR of the Glucagon ( Gcg ) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER T2 mice. Recombination efficiency in GCG + pancreatic α-cells and glucagon-like peptide 1 positive (GLP1 + ) enteroendocrine L-cells was measured in Gcg-CreER T2 ; Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Tamoxifen injection of Gcg-CreER T2 ; Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreER T2 allele were phenotypically normal. We successfully derived a Gcg-CreER T2 mouse line that expresses CreER T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Antitumor Activity of a Mesenchymal Stem Cell Line Stably Secreting a Tumor-Targeted TNF-Related Apoptosis-Inducing Ligand Fusion Protein

PubMed Central

Marini, Irene; Siegemund, Martin; Hutt, Meike; Kontermann, Roland E.; Pfizenmaier, Klaus

2017-01-01

Mesenchymal stem cells (MSCs) are currently exploited as gene delivery systems for transient in situ expression of cancer therapeutics. As an alternative to the prevailing viral expression, we here describe a murine MSC line stably expressing a therapeutic protein for up to 42 passages, yet fully maintaining MSC features. Because of superior antitumoral activity of hexavalent TNF-related apoptosis-inducing ligand (TRAIL) formats and the advantage of a tumor-targeted action, we choose expression of a dimeric EGFR-specific diabody single-chain TRAIL (Db-scTRAIL) as a model. The bioactivity of Db-scTRAIL produced from an isolated clone (MSC.TRAIL) was revealed from cell death induction in Colo205 cells treated with either culture supernatants from or cocultured with MSC.TRAIL. In vivo, therapeutic activity of MSC.TRAIL was shown upon peritumoral injection in a Colo205 xenograft tumor model. Best antitumor activity in vitro and in vivo was observed upon combined treatment of MSC.TRAIL with bortezomib. Importantly, in vivo combination treatment did not cause apparent hepatotoxicity, weight loss, or behavioral changes. The development of well characterized stocks of stable drug-producing human MSC lines has the potential to establish standardized protocols of cell-based therapy broadly applicable in cancer treatment. PMID:28553285

Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells.

PubMed

Kumakura, Shin-ichi; Tsutsui, Takeo W; Yagisawa, Junko; Barrett, J Carl; Tsutsui, Takeki

2005-04-01

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance.

PubMed

Hu, Hsien-Ming; Zhao, Xin; Kaushik, Swati; Robillard, Lilliane; Barthelet, Antoine; Lin, Kevin K; Shah, Khyati N; Simmons, Andy D; Raponi, Mitch; Harding, Thomas C; Bandyopadhyay, Sourav

2018-04-17

Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

PubMed Central

Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

2012-01-01

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

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

Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James

2004-12-23

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor bymore » expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.« less

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

NASA Astrophysics Data System (ADS)

Birsoy, Kıvanç; Possemato, Richard; Lorbeer, Franziska K.; Bayraktar, Erol C.; Thiru, Prathapan; Yucel, Burcu; Wang, Tim; Chen, Walter W.; Clish, Clary B.; Sabatini, David M.

2014-04-01

As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.

C. elegans GATA factors EGL-18 and ELT-6 function downstream of Wnt signaling to maintain the progenitor fate during larval asymmetric divisions of the seam cells.

PubMed

Gorrepati, Lakshmi; Thompson, Kenneth W; Eisenmann, David M

2013-05-01

The C. elegans seam cells are lateral epithelial cells arrayed in a single line from anterior to posterior that divide in an asymmetric, stem cell-like manner during larval development. These asymmetric divisions are regulated by Wnt signaling; in most divisions, the posterior daughter in which the Wnt pathway is activated maintains the progenitor seam fate, while the anterior daughter in which the Wnt pathway is not activated adopts a differentiated hypodermal fate. Using mRNA tagging and microarray analysis, we identified the functionally redundant GATA factor genes egl-18 and elt-6 as Wnt pathway targets in the larval seam cells. EGL-18 and ELT-6 have previously been shown to be required for initial seam cell specification in the embryo. We show that in larval seam cell asymmetric divisions, EGL-18 is expressed strongly in the posterior seam-fated daughter. egl-18 and elt-6 are necessary for larval seam cell specification, and for hypodermal to seam cell fate transformations induced by ectopic Wnt pathway overactivation. The TCF homolog POP-1 binds a site in the egl-18 promoter in vitro, and this site is necessary for robust seam cell expression in vivo. Finally, larval overexpression of EGL-18 is sufficient to drive expression of a seam marker in other hypodermal cells in wild-type animals, and in anterior hypodermal-fated daughters in a Wnt pathway-sensitized background. These data suggest that two GATA factors that are required for seam cell specification in the embryo independently of Wnt signaling are reused downstream of Wnt signaling to maintain the progenitor fate during stem cell-like divisions in larval development.

C. elegans GATA factors EGL-18 and ELT-6 function downstream of Wnt signaling to maintain the progenitor fate during larval asymmetric divisions of the seam cells

PubMed Central

Gorrepati, Lakshmi; Thompson, Kenneth W.; Eisenmann, David M.

2013-01-01

The C. elegans seam cells are lateral epithelial cells arrayed in a single line from anterior to posterior that divide in an asymmetric, stem cell-like manner during larval development. These asymmetric divisions are regulated by Wnt signaling; in most divisions, the posterior daughter in which the Wnt pathway is activated maintains the progenitor seam fate, while the anterior daughter in which the Wnt pathway is not activated adopts a differentiated hypodermal fate. Using mRNA tagging and microarray analysis, we identified the functionally redundant GATA factor genes egl-18 and elt-6 as Wnt pathway targets in the larval seam cells. EGL-18 and ELT-6 have previously been shown to be required for initial seam cell specification in the embryo. We show that in larval seam cell asymmetric divisions, EGL-18 is expressed strongly in the posterior seam-fated daughter. egl-18 and elt-6 are necessary for larval seam cell specification, and for hypodermal to seam cell fate transformations induced by ectopic Wnt pathway overactivation. The TCF homolog POP-1 binds a site in the egl-18 promoter in vitro, and this site is necessary for robust seam cell expression in vivo. Finally, larval overexpression of EGL-18 is sufficient to drive expression of a seam marker in other hypodermal cells in wild-type animals, and in anterior hypodermal-fated daughters in a Wnt pathway-sensitized background. These data suggest that two GATA factors that are required for seam cell specification in the embryo independently of Wnt signaling are reused downstream of Wnt signaling to maintain the progenitor fate during stem cell-like divisions in larval development. PMID:23633508

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein.

PubMed

Zeng, Zhao-Jun; Johansson, Erik; Hayashi, Amiko; Chavali, Pavithra L; Akrap, Nina; Yoshida, Takeshi; Kohno, Kimitoshi; Izumi, Hiroto; Funa, Keiko

2012-06-15

TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein

PubMed Central

Zeng, Zhao-jun; Johansson, Erik; Hayashi, Amiko; Chavali, Pavithra L.; Akrap, Nina; Yoshida, Takeshi; Kohno, Kimitoshi; Izumi, Hiroto; Funa, Keiko

2012-01-01

Summary TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis. PMID:23213445

Fabricating microfluidic valve master molds in SU-8 photoresist

NASA Astrophysics Data System (ADS)

Dy, Aaron J.; Cosmanescu, Alin; Sluka, James; Glazier, James A.; Stupack, Dwayne; Amarie, Dragos

2014-05-01

Multilayer soft lithography has become a powerful tool in analytical chemistry, biochemistry, material and life sciences, and medical research. Complex fluidic micro-circuits require reliable components that integrate easily into microchips. We introduce two novel approaches to master mold fabrication for constructing in-line micro-valves using SU-8. Our fabrication techniques enable robust and versatile integration of many lab-on-a-chip functions including filters, mixers, pumps, stream focusing and cell-culture chambers, with in-line valves. SU-8 created more robust valve master molds than the conventional positive photoresists used in multilayer soft lithography, but maintained the advantages of biocompatibility and rapid prototyping. As an example, we used valve master molds made of SU-8 to fabricate PDMS chips capable of precisely controlling beads or cells in solution.

The Pathway Analysis of Micrornas Regulated Drug-Resistant Responses in HeLa Cells.

PubMed

Yang, Yubo; Dai, Cuihong; Cai, Zhipeng; Hou, Aiju; Cheng, Dayou; Wu, Guanying; Li, Jing; Cui, Jie; Xu, Dechang

2016-03-01

Chemotherapy is the main strategy in the treatment of cancer; however, the development of drug-resistance is the obstacle in long-term treatment of cervical cancer. Cisplatin is one of the most common drugs used in cancer therapy. Recently, accumulating evidence suggests that miRNAs are involved in various bioactivities in oncogenesis. It is not unexpected that miRNAs play a key role in acquiring of drug-resistance in the progression of tumor. In this study, we induced and maintained four levels of cisplatin-resistant HeLa cell lines (HeLa/CR1, HeLa/CR2, HeLa/CR3, and HeLa/CR4). According to the previous studies and existing evidence, we selected five miRNAs (miR-183, miR-182, miR-30a, miR-15b, and miR-16) and their potential target mRNAs as our research targets. The real-time RT-PCR was adopted to detect the relative expression of miRNAs and their mRNAs. The results show that miR-182 and miR-15b were up-regulated in resistant cell lines, while miR-30a was significantly down-regulated. At the same time, their targets are related to drug resistance. Compared to their parent HeLa cell line, the expression of selected miRNAs in resistant cell lines altered. The alteration suggests that HeLa cell drug resistance is associated with distinct miRNAs, which indicates that miRNAs may be one of the therapy targets in the treatment of cervical cancer by sensitizing cell to chemotherapy. We suggested a possible network diagram based on the existing theory and the preliminary results of candidate miRNAs and their targets in HeLa cells during development of drug resistance.

Cytological characterization of a thermo-sensitive cytoplasmic male-sterile wheat line having K-type cytoplasm of Aegilops kotschyi

PubMed Central

Meng, Liying; Liu, Zihan; Zhang, Lingli; Hu, Gan; Song, Xiyue

2016-01-01

Male sterility is an important tool for obtaining crop heterosis. A thermo-sensitive cytoplasmic male-sterile (TCMS) line was developed recently using a new method based on tiller regeneration. In the present study, we explored the critical growth stages required to maintain thermo-sensitive male sterility in TCMS lines and found that fertility is associated with abnormal tapetal and microspore development. We investigated the fertility and cytology of temperature-treated plant anthers at various developmental stages. TCMS line KTM3315A exhibited thermo-sensitive male sterility in Zadoks growth stages 41–49 and 58–59. Morphologically, the line exhibited thermo-sensitive male sterility at 3–9 days before heading and at 3–6 days before flowering, and it was partially restored in three locations during spring and summer. TCMS line KTM3315A plants exhibited premature tapetal programmed cell death (PCD) from the early uninucleate stage of microspore development until the tapetal cells degraded completely. Microspore development was then blocked and the pollen abortion type was stainable abortion. Thus, male fertility in the line KTM3315A is sensitive to temperature and premature tapetal PCD is the main cause of pollen abortion, where it determines the starting period and affects male fertility conversion in K-type TCMS lines at certain temperatures. PMID:28163591

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.

Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?

PubMed Central

Saas, P; Walker, P R; Hahne, M; Quiquerez, A L; Schnuriger, V; Perrin, G; French, L; Van Meir, E G; de Tribolet, N; Tschopp, J; Dietrich, P Y

1997-01-01

Astrocytomas are among the most common brain tumors that are usually fatal in their malignant form. They appear to progress without significant impedance from the immune system, despite the presence of intratumoral T cell infiltration. To date, this has been thought to be the result of T cell immunosuppression induced by astrocytoma-derived cytokines. Here, we propose that cell contact-mediated events also play a role, since we demonstrate the in vivo expression of Fas ligand (FasL/CD95L) by human astrocytoma and the efficient killing of Fas-bearing cells by astrocytoma lines in vitro and by tumor cells ex vivo. Functional FasL is expressed by human, mouse, and rat astrocytoma and hence may be a general feature of this nonlymphoid tumor. In the brain, astrocytoma cells can potentially deliver a death signal to Fas+ cells which include infiltrating leukocytes and, paradoxically, astrocytoma cells themselves. The expression of FasL by astrocytoma cells may extend the processes that are postulated to occur in normal brain to maintain immune privilege, since we also show FasL expression by neurons. Overall, our findings suggest that FasL-induced apoptosis by astrocytoma cells may play a significant role in both immunosuppression and the regulation of tumor growth within the central nervous system. PMID:9077524

Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288.

PubMed

Heng, Shuangping; Wei, Chao; Jing, Bing; Wan, Zhengjie; Wen, Jing; Yi, Bin; Ma, Chaozhi; Tu, Jinxing; Fu, Tingdong; Shen, Jinxiong

2014-04-30

Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288. Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line "J163-4" are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity. The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

PubMed

Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant

PubMed Central

Aspe, Jonathan R.; Diaz Osterman, Carlos J.; Jutzy, Jessica M.S.; Deshields, Simone; Whang, Sonia; Wall, Nathan R.

2014-01-01

Background Current therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP) protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A) has been shown to block Survivin, inducing caspase activation and apoptosis. Methods In this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2) cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work. Results Here we show that exosomes collected in the absence of tetracycline (tet-off) from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines. Conclusion This exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas. PMID:24624263

Continuous cell lines from the Muscovy duck as potential replacement for primary cells in the production of avian vaccines.

PubMed

Jordan, Ingo; John, Katrin; Höwing, Kristin; Lohr, Verena; Penzes, Zoltán; Gubucz-Sombor, Erzsébet; Fu, Yan; Gao, Peng; Harder, Timm; Zádori, Zoltán; Sandig, Volker

2016-01-01

Veterinary vaccines contribute to food security, interrupt zoonotic transmissions, and help to maintain overall health in livestock. Although vaccines are usually cost-effective, their adoption depends on a multitude of factors. Because poultry vaccines are usually given to birds with a short life span, very low production cost per dose is one important challenge. Other hurdles are to ensure a consistent and reliable supply of very large number of doses, and to have flexible production processes to accommodate a range of different pathogens and dosage requirements. Most poultry vaccines are currently being produced on primary avian cells derived from chicken or waterfowl embryos. This production system is associated with high costs, logistic complexities, rigid intervals between harvest and production, and supply limitations. We investigated whether the continuous cell lines Cairina retina and CR.pIX may provide a substrate independent of primary cell cultures or embryonated eggs. Viruses examined for replication in these cell lines are strains associated with, or contained in vaccines against egg drop syndrome, Marek's disease, Newcastle disease, avian influenza, infectious bursal disease and Derzsy's disease. Each of the tested viruses required the development of unique conditions for replication that are described here and can be used to generate material for in vivo efficacy studies and to accelerate transfer of the processes to larger production volumes.

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status

PubMed Central

Floch, Renaud Le; Moura, Conceição Souto

2015-01-01

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function. To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via ‘Zinc-Finger Nucleases’. The three homozygous BSG−/− cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG−/− cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin. PMID:25894929

Time course field analysis of COMT-downregulated switchgrass: Lignification, recalcitrance, and rust susceptibility

DOE PAGES

Baxter, Holly L.; Mazarei, Mitra; Fu, Chunxiang; ...

2016-05-18

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38% more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2more » years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11-16% less lignin, 33-40% lower S/G (syringyl-to-guaiacyl) ratios, and 15-42% higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. Finally, these results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.« less

Time course field analysis of COMT-downregulated switchgrass: Lignification, recalcitrance, and rust susceptibility

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

Baxter, Holly L.; Mazarei, Mitra; Fu, Chunxiang

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38% more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2more » years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11-16% less lignin, 33-40% lower S/G (syringyl-to-guaiacyl) ratios, and 15-42% higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. Finally, these results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.« less

Possible involvement of persistent activity of the mammalian target of rapamycin pathway in the cisplatin resistance of AFP-producing gastric cancer cells.

PubMed

Kamata, Shigeyuki; Kishimoto, Takashi; Kobayashi, Soichi; Miyazaki, Masaru; Ishikura, Hiroshi

2007-07-01

AFP-producing gastric carcinoma (AFPGC) is a highly malignant variant of gastric cancer. An effective chemotherapy is needed to improve on the poor outcome of this disease. Survival signals activated by intracellular kinase networks could be involved in chemoresistance in malignant tumors. We investigated the role of a pivotal kinase pathway, the mammalian target of rapamycin complex 1 (mTORC1) pathway, in the effectiveness of chemotherapeutic agents in three AFPGC cell lines (GCIY, FU97 and Takigawa) as well as in four cell lines of conventional-type gastric carcinoma (CGC). AFPGC cells were generally resistant to multiple chemotherapeutic agents, including cisplatin, while CGC cells were generally sensitive. Downstream targets of mTORC1, including p70S6K and 4EBP1, were phosphorylated in all cell lines. Interestingly, cisplatin virtually abolished phosphorylation of p70S6K and 4EBP1 in CGC cells, while phosphorylation was maintained in cisplatin-treated AFPGC cells. The addition of rapamycin, an inhibitor of mTORC1, diminished the remaining activity of mTORC1 and significantly intensified the cytotoxic action of cisplatin in AFPGC cells. These results suggested that persistent activity of mTORC1 signals in cisplatin-treated AFPGC cells is involved in the mechanisms of cisplatin resistance in AFPGC. Finally, combined treatment of rapamycin and cisplatin significantly suppressed the subcutaneously implanted GCIY cells. In conclusion rapamycin may be a potential supplemental agent for the treatment of AFPGC when used in combination with cisplatin.

Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer.

PubMed

Witkiewicz, Agnieszka K; Borja, Nicholas A; Franco, Jorge; Brody, Jonathan R; Yeo, Charles J; Mansour, John; Choti, Michael A; McCue, Peter; Knudsen, Erik S

2015-06-30

Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance.

PubMed

Chen, J.; Goldsbrough, P. B.

1994-09-01

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.

Tumor-Specific Chromosome Mis-Segregation Controls Cancer Plasticity by Maintaining Tumor Heterogeneity

PubMed Central

Hu, Yuanjie; Ru, Ning; Xiao, Huasheng; Chaturbedi, Abhishek; Hoa, Neil T.; Tian, Xiao-Jun; Zhang, Hang; Ke, Chao; Yan, Fengrong; Nelson, Jodi; Li, Zhenzhi; Gramer, Robert; Yu, Liping; Siegel, Eric; Zhang, Xiaona; Jia, Zhenyu; Jadus, Martin R.; Limoli, Charles L.; Linskey, Mark E.; Xing, Jianhua; Zhou, Yi-Hong

2013-01-01

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers. PMID:24282558

Celecoxib enhances the radiosensitivity of HCT116 cells in a COX-2 independent manner by up-regulating BCCIP

PubMed Central

Xu, Xiao-Ting; Hu, Wen-Tao; Zhou, Ju-Ying; Tu, Yu

2017-01-01

It has been reported that celecoxib, a cyclooxygenase-2 (COX-2)-selective nonsteroidal anti-inflammatory drug (NSAID), regulates the radiosensitivity of several cancer cells. BCCIP (BRCA2 and CDKN1A interacting protein) plays a critical role in maintaining the critical functions of p53 in tumor suppression and response to therapy. However, whether the effect of celecoxib on the radiosensitivity of colorectal cancer (CRC) cells is dependent on BCCIP is largely unclear. In this study, we found that celecoxib enhanced the radiosensitivity of HeLa (a human cervical carcinoma cell line), A549 (a human lung carcinoma cell line), and HCT116 cells (a human CRC cells line). Among these cells, COX-2 expression was undetected in HCT116 cells. Treatment with celecoxib significantly increased BCCIP expression in COX-2 negative HCT116 cells. Knockdown of BCCIP obviously abrogated the enhanced radiosensitivity of HCT116 cells induced by celecoxib. A combination of celecoxib and irradiation treatment induced much more γ-H2AX foci formation, higher levels of radiation injury-related proteins phosphorylation, G2/M arrest, apoptosis, and p53 and p21 expression, and lower levels of Cyclin B1 in HCT116 cells than those in cells treated with irradiation alone. However, these changes were undetected in BCCIP-silenced HCT116 cells. Therefore, these data suggest that BCCIP gene may be a radiosensitivity-related gene in CRC. Celecoxib affects the functions of p53 and inhibits the recovery from the irradiation-induced injury by up-regulating the expression of BCCIP, and subsequently regulates the expressions of genes such as p21 and Cyclin B1 to enhance the radiosensitivity of HCT116 cells in a COX-2 independent manner. PMID:28386336

Conditioned medium: a new alternative for cryopreservation of equine umbilical cord mesenchymal stem cells.

PubMed

Maia, Leandro; Dias, Marianne Camargos; de Moraes, Carolina Nogueira; de Paula Freitas-Dell'Aqua, Camila; da Mota, Ligia S L Silveira; Santiloni, Valquíria; da Cruz Landim-Alvarenga, Fernanda

2017-03-01

Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process. © 2017 International Federation for Cell Biology.

The natural compound forskolin synergizes with dexamethasone to induce cell death in myeloma cells via BIM.

PubMed

Follin-Arbelet, Virginie; Misund, Kristine; Naderi, Elin Hallan; Ugland, Hege; Sundan, Anders; Blomhoff, Heidi Kiil

2015-08-26

We have previously demonstrated that activation of the cyclic adenosine monophosphate (cAMP) pathway kills multiple myeloma (MM) cells both in vitro and in vivo. In the present study we have investigated the potential of enhancing the killing of MM cell lines and primary MM cells by combining the cAMP-elevating compound forskolin with the commonly used MM therapeutic drugs melphalan, cyclophosphamide, doxorubicin, bortezomib and dexamethasone. We observed that forskolin potentiated the killing induced by all the tested agents as compared to treatment with the single agents alone. In particular, forskolin had a synergistic effect on the dexamethasone-responsive cell lines H929 and OM-2. By knocking down the proapoptotic BCL-2 family member BIM, we proved this protein to be involved in the synergistic induction of apoptosis by dexamethasone and forskolin. The ability of forskolin to maintain the killing of MM cells even at lower concentrations of the conventional agents suggests that forskolin may be used to diminish treatment-associated side effects. Our findings support a potential role of forskolin in combination with current conventional agents in the treatment of MM.

The natural compound forskolin synergizes with dexamethasone to induce cell death in myeloma cells via BIM

PubMed Central

Follin-Arbelet, Virginie; Misund, Kristine; Hallan Naderi, Elin; Ugland, Hege; Sundan, Anders; Kiil Blomhoff, Heidi

2015-01-01

We have previously demonstrated that activation of the cyclic adenosine monophosphate (cAMP) pathway kills multiple myeloma (MM) cells both in vitro and in vivo. In the present study we have investigated the potential of enhancing the killing of MM cell lines and primary MM cells by combining the cAMP-elevating compound forskolin with the commonly used MM therapeutic drugs melphalan, cyclophosphamide, doxorubicin, bortezomib and dexamethasone. We observed that forskolin potentiated the killing induced by all the tested agents as compared to treatment with the single agents alone. In particular, forskolin had a synergistic effect on the dexamethasone-responsive cell lines H929 and OM-2. By knocking down the proapoptotic BCL-2 family member BIM, we proved this protein to be involved in the synergistic induction of apoptosis by dexamethasone and forskolin. The ability of forskolin to maintain the killing of MM cells even at lower concentrations of the conventional agents suggests that forskolin may be used to diminish treatment-associated side effects. Our findings support a potential role of forskolin in combination with current conventional agents in the treatment of MM. PMID:26306624

Bone formation in vitro and in nude mice by human osteosarcoma cells.

PubMed

Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E

1995-01-01

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.

Protein-free culture of the human pancreatic cancer cell line, SUIT-2.

PubMed

Taniguchi, S; Iwamura, T; Kitamura, N; Yamanari, H; Kojima, A; Hidaka, K; Seguchi, K; Setoguchi, T

1994-12-01

A human pancreatic cancer cell line (SUIT-2), usually cultured in serum-supplemented medium (DMEM/FBS), was adapted to protein-free conditions using a 1:1 mixture of DMEM and Ham's F12 medium (DMEM/F12). The cells have been maintained in DMEM/F12 for more than 2 years, with over 50 passages. The SUIT-2 cells grew in DMEM/F12 with a doubling time of 35.7 h, which was similar to that in DMEM/FBS (35.0 h). The cellular morphology was similar in both media. Type IV collagenolytic activity was detected in the conditioned media from cells grown in DMEM/F12. The secretion of CEA and CA19-9 initially decreased in DMEM/F12. CEA was not detected after passage 5 (p5) but the concentration of CA19-9 did not decrease further after the first few serial passages in protein-free medium. Xenografts of SUIT-2 cells cultured in DMEM/F12 remained tumorigenic and could form metastatic tumors in nude mice. In conclusion, SUIT-2 cells grown in protein-free media continued to produce CA19-9 and type IV collagenase in vitro and formed metastatic tumors in vivo.

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

PubMed

Dani, Sergio U; Espindola, Rachel

2002-06-30

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

PubMed Central

Albright, Emily R.

2013-01-01

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34+ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34+ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34+ cells. However, in contrast to what occurs in primary CD34+ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34+ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed. PMID:23824798

Cellular homeostasis in fungi: impact on the aging process.

PubMed

Scheckhuber, Christian Q; Hamann, Andrea; Brust, Diana; Osiewacz, Heinz D

2012-01-01

Cellular quality control pathways are needed for maintaining the biological function of organisms. If these pathways become compromised, the results are usually highly detrimental. Functional impairments of cell components can lead to diseases and in extreme cases to organismal death. Dysfunction of cells can be induced by a number of toxic by-products that are formed during metabolic activity, like reactive oxygen and nitrogen species, for example. A key source of reactive oxygen species (ROS) are the organelles of oxidative phosphorylation, mitochondria. Therefore mitochondrial function is also directly affected by ROS, especially if there is a compromised ROS-scavenging capacity. Biological systems therefore depend on several lines of defence to counteract the toxic effects of ROS and other damaging agents. The first level is active at the molecular level and consists of various proteases that bind and degrade abnormally modified and / or aggregated mitochondrial proteins. The second level is concerned with maintaining the quality of whole mitochondria. Among the pathways of this level are mitochondrial dynamics and autophagy (mitophagy). Mitochondrial dynamics describes the time-dependent fusion and fission of mitochondria. It is argued that this kind of organellar dynamics has the power to restore the function of impaired organelles by content mixing with intact organelles. If the first and second lines of defence against damage fail and mitochondria become damaged too severely, there is the option to remove affected cells before they can elicit more damage to their surrounding environment by apoptosis. This form of programmed cell death is strictly regulated by a complex network of interacting components and can be divided into mitochondria-dependent and mitochondria-independent modes of action. In this review we give an overview on various biological quality control systems in fungi (yeasts and filamentous fungi) with an emphasis on autophagy (mitophagy) and apoptosis and how these pathways allow fungal organisms to maintain a balanced cellular homeostasis.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

PubMed

Fernando, Elizabeth H; Dicay, Michael; Stahl, Martin; Gordon, Marilyn H; Vegso, Andrew; Baggio, Cristiane; Alston, Laurie; Lopes, Fernando; Baker, Kristi; Hirota, Simon; McKay, Derek M; Vallance, Bruce; MacNaughton, Wallace K

2017-11-01

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner. Copyright © 2017 the American Physiological Society.

HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors.

PubMed

Weber, Maja; Knoefler, Ilka; Schleussner, Ekkehard; Markert, Udo R; Fitzgerald, Justine S

2013-01-01

JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Inflammatory memory sensitizes skin epithelial stem cells to tissue damage.

PubMed

Naik, Shruti; Larsen, Samantha B; Gomez, Nicholas C; Alaverdyan, Kirill; Sendoel, Ataman; Yuan, Shaopeng; Polak, Lisa; Kulukian, Anita; Chai, Sophia; Fuchs, Elaine

2017-10-26

The skin barrier is the body's first line of defence against environmental assaults, and is maintained by epithelial stem cells (EpSCs). Despite the vulnerability of EpSCs to inflammatory pressures, neither the primary response to inflammation nor its enduring consequences are well understood. Here we report a prolonged memory to acute inflammation that enables mouse EpSCs to hasten barrier restoration after subsequent tissue damage. This functional adaptation does not require skin-resident macrophages or T cells. Instead, EpSCs maintain chromosomal accessibility at key stress response genes that are activated by the primary stimulus. Upon a secondary challenge, genes governed by these domains are transcribed rapidly. Fuelling this memory is Aim2, which encodes an activator of the inflammasome. The absence of AIM2 or its downstream effectors, caspase-1 and interleukin-1β, erases the ability of EpSCs to recollect inflammation. Although EpSCs benefit from inflammatory tuning by heightening their responsiveness to subsequent stressors, this enhanced sensitivity probably increases their susceptibility to autoimmune and hyperproliferative disorders, including cancer.

Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

PubMed

Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

2014-01-15

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. © 2013 Elsevier Inc. All rights reserved.

High-Resolution Dual-Comb Spectroscopy with Ultra-Low Noise Frequency Combs

NASA Astrophysics Data System (ADS)

Hänsel, Wolfgang; Giunta, Michele; Beha, Katja; Perry, Adam J.; Holzwarth, R.

2017-06-01

Dual-comb spectroscopy is a powerful tool for fast broad-band spectroscopy due to the parallel interrogation of thousands of spectral lines. Here we report on the spectroscopic analysis of acetylene vapor in a pressurized gas cell using two ultra-low noise frequency combs with a repetition rate around 250 MHz. Optical referencing to a high-finesse cavity yields a sub-Hertz stability of all individual comb lines (including the virtual comb lines between 0 Hz and the carrier) and permits one to pick a small difference of repetition rate for the two frequency combs on the order of 300 Hz, thus representing an optical spectrum of 100 THz (˜3300 \\wn) within half the free spectral range (125 MHz). The transmission signal is derived straight from a photodetector and recorded with a high-resolution spectrum analyzer or digitized with a computer-controlled AD converter. The figure to the right shows a schematic of the experimental setup which is all fiber-coupled with polarization-maintaining fiber except for the spectroscopic cell. The graph on the lower right reveals a portion of the recorded radio-frequency spectrum which has been scaled to the optical domain. The location of the measured absorption coincides well with data taken from the HITRAN data base. Due to the intrinsic linewidth of all contributing comb lines, each sampling point in the transmission graph corresponds to the probing at an optical frequency with sub-Hertz resolution. This resolution is maintained in coherent wavelength conversion processes such as difference-frequency generation (DFG), sum-frequency generation (SFG) or non-linear broadening (self-phase modulation), and is therefore easily transferred to a wide spectral range from the mid infrared up to the visible spectrum.

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

PubMed Central

Feddersen, R M; Van Ness, B G

1990-01-01

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed. Images PMID:2153918

The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

PubMed Central

Mendez-Bermudez, Aaron; Hidalgo-Bravo, Alberto; Cotton, Victoria E.; Gravani, Athanasia; Jeyapalan, Jennie N.; Royle, Nicola J.

2012-01-01

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells. PMID:22989712

The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres.

PubMed

Mendez-Bermudez, Aaron; Hidalgo-Bravo, Alberto; Cotton, Victoria E; Gravani, Athanasia; Jeyapalan, Jennie N; Royle, Nicola J

2012-11-01

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN- ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN- ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.

Derivation and Characterization of Induced Pluripotent Stem Cells from Equine Fibroblasts

PubMed Central

Breton, Amandine; Sharma, Ruchi; Diaz, Andrea Catalina; Parham, Alea Gillian; Graham, Audrey; Neil, Claire; Whitelaw, Christopher Bruce; Milne, Elspeth

2013-01-01

Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine. PMID:22897112

Loss of insulin-like growth factor II imprinting is a hallmark associated with enhanced chemo/radiotherapy resistance in cancer stem cells.

PubMed

Zhao, Xin; Liu, Xiaoliang; Wang, Guanjun; Wen, Xue; Zhang, Xiaoying; Hoffman, Andrew R; Li, Wei; Hu, Ji-Fan; Cui, Jiuwei

2016-08-09

Insulin-like growth factor II (IGF2) is maternally imprinted in most tissues, but the epigenetic regulation of the gene in cancer stem cells (CSCs) has not been defined. To study the epigenetic mechanisms underlying self-renewal, we isolated CSCs and non-CSCs from colon cancer (HT29, HRT18, HCT116), hepatoma (Hep3B), breast cancer (MCF7) and prostate cancer (ASPC) cell lines. In HT29 and HRT18 cells that show loss of IGF2 imprinting (LOI), IGF2 was biallelically expressed in the isolated CSCs. Surprisingly, we also found loss of IGF2 imprinting in CSCs derived from cell lines HCT116 and ASPC that overall demonstrate maintenance of IGF2 imprinting. Using chromatin conformation capture (3C), we found that intrachromosomal looping between the IGF2 promoters and the imprinting control region (ICR) was abrogated in CSCs, in parallel with loss of IGF2 imprinting in these CSCs. Loss of imprinting led to increased IGF2 expression in CSCs, which have a higher rate of colony formation and greater resistance to chemotherapy and radiotherapy in vitro. These studies demonstrate that IGF2 LOI is a common feature in CSCs, even when the stem cells are derived from a cell line in which the general population of cells maintain IGF2 imprinting. This finding suggests that aberrant IGF2 imprinting may be an intrinsic epigenetic control mechanism that enhances stemness, self-renewal and chemo/radiotherapy resistance in cancer stem cells.

The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro

PubMed Central

Potter, Daniel R.; Jiang, John; Damiano, Edward R.

2009-01-01

Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent micro-particle image velocimetry (µ-PIV) in mouse cremaster-muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5–7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and since demonstrable evidence has emerged that standard cell-culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell-surface glycocalyx in vitro, we sought to determine if merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using µ-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days post-confluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial-cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial-cell function. PMID:19443840

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

PubMed Central

Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

2017-01-01

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

PubMed

Yamamoto, Hideaki; Tonello, Jane Marie; Sambuichi, Takanori; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

2018-01-01

New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Dohda, Takeaki; Kino-Oka, Masahiro

2018-03-01

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k L a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h -1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 10 9 cells L -1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018. © 2017 American Institute of Chemical Engineers.

Tobacco exposure results in increased E6 and E7 oncogene expression, DNA damage and mutation rates in cells maintaining episomal human papillomavirus 16 genomes.

PubMed

Wei, Lanlan; Griego, Anastacia M; Chu, Ming; Ozbun, Michelle A

2014-10-01

High-risk human papillomavirus (HR-HPV) infections are necessary but insufficient agents of cervical and other epithelial cancers. Epidemiological studies support a causal, but ill-defined, relationship between tobacco smoking and cervical malignancies. In this study, we used mainstream tobacco smoke condensate (MSTS-C) treatments of cervical cell lines that maintain either episomal or integrated HPV16 or HPV31 genomes to model tobacco smoke exposure to the cervical epithelium of the smoker. MSTS-C exposure caused a dose-dependent increase in viral genome replication and correspondingly higher early gene transcription in cells with episomal HPV genomes. However, MSTS-C exposure in cells with integrated HR-HPV genomes had no effect on genome copy number or early gene transcription. In cells with episomal HPV genomes, the MSTS-C-induced increases in E6 oncogene transcription led to decreased p53 protein levels and activity. As expected from loss of p53 activity in tobacco-exposed cells, DNA strand breaks were significantly higher but apoptosis was minimal compared with cells containing integrated viral genomes. Furthermore, DNA mutation frequencies were higher in surviving cells with HPV episomes. These findings provide increased understanding of tobacco smoke exposure risk in HPV infection and indicate tobacco smoking acts more directly to alter HR-HPV oncogene expression in cells that maintain episomal viral genomes. This suggests a more prominent role for tobacco smoke in earlier stages of HPV-related cancer progression. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Distinct Hypericum perforatum L. total extracts exert different antitumour activity on erythroleukemic K562 cells.

PubMed

Valletta, Elena; Rinaldi, Annamaria; Marini, Mario; Franzese, Ornella; Roscetti, Gianna

2018-05-22

Total flower extracts of Hypericum perforatum L. obtained with 3 different solvent systems were tested on tumour cell line cultures by comparing two groups of plants harvested in different times and places. The extracts, characterized according to the spectroscopic profile and the hypericin content, were tested on the growth and apoptotic death of K562 cells, a human erythroleukemic cell line. Growth and apoptosis were analysed by viable cell count, flow cytometry, and fluorescence microscopy at 6, 24, and 48 hr of culture following 1 hr exposure to the extracts under investigation. Here, we show that Hypericum extracts are able to reduce the growth of K562 cells and induce different degrees and kinetics of apoptosis according to the group of plants of origin. Also, we highlighted interesting differences in terms of efficacy among the extracts, with some samples losing their effectiveness along the culture time and others able to maintain or even increase their efficacy. Furthermore, the data herein obtained confirm the role of non hypericin compounds that are present in different proportions in the two plant groups and in the extracts analysed. Copyright © 2018 John Wiley & Sons, Ltd.

Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes.

PubMed

Dong, Xiangshu; Kim, Wan Kyu; Lim, Yong-Pyo; Kim, Yeon-Ki; Hur, Yoonkang

2013-02-01

We investigated the mechanism regulating cytoplasmic male sterility (CMS) in Brassica rapa ssp. pekinensis using floral bud transcriptome analyses of Ogura-CMS Chinese cabbage and its maintainer line in B. rapa 300-K oligomeric probe (Br300K) microarrays. Ogura-CMS Chinese cabbage produced few and infertile pollen grains on indehiscent anthers. Compared to the maintainer line, CMS plants had shorter filaments and plant growth, and delayed flowering and pollen development. In microarray analysis, 4646 genes showed different expression, depending on floral bud size, between Ogura-CMS and its maintainer line. We found 108 and 62 genes specifically expressed in Ogura-CMS and its maintainer line, respectively. Ogura-CMS line-specific genes included stress-related, redox-related, and B. rapa novel genes. In the maintainer line, genes related to pollen coat and germination were specifically expressed in floral buds longer than 3mm, suggesting insufficient expression of these genes in Ogura-CMS is directly related to dysfunctional pollen. In addition, many nuclear genes associated with auxin response, ATP synthesis, pollen development and stress response had delayed expression in Ogura-CMS plants compared to the maintainer line, which is consistent with the delay in growth and development of Ogura-CMS plants. Delayed expression may reduce pollen grain production and/or cause sterility, implying that mitochondrial, retrograde signaling delays nuclear gene expression. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

PubMed

Das, Mitali; Prasad, Shyam Babu; Yadav, Suresh Singh; Modi, Arusha; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

2015-12-01

Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

PubMed Central

Ravi, Dashnamoorthy; Chen, Yidong; Karia, Bijal; Brown, Adam; Gu, Ting Ting; Li, Jie; Carey, Mark S.; Hennessy, Bryan T.; Bishop, Alexander J. R.

2011-01-01

Background In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2. Principal Findings Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis. Conclusions Cancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression. PMID:21249227

Comparative transcript profiling of alloplasmic male-sterile lines revealed altered gene expression related to pollen development in rice (Oryza sativa L.).

PubMed

Hu, Jihong; Chen, Guanglong; Zhang, Hongyuan; Qian, Qian; Ding, Yi

2016-08-05

Cytoplasmic male sterility (CMS) is an ideal model for investigating the mitochondrial-nuclear interaction and down-regulated genes in CMS lines which might be the candidate genes for pollen development in rice. In this study, a set of rice alloplasmic sporophytic CMS lines was obtained by successive backcrossing of Meixiang B, with three different cytoplasmic types: D62A (D type), ZS97A (WA type) and XQZ-A (DA type). Using microarray, the anther transcript profiles of the three indica rice CMS lines revealed 622 differentially expressed genes (DEGs) in each of the three CMS lines compared with the maintainer line Meixiang B. GO and MapMan analysis indicated that these DEGs were mainly involved in lipid metabolism and cell wall organization. Compared with the gene expression of sporophytic and gametophytic CMS lines, 303 DEGs were identified and 56 of them were down-regulated in all the CMS lines of rice. These down-regulated DEGs in the CMS lines were found to be involved in tapetum or cell wall formation and their suppressed expression might be related to male sterility. Weighted gene co-expression network analysis (WGCNA) revealed that two modules were significantly associated with male sterility and many hub genes that were differentially expressed in the CMS lines. A large set of putative genes involved in anther development was identified in the present study. The results will give some information for the nuclear gene regulation by different cytoplasmic genotypes and provide a rich resource for further functional research on the pollen development in rice.

Regulation of proliferation and functioning of transplanted cells by using herpes simplex virus thymidine kinase gene in mice.

PubMed

Tsujimura, Mari; Kusamori, Kosuke; Oda, Chihiro; Miyazaki, Airi; Katsumi, Hidemasa; Sakane, Toshiyasu; Nishikawa, Makiya; Yamamoto, Akira

2018-04-10

Though cell transplantation is becoming an attractive therapeutic method, uncontrolled cell proliferation or overexpression of cellular functions could cause adverse effects. These unfavorable outcomes could be avoided by regulating the proliferation or functioning of transplanted cells. In this study, we used a combination of the herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, and ganciclovir (GCV) to control the proliferation and functioning of insulin-secreting cells after transplantation in diabetic mice. Mouse pancreatic β cell line MIN6 cells were selected as insulin-secreting cells for transfection with the HSVtk gene to obtain MIN6/HSVtk cells. Proliferation of MIN6/HSVtk cells was suppressed by GCV in a concentration-dependent manner; 0.25 μg/mL GCV maintained a constant number of MIN6/HSVtk cells for at least 16 days. MIN6 or MIN6/HSVtk cells were then transplanted to streptozotocin-induced diabetic mice. Mice transplanted with MIN6 cells exhibited hypoglycemia irrespective of GCV administration. In contrast, normal (around 150 mg/dL) blood glucose levels were maintained in mice transplanted with MIN6/HSVtk cells by a daily administration of 50 mg/kg of GCV. These results indicate that controlling the proliferation and functioning of HSVtk gene-expressing cells by GCV could greatly improve the usefulness and safety of cell-based therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Paclitaxel synergizes with exposure time adjusted CD22-targeting immunotoxins against B-cell malignancies.

PubMed

Müller, Fabian; Stookey, Stephanie; Cunningham, Tyler; Pastan, Ira

2017-05-09

CD22-targeted recombinant immunotoxins (rIT) are active in hairy cell leukemia or acute lymphoblastic leukemia (ALL), but not in mantle cell lymphoma (MCL) patients. The goal was to enhance rIT efficacy in vivo and to define a strong combination treatment. Activity of Moxetumomab pasudotox (Moxe) and LR combined with paclitaxel was tested against MCL cell lines in vitro and as bolus doses or continuous infusion in xenograft models. In the KOPN-8 ALL xenograft, Moxe or paclitaxel alone was active, but all mice died from leukemia; when combined, 60% of the mice achieved a sustained complete remission. Against MCL cells in vitro, LR was more active than Moxe and the cells had to be exposed to rIT for more than 24 hours for them to die. To maintain high blood levels in vivo, LR was administered continuously by 7-day pumps achieving a well-tolerated steady plasma concentration of 45 ng/ml. In JeKo-1 xenografts, continuously administered LR was 14-fold more active than bolus doses and the combination with paclitaxel additionally improved responses by 135-fold. Maintaining high rIT-plasma levels greatly improves responses in the JeKo-1 model and paclitaxel substantially enhances bolus and continuously infused rIT, supporting a clinical evaluation against B-cell malignancies.

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

PubMed

Kolbinger, Fiona R; Koeneke, Emily; Ridinger, Johannes; Heimburg, Tino; Müller, Michael; Bayer, Theresa; Sippl, Wolfgang; Jung, Manfred; Gunkel, Nikolas; Miller, Aubry K; Westermann, Frank; Witt, Olaf; Oehme, Ina

2018-06-09

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

PubMed Central

Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

2010-01-01

Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

Loss of EGFR confers acquired resistance to AZD9291 in an EGFR-mutant non-small cell lung cancer cell line with an epithelial-mesenchymal transition phenotype.

PubMed

Xu, Jing; Zhao, Xiaoting; He, Dengfeng; Wang, Jinghui; Li, Weiying; Liu, Yinghui; Ma, Li; Jiang, Mei; Teng, Yu; Wang, Ziyu; Gu, Meng; Wu, Jianbin; Wang, Yue; Yue, Wentao; Zhang, Shucai

2018-05-24

AZD9291 is an irreversible, small-molecule inhibitor which has potency against mutant EGFR- and T790M-resistant mutation. Despite the encouraging efficacy in clinical, the acquired resistance will finally occur. Further study will need to be done to identify the acquired resistance mechanisms and determine the next treatment. We established an AZD9291-resistant cell line (HCC827/AZDR) from parental HCC827 cell line through stepwise pulsed selection of AZD9291. The expression of EGFR and its downstream pathways were determined by western blot analysis or immunofluorescence assay. The sensitivity to indicated agents were evaluated by MTS. Compared with parental HCC827 cells, the HCC827/AZDR cells showed high resistance to AZD9291 and other EGFR-TKIs, and exhibited a mesenchymal-like phenotype. Almost complete loss of EGFR expression was observed in HCC827/AZDR cells. But the activation of downstream pathway, MAPK signaling, was found in HCC827/AZDR cells even in the presence of AZD9291. Inhibition of MAPK signaling had no effect on cell viability of HCC827/AZDR and could not reverse AZD9291 resistance because of the subsequent activation of AKT signaling. When treated with the combination of AKT and MAPK inhibitor, HCC827/AZDR showed remarkable growth inhibition. Loss of EGFR could be proposed as a potential acquired resistance mechanism of AZD9291 in EGFR-mutant NSCLC cells with an EMT phenotype. Despite the loss of EGFR, the activation of MAPK pathway which had crosstalk with AKT pathway could maintain the proliferation and survival of resistant cells. Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway.

PubMed

Reis, Pedro A A; Rosado, Gustavo L; Silva, Lucas A C; Oliveira, Luciana C; Oliveira, Lucas B; Costa, Maximiller D L; Alvim, Fátima C; Fontes, Elizabeth P B

2011-12-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.

The Binding Protein BiP Attenuates Stress-Induced Cell Death in Soybean via Modulation of the N-Rich Protein-Mediated Signaling Pathway1[C][W][OA

PubMed Central

Reis, Pedro A.A.; Rosado, Gustavo L.; Silva, Lucas A.C.; Oliveira, Luciana C.; Oliveira, Lucas B.; Costa, Maximiller D.L.; Alvim, Fátima C.; Fontes, Elizabeth P.B.

2011-01-01

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response. PMID:22007022

Synthesis and biological evaluation of some novel triazole hybrids of curcumin mimics and their selective anticancer activity against breast and prostate cancer cell lines.

PubMed

Mandalapu, Dhanaraju; Saini, Karan S; Gupta, Sonal; Sharma, Vikas; Yaseen Malik, Mohd; Chaturvedi, Swati; Bala, Veenu; Hamidullah; Thakur, Subhadra; Maikhuri, Jagdamba P; Wahajuddin, Muhammad; Konwar, Rituraj; Gupta, Gopal; Sharma, Vishnu Lal

2016-09-01

The anti-cancer property of curcumin, an active component of turmeric, is limited due to its poor solubility, stability and bioavailability. To enhance its efficacy, we designed a novel series of twenty-four monocarbonyl curcumin analogue-1,2,3-triazole conjugates and evaluated their anti-cancer activity towards endocrine related cancers. The new compounds (17-40) were synthesized through CuAAC click reaction and SAR analysis carried out. Out of these all, compound 17 showed most significant anti-cancer activity against prostate cancer cells with IC50 values of 8.8μM and 9.5μM in PC-3 and DU-145 cells, respectively. Another compound 26 showed significant anti-cancer activity against breast cancer cells with IC50 of 6μM, 10μM and 6.4μM in MCF-7, MDA-MB-231 and 4T1 cells, respectively while maintaining low toxicity towards non-cancer originated cell line, HEK-293. Compounds 17 and 26 arrested cell cycle and induced mitochondria-mediated apoptosis in cancer cells. Further, both of these compounds significantly down-regulated cell proliferation marker (PCNA), inhibited activation of cell survival protein (Akt phosphorylation), upregulated pro-apoptotic protein (Bax) and down-regulated anti-apoptotic protein (Bcl-2) in their respective cell lines. In addition, in vitro stability, solubility and plasma binding studies of the compounds 17 and 26 showed them to be metabolically stable. Thus, this study identified two new curcumin monocarbonyl-1,2,3-triazole conjugate compounds with more potent activity than curcumin against breast and prostate cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biological characterization of HIV type 1 envelope V3 regions from mothers and infants associated with perinatal transmission.

PubMed

Matala, E; Hahn, T; Yedavalli, V R; Ahmad, N

2001-12-10

Our previous study has shown that the human immunodeficiency virus type 1 (HIV-1) envelope V3 region minor genotypes of infected mothers were transmitted to their infants and predominated initially as a homogeneous virus population in the infants (Ahmad N, Baroudy BM, Baker RC, et al.: J Virol 1995;69:1001-1012). Here we have characterized the biological properties, including cellular tropism, replication efficiency, cytopathic effects, and coreceptor utilization, of these V3 region isolates from mothers and infants. Nineteen V3 region sequences from three mother-infant pairs, including the minor variants of mothers and the major variants of infants as characterized in our previous study, were reciprocally inserted into an HIV-1 infectious molecular clone, pNL4-3, and chimeric viruses were generated by DNA transfections into HeLa cells. Equal amounts of chimeric viruses were then used to infect T lymphocyte cell lines (A3.01 and MT-2), primary blood lymphocytes (PBLs), primary monocyte-derived macrophages (MDMs), and coreceptor cell lines. We found that the V3 region chimeras failed to replicate in T lymphocyte cell lines but replicated in MDMs and PBLs, albeit at reduced levels compared with R5 laboratory HIV-1 strains. In addition, the V3 region chimeras were able to infect the HOS-CD4(+)CCR5(+) cell line, suggesting CCR5 coreceptor utilization. Moreover, the V3 region chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium-inducing (NSI) phenotypes. In conclusion, the HIV-1 minor genotypes of infected mothers with macrophage-tropic and NSI or R5 phenotypes are transmitted to their infants and are initially maintained with the same properties.

An Ichor-dependent apical extracellular matrix regulates seamless tube shape and integrity

PubMed Central

Rosa, Jeffrey B.; Metzstein, Mark M.

2018-01-01

During sprouting angiogenesis in the vertebrate vascular system, and primary branching in the Drosophila tracheal system, specialized tip cells direct branch outgrowth and network formation. When tip cells lumenize, they form subcellular (seamless) tubes. How these seamless tubes are made, shaped and maintained remains poorly understood. Here we characterize a Drosophila mutant called ichor (ich), and show that ich is essential for the integrity and shape of seamless tubes in tracheal terminal cells. We find that Ich regulates seamless tubulogenesis via its role in promoting the formation of a mature apical extracellular matrix (aECM) lining the lumen of the seamless tubes. We determined that ich encodes a zinc finger protein (CG11966) that acts, as a transcriptional activator required for the expression of multiple aECM factors, including a novel membrane-anchored trypsin protease (CG8213). Thus, the integrity and shape of seamless tubes are regulated by the aECM that lines their lumens. PMID:29309404

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Targeting Metabolic Plasticity in Breast Cancer Cells via Mitochondrial Complex I Modulation

PubMed Central

Xu, Qijin; Biener-Ramanujan, Eva; Yang, Wei; Ramanujan, V Krishnan

2016-01-01

Purpose Heterogeneity commonly observed in clinical tumors stems both from the genetic diversity as well as from the differential metabolic adaptation of multiple cancer types during their struggle to maintain uncontrolled proliferation and invasion in vivo. This study aims to identify a potential metabolic window of such adaptation in aggressive human breast cancer cell lines. Methods With a multidisciplinary approach using high resolution imaging, cell metabolism assays, proteomic profiling and animal models of human tumor xenografts and via clinically-relevant, pharmacological approach for modulating mitochondrial complex I function in human breast cancer cell lines, we report a novel route to target metabolic plasticity in human breast cancer cells. Results By a systematic modulation of mitochondrial function and by mitigating metabolic switch phenotype in aggressive human breast cancer cells, we demonstrate that the resulting metabolic adaptation signatures can predictably decrease tumorigenic potential in vivo. Proteomic profiling of the metabolic adaptation in these cells further revealed novel protein-pathway interactograms highlighting the importance of antioxidant machinery in the observed metabolic adaptation. Conclusions Improved metabolic adaptation potential in aggressive human breast cancer cells contribute to improving mitochondrial function and reducing metabolic switch phenotype –which may be vital for targeting primary tumor growth in vivo. PMID:25677747

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

PubMed

Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

2016-08-01

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Experimental arthritis and uveitis in rats associated with Mycobacterium butyricum.

PubMed

Petty, R E; Hunt, D W; Mathers, D M; McCormick, A Q; Barker, H; Southwood, T R; Corson, L

1994-08-01

To determine if the anterior uveitis associated with adjuvant arthritis (AA) in the rat can be passively transferred with arthritis to syngeneic recipients using spleen cells or T cell lines prepared from animals given complete Freund's adjuvant (CFA) and Mycobacterium butyricum (M. butyricum) in incomplete Freund's adjuvant (IFA). Spleen cells from Lewis or Lewis SsN rats given IFA, CFA, type I collagen in IFA (CI-IFA), or type II collagen in IFA (CII-IFA) were administered to naive rats or rats treated with pertussis toxin or bacterial endotoxin. Three CD4+ T cell lines, propagated from CFA injected rats and maintained in vitro with M. butyricum (M-1), bovine proteoglycan (PR-1) or an extract of M. butyricum (MBE-1) were administered to naive or immunosuppressed rats. The arthritogenic and uveitogenic properties of these cell preparations and intradermal MBE-IFA, CII-IFA and intraperitoneal (ip) M. butyricum without adjuvant were evaluated. Uveitis was observed in 15/69 (22%) arthritic rats given CFA. Spleen cells prepared from CFA injected rats caused arthritis in 55 (82%) and uveitis in 2 (3%) of 67 cell recipients. Uveitis occurred in 2/6 cell recipients pretreated with bacterial endotoxin. Neither uveitis nor arthritis was observed in rats given IFA (0/6) or spleen cells prepared from rats given IFA (0/27), CI-IFA (0/6), or CII-IFA (0/28). CII-IFA produced polyarthritis in 5/6 rats, but no uveitis. CII-IFA induced arthritis associated uveitis in 1/15 animals receiving spleen cells from rats given CII-IFA, but not those given CI-IFA (0/3) or IFA (0/13). Uveitis was observed in one recipient of the M-1 T cell line and in 2 recipients of the PR-1 T cell line. Immunization with 400 micrograms of MBE-IFA induced uveitis but not arthritis in 3/11 animals. The MBE specific T cell line was neither arthritogenic nor uveitogenic. A high frequency (5/6) of uveitis accompanied arthritis in male Lewis rats given ip M. butyricum. Arthritis occurred in 4/10 female Lewis rats given ip M. butyricum and 2 arthritic animals also developed uveitis. Uveitis occurs infrequently in arthritic rats given spleen cells from CFA injected animals. The ip administration of M. butyricum constitutes a novel disease model in which the immunopathological relationships between arthritis and uveitis may be more reliably studied.

Hypoexpression and epigenetic regulation of candidate tumor suppressor gene CADM-2 in human prostate cancer.

PubMed

Chang, Guimin; Xu, Shuping; Dhir, Rajiv; Chandran, Uma; O'Keefe, Denise S; Greenberg, Norman M; Gingrich, Jeffrey R

2010-11-15

Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers. ©2010 AACR.

Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.

PubMed

Xie, Ying; Zhao, Xiaoe; Jia, Hongxiang; Ma, Baohua

2013-01-01

Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

PubMed

Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

2017-04-11

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor

PubMed Central

Esakky, P; Hansen, D A; Drury, A M; Cusumano, A; Moley, K H

2015-01-01

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line. PMID:27551479

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias.

PubMed

Parton, Angela; Forest, David; Kobayashi, Hiroshi; Dowell, Lori; Bayne, Christopher; Barnes, David

2007-02-01

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

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

Nishio, Sachiyo; Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503; Ohira, Takahito

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcriptionmore » compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.« less

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

PubMed

Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

2011-08-01

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

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.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

PubMed Central

Augustsson, Per; Karlsen, Jonas T.; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-01-01

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size. PMID:27180912

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

PubMed

Augustsson, Per; Karlsen, Jonas T; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-05-16

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

Mutant IDH1 is required for IDH1 mutated tumor cell growth

PubMed Central

Jin, Genglin; Pirozzi, Christopher J.; Chen, Lee H.; Lopez, Giselle Y.; Duncan, Christopher G.; Feng, Jie; Spasojevic, Ivan; Bigner, Darell D.; He, Yiping; Yan, Hai

2012-01-01

Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1R132C heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target. PMID:22885298

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

PubMed Central

Millonigg, Sophia; Eckmann, Christian R.

2014-01-01

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation. PMID:25254367

Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

PubMed Central

Zhu, Wei; Kuo, Dwight; Nathanson, Jason; Satoh, Akira; Pao, Gerald M.; Yeo, Gene W.; Bryant, Susan V.; Voss, S. Randal; Gardiner, David M.; Hunter, Tony

2012-01-01

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration. PMID:22913491

Anodically bonded submicron microfluidic chambers.

PubMed

Dimov, S; Bennett, R G; Córcoles, A; Levitin, L V; Ilic, B; Verbridge, S S; Saunders, J; Casey, A; Parpia, J M

2010-01-01

We demonstrate the use of anodic bonding to fabricate cells with characteristic size as large as 7 x 10 mm(2), with height of approximately 640 nm, and without any internal support structure. The cells were fabricated from Hoya SD-2 glass and silicon wafers, each with 3 mm thickness to maintain dimensional stability under internal pressure. Bonding was carried out at 350 degrees C and 450 V with an electrode structure that excluded the electric field from the open region. We detail fabrication and characterization steps and also discuss the design of the fill line for access to the cavity.

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Kakeda, M; Yoneya, T; Tsumura, H; Ohashi, H; Yamaguchi, Y; Motoki, K; Sudo, T; Mori, K J

1996-09-01

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

PubMed

Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

2016-05-01

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

New pharmacological strategies in rheumatic diseases.

PubMed

Schiotis, R E; Buzoianu, A D; Mureșanu, D F; Suciu, S

2016-01-01

Targeting the pathogenic pathway of chronic inflammation represents an unmet challenge for controlling disease activity, preventing functional disability, and maintaining an adequate quality of life in patients with rheumatic diseases. Abatacept, a novel molecule that inhibits co-stimulation signal, induces an inhibitory effect on the T-cells. This will further interfere with the activity of several cell lines, leading to the normalization of the immune response. In the latest years, abatacept has been extensively investigated in studies of rheumatoid arthritis for which it was recently approved as a second line biologic treatment in Romania. This review presents the clinical efficacy of abatacept in several rheumatic diseases and highlights the safety profile of this biological agent. Abbreviations : ACR = American College of Rheumatology, ADR = Adverse drug reaction, APC = antigen presenting cell, ApS = psoriatic arthritis, CRP = C reactive protein, CTLA-4 = Cytotoxic T-Cell Lymphocyte Antigen-4, DAS = Disease activity score, DMARDs = Disease modifying antirheumatic drugs, EMA = European Medicine Agency, EULAR = European League Against Rheumatism, FDA = Food and Drugs Administration, HBV = Hepatitis B virus, JIA = Juvenile Idiopathic Arthritis, LDA = low disease activity (LDA), MRI = magnetic resonance imaging (MRI), MTX = methotrexate, RA = rheumatoid arthritis, RCT = randomized controlled trial, SS = Sjogren's syndrome, TCR = T cell receptor.

Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors.

PubMed

Malakootian, Mahshid; Mirzadeh Azad, Fatemeh; Fouani, Youssef; Taheri Bajgan, Elham; Saberi, Hooshang; Mowla, Seyed Javad

2018-06-01

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

Human embryonic stem cell lines model experimental human cytomegalovirus latency.

PubMed

Penkert, Rhiannon R; Kalejta, Robert F

2013-05-28

Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

PubMed Central

2011-01-01

Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies. PMID:22040608

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

Molecular-aided selection of male sterility for hybrid development in onion

USDA-ARS?s Scientific Manuscript database

Maintainer lines are used to seed propagate male-sterile lines for the development of hybrid-onion cultivars. Selection of maintainer lines is more efficient using molecular markers that distinguish cytoplasms and genotypes at the nuclear male-fertility restoration (Ms) locus. Onion cytoplasms can b...

MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

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

Liu, Lian, E-mail: tounao@126.com; Institute of Immunology, School of Medicine, Shandong University, Jinan 250012; Zhang, Xin

We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even whenmore » it fails to activate MAPK as expected.« less

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

PubMed

Chiba, Tetsuhiro; Kita, Kaoru; Zheng, Yun-Wen; Yokosuka, Osamu; Saisho, Hiromitsu; Iwama, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2006-07-01

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Intratumoral heterogeneity in EGFR mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition

PubMed Central

Soucheray, Margaret; Capelletti, Marzia; Pulido, Inés; Kuang, Yanan; Paweletz, Cloud P.; Becker, Jeffrey H.; Kikuchi, Eiki; Xu, Chunxiao; Patel, Tarun B.; Al-shahrour, Fatima; Carretero, Julián; Wong, Kwok-Kin; Jänne, Pasi A.; Shapiro, Geoffrey I.; Shimamura, Takeshi

2015-01-01

Non-small cell lung cancers (NSCLC) that have developed resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated-EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT, but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared to cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly develops EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele is lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. PMID:26282169

Trivial role for NSMCE2 during in vitro proliferation and differentiation of male germline stem cells.

PubMed

Zheng, Yi; Jongejan, Aldo; Mulder, Callista L; Mastenbroek, Sebastiaan; Repping, Sjoerd; Wang, Yinghua; Li, Jinsong; Hamer, Geert

2017-09-01

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using in vitro cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an Nsmce2 -/- GS cell line. Using this Nsmce2 -/- GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of Sgsm1 appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline. © 2017 Society for Reproduction and Fertility.

Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-08-01

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.

Pomegranate Polyphenols Downregulate Expression of Androgen Synthesizing Genes in Human Prostate Cancer Cells Overexpressing the Androgen Receptor

PubMed Central

Hong, Mee Young; Seeram, Navindra P.; Heber, David

2008-01-01

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state where they progress in the absence of circulating testosterone leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In the present study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen synthesizing enzymes and the AR. We measured expression of the HSD3B2, AKR1C3 and SRD5A1 genes for the respective androgen synthesizing enzymes in LNCaP, LNCaP-AR, and DU-145 human prostate cancer cells. A two-fold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P =.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen synthesis enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is upregulated. PMID:18479901

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

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

Kim, In Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology; Kim, Seo Yoen

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cellsmore » than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.« less

Pseudoislets as primary islet replacements for research: report on a symposium at King's College London, London UK.

PubMed

Persaud, Shanta J; Arden, Catherine; Bergsten, Peter; Bone, Adrian J; Brown, James; Dunmore, Simon; Harrison, Moira; Hauge-Evans, Astrid; Kelly, Catriona; King, Aileen; Maffucci, Tania; Marriott, Claire E; McClenaghan, Neville; Morgan, Noel G; Reers, Christina; Russell, Mark A; Turner, Mark D; Willoughby, Emma; Younis, Mustafa Y G; Zhi, Z L; Jones, Peter M

2010-01-01

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

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

Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S.

1994-09-01

Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viralmore » DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.« less

Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

PubMed

Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

2010-11-01

The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton.

PubMed

Yang, Peng; Han, Jinfeng; Huang, Jinling

2014-01-01

Cytoplasmic male sterility (CMS) is the failure to produce functional pollen, which is inherited maternally. And it is known that anther development is modulated through complicated interactions between nuclear and mitochondrial genes in sporophytic and gametophytic tissues. However, an unbiased transcriptome sequencing analysis of CMS in cotton is currently lacking in the literature. This study compared differentially expressed (DE) genes of floral buds at the sporogenous cells stage (SS) and microsporocyte stage (MS) (the two most important stages for pollen abortion in JA-CMS) between JA-CMS and its fertile maintainer line JB cotton plants, using the Illumina HiSeq 2000 sequencing platform. A total of 709 (1.8%) DE genes including 293 up-regulated and 416 down-regulated genes were identified in JA-CMS line comparing with its maintainer line at the SS stage, and 644 (1.6%) DE genes with 263 up-regulated and 381 down-regulated genes were detected at the MS stage. By comparing the two stages in the same material, there were 8 up-regulated and 9 down-regulated DE genes in JA-CMS line and 29 up-regulated and 9 down-regulated DE genes in JB maintainer line at the MS stage. Quantitative RT-PCR was used to validate 7 randomly selected DE genes. Bioinformatics analysis revealed that genes involved in reduction-oxidation reactions and alpha-linolenic acid metabolism were down-regulated, while genes pertaining to photosynthesis and flavonoid biosynthesis were up-regulated in JA-CMS floral buds compared with their JB counterparts at the SS and/or MS stages. All these four biological processes play important roles in reactive oxygen species (ROS) homeostasis, which may be an important factor contributing to the sterile trait of JA-CMS. Further experiments are warranted to elucidate molecular mechanisms of these genes that lead to CMS.

Transcriptome Sequencing and De Novo Analysis of Cytoplasmic Male Sterility and Maintenance in JA-CMS Cotton

PubMed Central

Yang, Peng; Han, Jinfeng; Huang, Jinling

2014-01-01

Cytoplasmic male sterility (CMS) is the failure to produce functional pollen, which is inherited maternally. And it is known that anther development is modulated through complicated interactions between nuclear and mitochondrial genes in sporophytic and gametophytic tissues. However, an unbiased transcriptome sequencing analysis of CMS in cotton is currently lacking in the literature. This study compared differentially expressed (DE) genes of floral buds at the sporogenous cells stage (SS) and microsporocyte stage (MS) (the two most important stages for pollen abortion in JA-CMS) between JA-CMS and its fertile maintainer line JB cotton plants, using the Illumina HiSeq 2000 sequencing platform. A total of 709 (1.8%) DE genes including 293 up-regulated and 416 down-regulated genes were identified in JA-CMS line comparing with its maintainer line at the SS stage, and 644 (1.6%) DE genes with 263 up-regulated and 381 down-regulated genes were detected at the MS stage. By comparing the two stages in the same material, there were 8 up-regulated and 9 down-regulated DE genes in JA-CMS line and 29 up-regulated and 9 down-regulated DE genes in JB maintainer line at the MS stage. Quantitative RT-PCR was used to validate 7 randomly selected DE genes. Bioinformatics analysis revealed that genes involved in reduction-oxidation reactions and alpha-linolenic acid metabolism were down-regulated, while genes pertaining to photosynthesis and flavonoid biosynthesis were up-regulated in JA-CMS floral buds compared with their JB counterparts at the SS and/or MS stages. All these four biological processes play important roles in reactive oxygen species (ROS) homeostasis, which may be an important factor contributing to the sterile trait of JA-CMS. Further experiments are warranted to elucidate molecular mechanisms of these genes that lead to CMS. PMID:25372034

Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.

PubMed

Zheng, Liming; Zhai, Yuanxin; Li, Na; Wu, Chongyang; Zhu, Haijing; Wei, Zhuying; Bai, Chunling; Li, Guangpeng; Hua, Jinlian

2016-04-01

Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation, which modulates DNA methylation and gene transcription. DNA methylation and histone methylation are critical elements in self-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells able to achieve intergenerational transfer of genetic information, which is accomplished through differentiated sperm cells. However, numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1. An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay. The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in nuclear location of that methylation marker. While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells, it was present in only particular locations in mGSCs. H3K27me3 was distributed surrounding the edges of nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines. Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment. © 2016 John Wiley & Sons Ltd.

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

PubMed Central

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

Human Nanog pseudogene8 promotes the proliferation of gastrointestinal cancer cells

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

Uchino, Keita, E-mail: uchino13@intmed1.med.kyushu-u.ac.jp; Hirano, Gen; Hirahashi, Minako

2012-09-10

There is emerging evidence that human solid tumor cells originate from cancer stem cells (CSCs). In cancer cell lines, tumor-initiating CSCs are mainly found in the side population (SP) that has the capacity to extrude dyes such as Hoechst 33342. We found that Nanog is expressed specifically in SP cells of human gastrointestinal (GI) cancer cells. Nucleotide sequencing revealed that NanogP8 but not Nanog was expressed in GI cancer cells. Transfection of NanogP8 into GI cancer cell lines promoted cell proliferation, while its inhibition by anti-Nanog siRNA suppressed the proliferation. Immunohistochemical staining of primary GI cancer tissues revealed NanogP8 proteinmore » to be strongly expressed in 3 out of 60 cases. In these cases, NanogP8 was found especially in an infiltrative part of the tumor, in proliferating cells with Ki67 expression. These data suggest that NanogP8 is involved in GI cancer development in a fraction of patients, in whom it presumably acts by supporting CSC proliferation. -- Highlights: Black-Right-Pointing-Pointer Nanog maintains pluripotency by regulating embryonic stem cells differentiation. Black-Right-Pointing-Pointer Nanog is expressed in cancer stem cells of human gastrointestinal cancer cells. Black-Right-Pointing-Pointer Nucleotide sequencing revealed that Nanog pseudogene8 but not Nanog was expressed. Black-Right-Pointing-Pointer Nanog pseudogene8 promotes cancer stem cells proliferation. Black-Right-Pointing-Pointer Nanog pseudogene8 is involved in gastrointestinal cancer development.« less

Insights into the protective role of solid lipid nanoparticles on rosmarinic acid bioactivity during exposure to simulated gastrointestinal conditions.

PubMed

Madureira, Ana Raquel; Campos, Débora A; Oliveira, Ana; Sarmento, Bruno; Pintado, Maria Manuela; Gomes, Ana Maria

2016-03-01

The evaluation of the digestion effects on bioactive solid lipid nanoparticles (SLN) was performed. For this purpose, witepsol and carnauba SLN loaded with rosmarinic acid (RA) were exposed to the simulated gastrointestinal tract (GIT) conditions prevailing in stomach and small intestine. The simulation of intestinal epithelium was made with a dialysis bag and intestinal cell culture lines. Changes on SLN physical properties, RA release and absorption profiles were followed at each step. Combination of digestion pH and enzymes showed a significant effect upon SLN physical properties. Zeta potential values increased at stomach conditions and decreased at small intestine simulation. Also, at intestine, SLN increased their sizes and released 40-60% of RA, maintaining its initial antioxidant activity values. Sustained release of 40% of RA from SLN was also observed in dialysis tube. At CaCo-2 cell line, both types of SLN showed similar absorbed RA % (ca. 30%). Nevertheless, in CaCo-2/HT29x mix cell lines, for carnauba SLN a lower adsorption RA % was observed than for witepsol SLN. Solid lipid nanoparticles protected RA bioactivity (in terms of antioxidant activity) until reaching the intestine. A controlled release of RA from SLN was achieved and a significant absorption was observed at intestinal cells. Overall, SLN produced with witepsol showed a higher stability than carnauba SLN. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

PubMed Central

Nishihara, Kana; Huang, Ruili; Zhao, Jinghua; Shahane, Sampada A.; Witt, Kristine L.; Smith-Roe, Stephanie L.; Tice, Raymond R.; Takeda, Shunichi; Xia, Menghang

2016-01-01

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 −/−/RAD54 −/− and REV3 −/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA. PMID:26243743

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

PubMed

Pi, Woojin; Ryu, Jae-Sook; Roh, Jaesook

2011-09-01

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

Cis activation of the c-myc gene in bovine papilloma virus type 1/human c-myc hybrid plasmids

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

Modjtahedi, N.; Feunteun, J.; Brison, O.

1988-01-01

The c-myc gene amplification observed in human tumors is likely to represent an activation mechanism aiming at an increased transcription level. In order to evaluate the biological significance of this amplification in the malignant transformation the authors designed an experimental model that could possibly mimic this situation in vitro. They have constructed a series of plasmids which physically link the human c-myc gene to the bovine papilloma virus type 1 genome (BPV1) and therefore should be maintained as amplified episomes upon transformation of rodent cells. Anticipating that the high copy number will bring about the immortalizing capacity of the c-mycmore » gene, the constructions were introduced into primary rat embryo cells. Immortal cell lines were established by transfection of the hybrid plasmids carrying either the complete BPV1 genome or the transforming region of the viral genome. The BPV1 DNA alone or the c-myc gene alone has no activity in this assay. The analysis of the established cell lines demonstrates that the transfected plasmids are present not as free copies as anticipated but rather integrated as tandem repeats. They present data which strongly suggest that the immortalization capacity of the hybrid plasmids reflects the activation of the c-myc gene by the transactivable BPV1 enhancer. Although both the BPV1 early genes and the c-myc gene are actively transcribed, most of the cell lines do not display a transformed phenotype.« less

Effects of stress waves on cells

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

Campbell, H L; Da Silva, L B; Visuri, S R

Laser induced stress waves are being used in a variety of medical applications, including drug delivery and targeted tissue disruption. Stress waves can also be an undesirable side effect in laser procedures such as ophthalmology and angioplasty. Thus, a study of the effects of stress waves on a cellular level is useful. Thermoelastic stress waves were produced using a Q-switched frequency-doubled Nd:YAG laser (@.=532nm) with a pulse duration of 4 ns. The laser radiation was delivered to an absorbing media. A thermoelastic stress wave was produced in the absorbing media and propagated into plated cells. The energy per pulse deliveredmore » to a sample and the spot size were varied. Stress waves were quantified. We assayed for cell viability and damage using two methods. The laser parameters within which cells maintain viability were investigated and thresholds for cell damage were defined. A comparison of cell damage thresholds for different cell lines was made.« less

CD4 expression on EL4 cells as an epiphenomenon of retroviral transduction and selection.

PubMed

Logan, Grant J; Spinoulas, Afroditi; Alexander, Stephen I; Smythe, Jason A; Alexander, Ian E

2004-04-01

The EL4 murine tumour cell line, isolated from a chemically induced lymphoma over 50 years ago, has been extensively exploited in immunological research. The conclusions drawn from many of these studies have been based on the presumption that EL4 cells maintain a stable phenotype during experimental manipulation. To the contrary, we have observed 100-fold greater expression of cell surface CD4 (CD4(high)) on a subpopulation of EL4 cells following retroviral transduction and G418 selection when compared with unmodified populations. Although the mechanism responsible for this effect remains to be elucidated, the unexpected expression of CD4, a molecule that functions as both a coreceptor with the T-cell receptor and ligand for the pro-inflammatory cytokine IL-16, has the potential to influence experimental outcomes. Upregulation of CD4 should be excluded when EL4 cells are utilized in experiments requiring a consistent immuno-phenotype.

Development of human epithelial cell systems for radiation risk assessment

NASA Astrophysics Data System (ADS)

Yang, C. H.; Craise, L. M.

1994-10-01

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

Development of human epithelial cell systems for radiation risk assessment

NASA Technical Reports Server (NTRS)

Yang, C. H.; Craise, L. M.

1994-01-01

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

Neural stem cells induce the formation of their physical niche during organogenesis

PubMed Central

Riebesehl, Bea F; Ambrosio, Elizabeth M; Stolper, Julian S; Lischik, Colin Q; Dross, Nicolas

2017-01-01

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches. PMID:28950935

Generation of Integration-Free Induced Pluripotent Stem Cells from Urine-Derived Cells Isolated from Individuals with Down Syndrome.

PubMed

M Lee, Young; Zampieri, Bruna L; Scott-McKean, Jonah J; Johnson, Mark W; Costa, Alberto C S

2017-06-01

Down syndrome (DS) is a genetic disorder caused by trisomy 21 (T21). Over the past two decades, the use of mouse models has led to significant advances in the understanding of mechanisms underlying various phenotypic features and comorbidities secondary to T21 and even informed the design of clinical trials aimed at enhancing the cognitive abilities of persons with DS. In spite of its success, this approach has been plagued by all the typical limitations of rodent modeling of human disorders and diseases. Recently, several laboratories have succeeded in producing T21 human induced pluripotent stem cells (T21-iPSCs) from individuals with DS, which is emerging as a promising complementary tool for the study of DS. Here, we describe the method by which we generated 10 T21-iPSC lines from epithelial cells in urine samples, presumably from kidney epithelial origin, using nonintegrating episomal vectors. We also show that these iPSCs maintain chromosomal stability for well over 20 passages and are more sensitive to proteotoxic stress than euploid iPSCs. Furthermore, these iPSC lines can be differentiated into glutamatergic neurons and cardiomyocytes. By culturing urine-derived cells and maximizing the efficiency of episomal vector transfection, we have been able to generate iPSCs noninvasively and effectively from participants with DS in an ongoing clinical trial, and thus address most shortcomings of previously generated T21-iPSC lines. These techniques should extend the application of iPSCs in modeling DS and other neurodevelopmental and neurodegenerative disorders, and may lead to future human cell-based platforms for high-throughput drug screening. Stem Cells Translational Medicine 2017;6:1465-1476. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

PubMed Central

van den Broeck, Hetty C; van Herpen, Teun WJM; Schuit, Cees; Salentijn, Elma MJ; Dekking, Liesbeth; Bosch, Dirk; Hamer, Rob J; Smulders, Marinus JM; Gilissen, Ludovicus JWJ; van der Meer, Ingrid M

2009-01-01

Background Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD). Results The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties. Conclusion The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties. PMID:19351412

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

PubMed Central

Sadikot, Takrima; Swink, Megan; Eskew, Jeffery D.; Brown, Douglas; Zhao, Huiping; Kusuma, Bhaskar R.; Rajewski, Roger A.; Blagg, Brian S. J.; Matts, Robert L.; Holzbeierlein, Jeffrey M.

2013-01-01

Abstract The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu. PMID:24127661

Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

PubMed

Lee, Myungook; Ahn, Jong Il; Ahn, Ji Yeon; Yang, Woo Sub; Hubbell, Jeffrey A; Lim, Jeong Mook; Lee, Seung Tae

2017-11-01

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017. © 2016 Wiley Periodicals, Inc.

Sampling-based Bayesian approaches reveal the importance of quasi-bistable behavior in cellular decision processes on the example of the MAPK signaling pathway in PC-12 cell lines.

PubMed

Jensch, Antje; Thomaseth, Caterina; Radde, Nicole E

2017-01-25

Positive and negative feedback loops are ubiquitous motifs in biochemical signaling pathways. The mitogen-activated protein kinase (MAPK) pathway module is part of many distinct signaling networks and comprises several of these motifs, whose functioning depends on the cell line at hand and on the particular context. The maintainance of specificity of the response of the MAPK module to distinct stimuli has become a key paradigm especially in PC-12 cells, where the same module leads to different cell fates, depending on the stimulating growth factor. This cell fate is regulated by differences in the ERK (MAPK) activation profile, which shows a transient response upon stimulation with EGF, while the response is sustained in case of NGF. This behavior was explained by different effective network topologies. It is widely believed that this sustained response requires a bistable system. In this study we present a sampling-based Bayesian model analysis on a dataset, in which PC-12 cells have been stimulated with different growth factors. This is combined with novel analysis methods to investigate the role of feedback interconnections to shape ERK response. Results strongly suggest that, besides bistability, an additional effect called quasi-bistability can contribute to explain the observed responses of the system to different stimuli. Quasi-bistability is the ability of a monostable system to maintain two distinct states over a long time period upon a transient signal, which is also related to positive feedback, but cannot be detected by standard steady state analysis methods. Although applied on a specific example, our framework is generic enough to be also relevant for other regulatory network modeling studies that comprise positive feedback to explain cellular decision making processes. Overall, this study advices to focus not only on steady states, but also to take transient behavior into account in the analysis.

Comparative human cellular radiosensitivity: I. The effect of SV40 transformation and immortalisation on the gamma-irradiation survival of skin derived fibroblasts from normal individuals and from ataxia-telangiectasia patients and heterozygotes.

PubMed

Arlett, C F; Green, M H; Priestley, A; Harcourt, S A; Mayne, L V

1988-12-01

We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and D, estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but D is more realistic when comparing shouldered and non-shouldered data.

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

PubMed

Piulats, Jose M; Kondo, Jumpei; Endo, Hiroko; Ono, Hiromasa; Hagihara, Takeshi; Okuyama, Hiroaki; Nishizawa, Yasuko; Tomita, Yasuhiko; Ohue, Masayuki; Okita, Kouki; Oyama, Hidejiro; Bono, Hidemasa; Masuko, Takashi; Inoue, Masahiro

2018-03-23

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

NASA Technical Reports Server (NTRS)

Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.

1999-01-01

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Potential of Equine Herpesvirus 1 as a Vector for Immunization

PubMed Central

Trapp, Sascha; von Einem, Jens; Hofmann, Helga; Köstler, Josef; Wild, Jens; Wagner, Ralf; Beer, Martin; Osterrieder, Nikolaus

2005-01-01

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells. PMID:15827159

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins.

PubMed

Kozono, Takuma; Tadahira, Kazuko; Okumura, Wataru; Itai, Nao; Tamura-Nakano, Miwa; Dohi, Taeko; Tonozuka, Takashi; Nishikawa, Atsushi

2018-06-06

Jaw1/LRMP is characterized as a type II integral membrane protein that is localized to endoplasmic reticulum (ER), however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with KASH proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with SUN proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules.

Electrical contact structures for solid oxide electrolyte fuel cell

DOEpatents

Isenberg, Arnold O.

1984-01-01

An improved electrical output connection means is provided for a high temperature solid oxide electrolyte type fuel cell generator. The electrical connection of the fuel cell electrodes to the electrical output bus, which is brought through the generator housing to be connected to an electrical load line maintains a highly uniform temperature distribution. The electrical connection means includes an electrode bus which is spaced parallel to the output bus with a plurality of symmetrically spaced transversely extending conductors extending between the electrode bus and the output bus, with thermal insulation means provided about the transverse conductors between the spaced apart buses. Single or plural stages of the insulated transversely extending conductors can be provided within the high temperatures regions of the fuel cell generator to provide highly homogeneous temperature distribution over the contacting surfaces.

CD22 and Siglec-G in B cell function and tolerance

PubMed Central

Poe, Jonathan C.; Tedder, Thomas F.

2012-01-01

The immune system has evolved into two main arms, the primitive innate arm that is the first line of defense but relatively short-lived and broad acting, and the advanced adaptive arm that generates immunologic “memory” allowing rapid, specific recall responses. T cell-independent type-2 (TI-2) antigens (Ags) invoke innate immune responses. However, due to its “at the ready” nature, how the innate arm of the immune system maintains tolerance to potentially abundant host TI-2 Ags remains elusive. Therefore, it is important to define the mechanisms that establish innate immune tolerance. This review highlights recent insights into B cell tolerance to theoretical self TI-2 Ags, and examines how the B cell-restricted Siglecs, CD22 and Siglec-G, might contribute to this process. PMID:22677186

Establishment of primary cell cultures from fish calcified tissues

PubMed Central

Marques, Cátia L.; Rafael, Marta S.; Cancela, M. Leonor

2007-01-01

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case. PMID:19002990

The preservation of living cells with biocompatible microparticles

NASA Astrophysics Data System (ADS)

Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

2016-07-01

Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

Dormancy activation mechanism of oral cavity cancer stem cells.

PubMed

Chen, Xiang; Li, Xin; Zhao, Baohong; Shang, Dehao; Zhong, Ming; Deng, Chunfu; Jia, Xinshan

2015-07-01

Radiotherapy and chemotherapy are targeted primarily at rapidly proliferating cancer cells and are unable to eliminate cancer stem cells in the G0 phase. Thus, these treatments cannot prevent the recurrence and metastasis of cancer. Understanding the mechanisms by which cancer stem cells are maintained in the dormant G0 phase, and how they become active is key to developing new cancer therapies. The current study found that the anti-cancer drug 5-fluorouracil, acting on the oral squamous cell carcinoma KB cell line, selectively killed proliferating cells while sparing cells in the G0 phase. Bisulfite sequencing PCR showed that demethylation of the Sox2 promoter led to the expression of Sox2. This then resulted in the transformation of cancer stem cells from the G0 phase to the division stage and suggested that the transformation of cancer stem cells from the G0 phase to the division stage is closely related to an epigenetic modification of the cell.

Basics of PD-1 in self-tolerance, infection, and cancer immunity.

PubMed

Chikuma, Shunsuke

2016-06-01

Successful cancer treatment requires understanding host immune response against tumor cells. PD-1 belongs to the CD28 superfamily of receptors that work as "checkpoints" of immune activation. PD-1 maintains immune self-tolerance to prevent autoimmunity and controls T-cell reaction during infection to prevent excessive tissue damage. Tumor cells that arise from normal tissue acquire mutations that can be targeted by lymphocytes. Accumulating lines of evidence suggest that tumor cells evade host immune attack by expressing physiological PD-1 ligands and stimulating PD-1 on the lymphocytes. Based on this idea, researchers have successfully demonstrated that systemic administration of monoclonal antibodies that inhibit the binding of PD-1 to the ligands reactivated T cells and augmented the anti-cancer immune response. In this review, I summarize the basics of T-cell biology and its regulation by PD-1 and discuss the current understanding and questions about this multifaceted molecule.

Phenotypically silent Cre recombination within the postnatal ventricular conduction system.

PubMed

Bhattacharyya, Samadrita; Bhakta, Minoti; Munshi, Nikhil Vilas

2017-01-01

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3' untranslated region (3'UTR). Here we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

PubMed

An, Jeung Hee; Kim, Seung U; Park, Mi-Kyung; Choi, Jeong Woo

2015-10-01

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Transforming growth factor-beta1 transcriptionally activates CD34 and prevents induced differentiation of TF-1 cells in the absence of any cell-cycle effects.

PubMed

Marone, M; Scambia, G; Bonanno, G; Rutella, S; de Ritis, D; Guidi, F; Leone, G; Pierelli, L

2002-01-01

A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-beta1 has been shown to activate a TGF-beta-activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-beta1 and CD34 overexpression. Our data demonstrate that TGF-beta1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents.

Induction of antigen-specific cytotoxic T-cell response by dendritic cells generated from ecto-mesenchymal stem cells infected with an adenovirus containing the MAGE-D4a gene.

PubMed

Hu, Shijie; Li, Bing; Shen, Xuefeng; Zhang, Rui; Gao, Dakuan; Guo, Qingdong; Jin, Yan; Fei, Zhou

2016-04-01

The present study aimed to investigate the feasibility of using ecto-mesenchymal stem cell (EMSC)-derived dendritic cells (DCs) for glioma immunotherapy following infection by a recombinant adenovirus containing the melanoma-associated antigen D4a (MAGE-D4a) gene. The ex vivo cultured EMSCs were infected by the adenoviral plasmid containing MAGE-D4a (pAd/MAGE-D4a). Efficiency of transfection was evaluated through the detection of green fluorescent protein-marked MAGE-D4a. The MAGE-EMSCs were induced to differentiate into DCs, termed as MAGE-EMSCs-DCs. The morphology was subsequently analyzed under a microscope, and methyl thiazolyl tetrazolium (MTT) and interferon-γ (IFN-γ) assays were performed to analyze the cytotoxicity of the MAGE-EMSC-DCs on the human glioma U251 cell line. Following purification by magnetic-activated cell sorting, the EMSCs grew into swirls, with a long spindle shape and were fibroblast-like. The gene transfected with recombinant adenovirus vectors maintained high and stable expression levels of MAGE-D4a, and its efficiency was increased in a multiplicity of infection-dependent manner. The results of the MTT assay indicated that the T cells, primed by the recombinant MAGE-D4a-infected EMSC-DCs in vitro , recognized MAGE-D4a-expressing tumor cell lines in a human leukocyte antigen class I-restricted manner, and evoked a higher cytotoxic T cell (CTL) response. The CTL response induced by the MAGE-EMSC-DCs, co-cultured with the U251 cells for 24 h, produced 765.0 pg/ml IFN-γ, which was significantly greater when compared to the control wells. T lymphocytes stimulated by MAGE-EMSC-DCs evoke a higher CTL response to human glioma cell lines, and may serve as a promising therapeutic modality for the treatment of MAGE-D4a-expressing glioma.

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

PubMed Central

Velugula‐Yellela, Sai Rashmika; Williams, Abasha; Trunfio, Nicholas; Hsu, Chih‐Jung; Chavez, Brittany; Yoon, Seongkyu

2017-01-01

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 106 cells‐d/mL, while maintaining specific antibody production (Qp > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018 PMID:29086492

Development of an automated high temperature valveless injection system for on-line gas chromatography

NASA Astrophysics Data System (ADS)

Kreisberg, N. M.; Worton, D. R.; Zhao, Y.; Isaacman, G.; Goldstein, A. H.; Hering, S. V.

2014-07-01

A reliable method of sample introduction is presented for on-line gas chromatography with a special application to in-situ field portable atmospheric sampling instruments. A traditional multi-port valve is replaced with a controlled pressure switching device that offers the advantage of long term reliability and stable sample transfer efficiency. An engineering design model is presented and tested that allows customizing the interface for other applications. Flow model accuracy is within measurement accuracy (1%) when parameters are tuned for an ambient detector and 15% accurate when applied to a vacuum based detector. Laboratory comparisons made between the two methods of sample introduction using a thermal desorption aerosol gas chromatograph (TAG) show approximately three times greater reproducibility maintained over the equivalent of a week of continuous sampling. Field performance results for two versions of the valveless interface used in the in-situ instrument demonstrate minimal trending and a zero failure rate during field deployments ranging up to four weeks of continuous sampling. Extension of the VLI to dual collection cells is presented with less than 3% cell-to-cell carry-over.

Cycling to Maintain and Improve Fitness: Line-1 Modes of Nuclear Entrance and Retrotransposition.

PubMed

Mita, Paolo; Boeke, Jef D

2018-04-01

The LINE-1/L1 retrotransposon is a transposable element still active in the human genome. Most retrotransposons in the genome are inactive or repressed by several host mechanisms. In specific contexts, active L1 retrotransposons may evade repression and copy themselves into new genomic loci. Despite a general knowledge of the L1 life cycle, little was known about the dynamics of L1 proteins and function during the different stages of the host cell cycle. Our work highlighted a well-orchestrated localization of L1 proteins and mRNA that take advantage of mitotic nuclear membrane breakdown. Once in the nucleus, L1 ribonucleoproteins (RNPs) are able to retrotranspose during the S phase when L1 retrotransposition peaks. Our conclusions highlight previously unappreciated features of the L1 life cycle, such as the differences between cytoplasmic and nuclear RNPs and the cycling of L1 ORF1 protein and L1 activity during progression through the cell cycle. These new observations are discussed here in light of the evolutionary arms race between L1 retrotransposons and the host cell.

3D gut-liver chip with a PK model for prediction of first-pass metabolism.

PubMed

Lee, Dong Wook; Ha, Sang Keun; Choi, Inwook; Sung, Jong Hwan

2017-11-07

Accurate prediction of first-pass metabolism is essential for improving the time and cost efficiency of drug development process. Here, we have developed a microfluidic gut-liver co-culture chip that aims to reproduce the first-pass metabolism of oral drugs. This chip consists of two separate layers for gut (Caco-2) and liver (HepG2) cell lines, where cells can be co-cultured in both 2D and 3D forms. Both cell lines were maintained well in the chip, verified by confocal microscopy and measurement of hepatic enzyme activity. We investigated the PK profile of paracetamol in the chip, and corresponding PK model was constructed, which was used to predict PK profiles for different chip design parameters. Simulation results implied that a larger absorption surface area and a higher metabolic capacity are required to reproduce the in vivo PK profile of paracetamol more accurately. Our study suggests the possibility of reproducing the human PK profile on a chip, contributing to accurate prediction of pharmacological effect of drugs.

Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.

PubMed

Barrett, Robert; Ornelas, Loren; Yeager, Nicole; Mandefro, Berhan; Sahabian, Anais; Lenaeus, Lindsay; Targan, Stephan R; Svendsen, Clive N; Sareen, Dhruv

2014-12-01

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblast-derived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications. ©AlphaMed Press.

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

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features

PubMed Central

Li, Xiaoran; Zhong, Yali; Lu, Jie; Axcrona, Karol; Eide, Lars; Syljuåsen, Randi G.; Peng, Qian; Wang, Junbai; Zhang, Hongquan; Goscinski, Mariusz Adam; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features. PMID:27248169

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

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

Silva, Patricio; Soto, Nicolás; Díaz, Jorge

2015-08-21

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5more » is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.« less

Pax6 influences expression patterns of genes involved in neuro- degeneration.

PubMed

Mishra, Suman; Maurya, Shashank Kumar; Srivastava, Khushboo; Shukla, Sachin; Mishra, Rajnikant

2015-10-01

Pax6, a highly conserved multifunctional transcription factor, has been critical for neurogenesis and neuronal plasticity. It is presumed that if level of Pax6 approaches either low or null, critical genes responsible for maintaining functional status of neurons or glia would be modulated. Therefore, it has been intended to explore possibility of either direct or indirect influence of Pax6 in neurodegeneration. The cell lines having origin of murine embryonic fibroblast (Pax6-non expressing, NIH3T3-cell line), murine neuroblastoma (Pax6-expressing brain-derived, Neuro-2a-cell line), and human glioblastoma-astrocytoma (U87MG) were cultured and maintained in a CO2 incubator at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. The knockdown of endogenous Pax6 in Neuro-2a cells was achieved through siRNA based gene knock-down approach. The efficiency and validation of knock-down was done by real time PCR. The knock-down of Pax6 was successfully achieved. The levels of expression of transcripts of some of the proposed putative markers of neurodegeneration like Pax6, S100β, GFAP, BDNF, NGN2, p73α, p73δ, LDH, SOD, and Catalase were analyzed in Pax6 knockdown condition for analysis of role of Pax6 in neurodegeneration. Since the Pax6 has been proposed to bind to promoter sequences of catalase, and catalase suppresses TGFβ, relative lower levels of catalase in Neuro-2a and U-87MG as compared to NIH-3T3 indicates a possible progressive dominant negative impact of Pax6. However, presence of SOD and LDH indicates alternative protective mechanism. Presence of BDNF and TGFβ indicates association between them in glioblastoma-astrocytoma. Therefore, Pax6 seems to be involved directly with p53 and TGFβ mediated pathways and indirectly with redox-sensitive pathway regulation. The neurodegenerative markers S100β, GFAP, BDNF, NGN2, p73α, p73δ, observed downregulated in Pax6 knockdown condition suggest Pax6-mediated regulation of these markers. Observations enlighten Pax6-mediated influences on cascades of genes involved in growth, differentiation and maturation of neurons and glia.

Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

PubMed

Sanders, Barbara P; Oakes, Isabel de los Rios; van Hoek, Vladimir; Liu, Ying; Marissen, Wilfred; Minor, Philip D; Wimmer, Eckard; Schuitemaker, Hanneke; Custers, Jerome H H V; Macadam, Andrew; Cello, Jeronimo; Edo-Matas, Diana

2015-11-27

As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (p<0.0001) on the PER.C6(®) cell platform as compared to the Vero cell platform in infectious titer and D-antigen content. Furthermore, Sabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and maintaining poliovirus eradication. Copyright © 2015 Elsevier Ltd. All rights reserved.

CXCL12/CXCR4 pathway is activated by oncogenic JAK2 in a PI3K-dependent manner

PubMed Central

Abdelouahab, Hadjer; Zhang, Yanyan; Wittner, Monika; Oishi, Shinya; Fujii, Nobutaka; Besancenot, Rodolphe; Plo, Isabelle; Ribrag, Vincent; Solary, Eric; Vainchenker, William; Barosi, Giovanni; Louache, Fawzia

2017-01-01

JAK2 activation is the driver mechanism in BCR-ABL-negative myeloproliferative neoplasms (MPN). These diseases are characterized by an abnormal retention of hematopoietic stem cells within the bone marrow microenvironment and their increased trafficking to extramedullary sites. The CXCL12/CXCR4 axis plays a central role in hematopoietic stem cell/ progenitor trafficking and retention in hematopoietic sites. The present study explores the crosstalk between JAK2 and CXCL12/CXCR4 signaling pathways in MPN. We show that JAK2, activated by either MPL-W515L expression or cytokine stimulation, cooperates with CXCL12/CXCR4 signaling to increase the chemotactic response of human cell lines and primary CD34+ cells through an increased phosphatidylinositol-3-kinase (PI3K) signaling. Accordingly, primary myelofibrosis (MF) patient cells demonstrate an increased CXCL12-induced chemotaxis when compared to controls. JAK2 inhibition by knock down or chemical inhibitors decreases this effect in MPL-W515L expressing cell lines and reduces the CXCL12/CXCR4 signaling in some patient primary cells. Taken together, these data indicate that CXCL12/CXCR4 pathway is overactivated in MF patients by oncogenic JAK2 that maintains high PI3K signaling over the threshold required for CXCR4 activation. These results suggest that inhibition of this crosstalk may contribute to the therapeutic effects of JAK2 inhibitors. PMID:28903325

AML sensitivity to YM155 is modulated through AKT and Mcl-1

PubMed Central

de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J.; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R.; Chen, Chien-Shing

2015-01-01

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound’s ability to downregulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

PubMed

Rodriguez, A M; Graef, A J; LeVine, D N; Cohen, I R; Modiano, J F; Kim, J-H

2015-01-01

Sphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells. S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

PubMed

Essone, Jean Claude Biteghe Bi; N'Dilimabaka, Nadine; Ondzaga, Julien; Lekana-Douki, Jean Bernard; Mba, Dieudonné Nkoghe; Deloron, Philippe; Mazier, Dominique; Gay, Frédrérick; Touré Ndouo, Fousseyni S

2017-06-27

Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells.

PubMed

Kobayashi, Yuki; Hayashi, Ryuhei; Quantock, Andrew J; Nishida, Kohji

2017-12-01

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Simultaneous targeting of EGFR, HER2, and HER4 by afatinib overcomes intrinsic and acquired cetuximab resistance in head and neck squamous cell carcinoma cell lines.

PubMed

De Pauw, Ines; Lardon, Filip; Van den Bossche, Jolien; Baysal, Hasan; Fransen, Erik; Deschoolmeester, Vanessa; Pauwels, Patrick; Peeters, Marc; Vermorken, Jan Baptist; Wouters, An

2018-06-01

The epidermal growth factor receptor (EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR-targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti-EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first-generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab-sensitive, intrinsically cetuximab-resistant, and acquired cetuximab-resistant HNSCC cell lines, independent of the HPV status. However, cross-resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G 0 /G 1 cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Rewiring the taste system.

PubMed

Lee, Hojoon; Macpherson, Lindsey J; Parada, Camilo A; Zuker, Charles S; Ryba, Nicholas J P

2017-08-17

In mammals, taste buds typically contain 50-100 tightly packed taste-receptor cells (TRCs), representing all five basic qualities: sweet, sour, bitter, salty and umami. Notably, mature taste cells have life spans of only 5-20 days and, consequently, are constantly replenished by differentiation of taste stem cells. Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (that is, ensuring that a labelled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, and so on), we examined how new connections are specified to retain fidelity of signal transmission. Here we show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (SEMA3A and SEMA7A). We demonstrate that targeted expression of SEMA3A or SEMA7A in different classes of TRCs produces peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered mice with bitter neurons that now responded to sweet tastants, sweet neurons that responded to bitter or sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labelled-line sensory circuit preserves signalling integrity despite rapid and stochastic turnover of receptor cells.

Rewiring the Taste System

PubMed Central

Lee, Hojoon; Macpherson, Lindsey J.; Parada, Camilo A.; Zuker, Charles S.; Ryba, Nicholas J.P.

2018-01-01

In mammals, taste buds typically contain 50-100 tightly packed taste receptor cells (TRCs) representing all five basic qualities: sweet, sour, bitter, salty and umami1,2. Notably, mature taste cells have life spans of only 5-20 days, and consequently, are constantly replenished by differentiation of taste stem cells3. Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (i.e. ensuring that a labeled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, etc.), we examined how new connections are specified to retain fidelity of signal transmission. Our results show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (Sema3A and Sema7A)4-6. Here, we demonstrate that targeted expression of Sema3A or Sema7A in different classes of TRCs produce peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered animals whereby bitter neurons now respond to sweet tastants, sweet neurons respond to bitter, or with sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labeled-line sensory circuit preserves signaling integrity despite rapid and stochastic turnover of receptor cells. PMID:28792937

Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

PubMed

Zhang, Wenlin; Ogando, Diego G; Kim, Edward T; Choi, Moon-Jung; Li, Hongde; Tenessen, Jason M; Bonanno, Joseph A

2017-07-01

To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). The immortalized Slc4a11+/+ and Slc4a11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs. This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11-/- MCECs. Furthermore, Slc4a11-/- MCECs recapitulate the glutaminolysis defects observed in Slc4a11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.

The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment.

PubMed

Wang, Juan; Wei, Yun; Zhao, Shasha; Zhou, Ying; He, Wei; Zhang, Yang; Deng, Wensheng

2017-01-01

Mammalian cells are very important experimental materials and widely used in biological and medical research fields. It is often required that mammalian cells are transported from one laboratory to another to meet with various researches. Conventional methods for cell shipment are laborious and costive despite of maintaining high viability. In this study we aimed to develop a simple and low-cost method for cell shipment by investigating the viabilities of different cell lines treated at different temperatures. We show that the viability of mammalian cells incubated at 1°C or 5°C significantly reduced when compared with that at 16°C or 22°C. Colony formation assays revealed that preservation of mammalian cells at 1°C or 5°C led to a poorer recovery than that at 16°C or 22°C. The data from proliferation and apoptotic assays confirmed that M2 cells could continue to proliferate at 16°C or 22°C, but massive death was caused by apoptosis at 1°C or 5°C. The morphology of mammalian cells treated under hypothermia showed little difference from that of the untreated cells. Quantitative RT-PCR and alkaline phosphatase staining confirmed that hypothermic treatment did not change the identity of mouse embryonic stem cells. A case study showed that mammalian cells directly suspended in culture medium were able to be shipped for long distance and maintained a high level of viability and recovery. Our findings not only broaden the understanding to the effect of hypothermia on the viability of mammalian cells, but also provide an alternative approach for cell shipment.

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

PubMed Central

Frattini, M G; Lim, H B; Laimins, L A

1996-01-01

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8610168

Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells.

PubMed

Kanno, Sanae; Hirano, Seishiro; Kayama, Fujio

2004-03-01

Phytoestrogens and environmental estrogens, which have in part some structural similarity to 17beta-estradiol, are reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decreases after menopause and the estrogen deficiency results in bone loss. In this study, we report the effects of phytoestrogens (genistein, daidzein, and coumestrol) and environmental estrogens (bisphenol A (BPA), p-n-nonylphenol (NP) and bis(2-ethylhexyl)phthalate (DEHP)) on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. Coumestrol (10(-10) to 10(-6)M) slightly enhanced cell proliferation, while neither the other phytoestrogens (daidzein, genistein) nor environmental estrogens increased cell proliferation. Alkaline phosphatase (ALP) activity and cellular calcium (Ca) and phosphorus (P) contents were increased by phytoestrogens and BPA; however, neither NP nor DEHP affected those osteoblastic indicators. The effects of estrogenic potency, using the cell proliferation of MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line, indicate that coumestrol has the highest estrogenic potency among those phytoestrogens and environmental estrogens. The estrogenic potency of NP and DEHP were lower than the others. In conclusion, phytoestrogens, such as coumestrol, genistein and daidzein, and BPA increased ALP activity and enhanced bone mineralization in MC3T3-E1 cells, suggesting that not only phytoestrogen but also BPA, an environmental estrogen, is implicated in bone metabolism.

Isolation and characterization of the trophectoderm from the Arabian camel (Camelus dromedarius).

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Elsafadi, Mona; Moumen, Abdullah F; Alzahrani, Faisal A; Mahmood, Amer; Alfayez, Musaad; Alowaimer, Abdullah N

2017-09-01

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels. Copyright © 2017 Elsevier Ltd. All rights reserved.

Anthothecol-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell growth by modulating sonic hedgehog pathway.

PubMed

Verma, Raj Kumar; Yu, Wei; Singh, Surya Pratap; Shankar, Sharmila; Srivastava, Rakesh K

2015-11-01

Anthothecol, a limonoid isolated from plant Khaya anthotheca (Meliaceae), is an antimalarial compound. The objectives of this study were to examine the molecular mechanisms by which anthothecol-encapsulated PLGA-nanoparticles (Antho-NPs) regulate the behavior of pancreatic cancer stem cells (CSCs). Antho-NPs inhibited cell proliferation and colony formation, and induced apoptosis in pancreatic CSCs and cancer cell lines, but had no effects on human normal pancreatic ductal epithelial cells. Antho-NPs inhibited self-renewal capacity of pancreatic CSCs isolated from human and Kras(G12D) mice. Furthermore, antho-NPs suppressed cell motility, migration and invasion by up-regulating E-cadherin and inhibiting N-cadherin and Zeb1. In addition, Antho-NPs inhibited pluripotency maintaining factors and stem cell markers, suggesting their inhibitory role on CSC population. Anthothecol disrupted binding of Gli to DNA, and inhibited Gli transcription and Gli target genes. Our studies establish preclinical significance of Antho-NPs for the treatment and/or prevention of pancreatic cancer. Despite medical advances, the prognosis of pancreatic cancer remains poor. The search for an effective treatment has been under intensive research for some time. In this article, the authors investigated the efficacy and mechanism of anthothecol (an antimalarial compound), encapsulated by PLGA nanoparticles (Antho-NPs), against pancreatic cancer cell lines. It was found that Antho-NPs acted via the Sonic hedgehog signaling pathway and inhibited cancer stem cell growth. These results have provided important basis for further clinical trials. Copyright © 2015 Elsevier Inc. All rights reserved.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy

PubMed Central

Wilkinson, Daniel S.; Ghosh, Debjani; Nickle, Rebecca A.; Moorman, Cody D.; Mannie, Mark D.

2017-01-01

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible “iTregs” because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy. PMID:29312311

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

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

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk; Flatt, Peter R.; McClenaghan, Neville H.

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mMmore » glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.« less

IL-3 induces apoptosis in a ras-transformed myeloid cell line.

PubMed

Ahmed, N; Anderson, S M; Berridge, M V

1999-04-01

Growth factors promote cell survival and proliferation. Homeostasis is maintained by programmed cell death which occurs when the growth stimulus is withdrawn, in response to negative growth regulators such as interferons, TNF-alpha and CD95 ligand, or following differentiation. Although acutely-transforming oncogenes often overcome the need for growth factors, growth regulatory cytokines can influence proliferative responses of transformed cells. In this study we investigated the effects of IL-3 on the proliferative responses of parental bone marrow-derived 32D cells and cells transformed with ras and abl oncogenes. We show that treatment of ras-transformed 32D cells with IL-3 reduced proliferative responses and decreased colony-forming ability. These effects were exacerbated in the absence of serum and associated with inhibition of tyrosine kinase activity, down-regulation of RAS and MYC expression, and induction of apoptosis as indicated by DNA fragmentation. In contrast, treatment of parental 32D cells with IL-3, which is obligatory for cell survival and proliferation, increased tyrosine kinase activity, upregulated MYC and RAS expression and maintained DNA integrity. With abl-transformed cells, proliferation and colony-forming ability were also inhibited by IL-3. Tyrosine kinase activity and MYC expression were reduced, but early apoptosis was not evident. Calcium uptake however, was stimulated by IL-3 in both parental and oncogene-transformed cells. These results suggest that threshold levels of tyrosine kinase activity are necessary for cell survival and proliferation and that with ras-transformed cells, IL-3 treatment may result in this threshold being breached. We conclude that in some situations, growth-promoting cytokines can inhibit proliferation of transformed cells and induce cell death by apoptosis.

Human embryonic stem cell research: implications from an ethical and legal standpoint.

PubMed

Trepagnier, D M

2000-12-01

The purpose of this paper is to discuss the ethical and legal implications of one of the newest and most controversial medical breakthroughs. Stem cell research has been performed on mice for many years, but human embryonic stem cells are believed by scientists to be the basis for possible treatments and/or cures to many diseases affecting millions of people around the world. In order to perform research on human embryonic stem cells, numerous ethical issues must be addressed. Guidelines and protocols can be established in order to allow scientists to pursue new medical advances while maintaining the highest ethical standards in the use of human embryos. An alternative to using embryos is adult stem cells which have recently proven to be more versatile than previously believed. Opposing views will always be encountered when facing new science technologies. Where should the ethical line be drawn?

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

PubMed

Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

2015-06-01

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Biological effects of low-dose-rate irradiation of pancreatic carcinoma cells in vitro using 125I seeds

PubMed Central

Wang, Zhong-Min; Lu, Jian; Zhang, Li-Yun; Lin, Xiao-Zhu; Chen, Ke-Min; Chen, Zhi-Jin; Liu, Fen-Ju; Yan, Fu-Hua; Teng, Gao-Jun; Mao, Ai-Wu

2015-01-01

AIM: To determine the mechanism of the radiation-induced biological effects of 125I seeds on pancreatic carcinoma cells in vitro. METHODS: SW1990 and PANC-1 pancreatic cancer cell lines were cultured in DMEM in a suitable environment. Gray’s model of iodine-125 (125I) seed irradiation was used. In vitro, exponential phase SW1990, and PANC-1 cells were exposed to 0, 2, 4, 6, and 8 Gy using 125I radioactive seeds, with an initial dose rate of 12.13 cGy/h. A clonogenic survival experiment was performed to observe the ability of the cells to maintain their clonogenic capacity and to form colonies. Cell-cycle and apoptosis analyses were conducted to detect the apoptosis percentage in the SW1990 and PANC-1 cells. DNA synthesis was measured via a tritiated thymidine (3H-TdR) incorporation experiment. After continuous low-dose-rate irradiation with 125I radioactive seeds, the survival fractions at 2 Gy (SF2), percentage apoptosis, and cell cycle phases of the SW1990 and PANC-1 pancreatic cancer cell lines were calculated and compared. RESULTS: The survival fractions of the PANC-1 and SW1990 cells irradiated with 125I seeds decreased exponentially as the dose increased. No significant difference in SF2 was observed between SW1990 and PANC-1 cells (0.766 ± 0.063 vs 0.729 ± 0.045, P < 0.05). The 125I seeds induced a higher percentage of apoptosis than that observed in the control in both the SW1990 and PANC-1 cells. The rate of apoptosis increased with increasing radiation dosage. The percentage of apoptosis was slightly higher in the SW1990 cells than in the PANC-1 cells. Dose-dependent G2/M cell-cycle arrest was observed after 125I seed irradiation, with a peak value at 6 Gy. As the dose increased, the percentage of G2/M cell cycle arrest increased in both cell lines, whereas the rate of DNA incorporation decreased. In the 3H-TdR incorporation experiment, the dosimetry results of both the SW1990 and PANC-1 cells decreased as the radiation dose increased, with a minimum at 6 Gy. There were no significant differences in the dosimetry results of the two cell lines when they were exposed to the same dose of radiation. CONCLUSION: The pancreatic cancer cell-killing effects induced by 125I radioactive seeds mainly occurred via apoptosis and G2/M cell cycle arrest. PMID:25741139

Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock

PubMed Central

Webb, Alexis B; Lengyel, Iván M; Jörg, David J; Valentin, Guillaume; Jülicher, Frank; Morelli, Luis G; Oates, Andrew C

2016-01-01

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a “segmentation clock”. This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics. DOI: http://dx.doi.org/10.7554/eLife.08438.001 PMID:26880542

Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

PubMed

Avgerinos, G C; Drapeau, D; Socolow, J S; Mao, J I; Hsiao, K; Broeze, R J

1990-01-01

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

PubMed

Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

2015-01-01

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

CheckMate 141: 1-Year Update and Subgroup Analysis of Nivolumab as First-Line Therapy in Patients with Recurrent/Metastatic Head and Neck Cancer.

PubMed

Gillison, Maura L; Blumenschein, George; Fayette, Jerome; Guigay, Joel; Colevas, A Dimitrios; Licitra, Lisa; Harrington, Kevin J; Kasper, Stefan; Vokes, Everett E; Even, Caroline; Worden, Francis; Saba, Nabil F; Iglesias Docampo, Lara Carmen; Haddad, Robert; Rordorf, Tamara; Kiyota, Naomi; Tahara, Makoto; Monga, Manish; Lynch, Mark; Li, Li; Ferris, Robert L

2018-06-04

Nivolumab significantly improved overall survival (OS) vs investigator's choice (IC) of chemotherapy at the primary analysis of randomized, open-label, phase 3 CheckMate 141 in patients with recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN). Here, we report that OS benefit with nivolumab was maintained at a minimum follow-up of 11.4 months. Further, OS benefit with nivolumab vs IC was also noted among patients who received first-line treatment for R/M SCCHN after progressing on platinum therapy for locally advanced disease in the adjuvant or primary (i.e., with radiation) setting. The Oncologist 2018. © AlphaMed Press 2018.

Base-line O sub 2 extraction influences cerebral blood flow response to hematocrit

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

Hudak, M.L.; Tang, Yuilin; Massik, J.

1988-01-01

The authors have shown that the fall in cerebral blood flow (CBF) as hematocrit (Hct) rises is due to the independent effects of increasing red blood cell (RBC) concentration and arterial O{sub 2} content (Ca{sub O{sub 2}}). In the present study, they tested the hypothesis that the magnitude of the effect of RBC concentration depends on the base-line cerebral fractional oxygen extraction (E). Pentobarbital-anesthetized 1- to 7-day-old sheep were first exchange transfused with plasma to lower Hct to 20%. Base-line E was set to either high or low levels by induction of hypocarbia, or hypercarbia. A second isovolemic exchange transfusionmore » with pure methemoglobin-containing adult sheep red cells then raised Hct with no significant increase in Ca{sub O{sub 2}}. Pa{sub CO{sub 2}} was maintained and other variables with potential effect on CBF did not change. CBF corrected for any individual alteration in CMRo{sub 2}. This study supports the hypothesis that the magnitude of the decline in CBF secondary to an increase in RBC concentration depends on the initial E. The effect of RBC concentration on CBF is greatest when E is low.« less

Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

PubMed

Soh, JunYi; Chueng, Adeline; Adio, Aminat; Cooper, Alan J; Birch, Brian R; Lwaleed, Bashir A

2013-04-01

Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines.

PubMed

Adam, Aziza A A; van der Mark, Vincent A; Donkers, Joanne M; Wildenberg, Manon E; Oude Elferink, Ronald P J; Chamuleau, Robert A F M; Hoekstra, Ruurdtje

2018-01-01

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

PubMed

Kalb, Daniel M; Fencl, Frank A; Woods, Travis A; Swanson, August; Maestas, Gian C; Juárez, Jaime J; Edwards, Bruce S; Shreve, Andrew P; Graves, Steven W

2017-09-19

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity

PubMed Central

Mattis, Virginia B.; Tom, Colton; Akimov, Sergey; Saeedian, Jasmine; Østergaard, Michael E.; Southwell, Amber L.; Doty, Crystal N.; Ornelas, Loren; Sahabian, Anais; Lenaeus, Lindsay; Mandefro, Berhan; Sareen, Dhruv; Arjomand, Jamshid; Hayden, Michael R.; Ross, Christopher A.; Svendsen, Clive N.

2015-01-01

Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of ‘persistent’ neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics. PMID:25740845

Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

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

Kito, Hiroaki; Yamazaki, Daiju; Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto

Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cellmore » turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.« less

[Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system].

PubMed

Luo, Shun-Tao; Tian, Wen-Hong; Wang, Gang; Dong, Xiao-Yan; Yang, Li; Wu, Xiao-Bing

2009-11-01

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.

Robust imaging and gene delivery to study human lymphoblastoid cell lines.

PubMed

Jolly, Lachlan A; Sun, Ying; Carroll, Renée; Homan, Claire C; Gecz, Jozef

2018-06-20

Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.

Effectiveness of Provider Education Followed by Computerized Provider Order Entry Alerts in Reducing Inappropriate Red Blood Cell Transfusion.

PubMed

Patel, Vijay M; Rains, Anna W; Clark, Christopher T

2016-01-01

To reduce the rate of inappropriate red blood cell transfusion, a provider education program, followed by alerts in the computerized provider order entry system (CPOE), was established to encourage AABB transfusion guidelines. Metrics were established for nonemergent inpatient transfusions. Service lines with high order volume were targeted with formal education regarding AABB 2012 transfusion guidelines. Transfusion orders were reviewed in real time with email communications sent to ordering providers falling outside of AABB recommendations. After 12 months of provider education, alerts were activated in CPOE. With provider education alone, the incidence of pretransfusion hemoglobin levels greater than 8 g/dL decreased from 16.64% to 6.36%, posttransfusion hemoglobin levels greater than 10 g/dL from 14.03% to 3.78%, and number of nonemergent two-unit red blood cell orders from 45.26% to 22.66%. Red blood cell utilization decreased by 13%. No additional significant reduction in nonemergent two-unit orders was observed with CPOE alerts. Provider education, an effective and low-cost method, should be considered as a first-line method for reducing inappropriate red blood cell transfusion rates in stable adult inpatients. Alerts in the computerized order entry system did not significantly lower the percentage of two-unit red blood cells orders but may help to maintain educational efforts.

Circadian Timing in the Lung; A Specific Role for Bronchiolar Epithelial Cells

PubMed Central

Gibbs, J. E.; Beesley, S.; Plumb, J.; Singh, D.; Farrow, S.; Ray, D. W.; Loudon, A. S. I.

2015-01-01

In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology. PMID:18787022

Long-term Culture and Cloning of Primary Human Bronchial Basal Cells that Maintain Multipotent Differentiation Capacity and CFTR Channel Function.

PubMed

Peters-Hall, Jennifer Ruth; Coquelin, Melissa L; Torres, Michael J; LaRanger, Ryan; Alabi, Busola Ruth; Sho, Sei; Calva-Moreno, Jose Francisco; Thomas, Philip J; Shay, Jerry William

2018-05-03

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome editing tools. Recently, conditional reprogramming of cells (CRC) with a ROCK inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the lifespan of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial growth medium instead of F-medium and 2% oxygen instead of 21% oxygen, that extend HBEC lifespan while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof of concept, CRISPR/Cas9 genome editing and cloning was used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals.

PubMed

Powell, Robin H; Behnke, Michael S

2017-05-15

Recent years have seen significant developments in the ability to continuously propagate organoids derived from intestinal crypts. These advancements have been applied to mouse and human samples providing models for gastrointestinal tissue development and disease. We adapt these methods for the propagation of intestinal organoids (enteroids) from various large farm and small companion (LF/SC) animals, including cat, dog, cow, horse, pig, sheep and chicken. We show that LF/SC enteroids propagate and expand in L-WRN conditioned media containing signaling factors Wnt3a, R-spondin-3, and Noggin (WRN). Multiple successful isolations were achieved for each species, and the growth of LF/SC enteroids was maintained to high passage number. LF/SC enteroids expressed crypt stem cell marker LGR5 and low levels of mesenchymal marker VIM. Labeling with EdU also showed distinct regions of cell proliferation within the enteroids marking crypt-like regions. The ability to grow and maintain LF/SC enteroid cell lines provides additional models for the study of gastrointestinal developmental biology as well as platforms for the study of host-pathogen interactions between intestinal cells and zoonotic enteric pathogens of medical importance. © 2017. Published by The Company of Biologists Ltd.

Third-line treatment with second-generation tyrosine kinase inhibitors (dasatinib or nilotinib) in patients with chronic myeloid leukemia after two prior TKIs: real-life data on a single center experience along with the review of the literature.

PubMed

Ongoren, Seniz; Eskazan, Ahmet Emre; Suzan, Veysel; Savci, Sercan; Erdogan Ozunal, Isil; Berk, Selin; Yalniz, Fevzi Fırat; Elverdi, Tugrul; Salihoglu, Ayse; Erbilgin, Yucel; Iseri, Sibel Aylin; Ar, Muhlis Cem; Baslar, Zafer; Aydin, Yildiz; Tuzuner, Nukhet; Ozbek, Ugur; Soysal, Teoman

2018-05-01

Newer tyrosine kinase inhibitors (TKIs) (bosutinib, ponatinib) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be utilized as a salvage therapy in patients with chronic myeloid leukemia (CML) who failed two lines (imatinib → nilotinib or imatinib → dasatinib) of TKI therapy. However, these TKIs are not available in many countries and not all patients can undergo allo-HSCT. In this study, CML patients who received dasatinib or nilotinib as a third-line treatment were retrospectively evaluated. Out of 209 patients, third-line dasatinib/nilotinib was administered in 21. During the follow-up, 16 out of 21 patients gained and/or maintained an optimal response, and 4 patients died due to progression. Seventeen patients were alive at the time of the analysis, of which 13 were still on TKI, whereas 4 patients quit treatment. In patients failing two lines of TKI, dasatinib or nilotinib can be beneficial and safely administered as a third-line treatment especially in nations with restricted resources.

HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion

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

Ball, Andrew J.; Abrahamsson, Annelie E.; Tyrberg, Bjoern

2007-04-06

To examine the mechanism by which growth-stimulated pancreatic {beta}-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and {beta}lox5, a cell line derived from human {beta}-cells which progressively dedifferentiated in culture. MIN6/{beta}lox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that {beta}lox5 expresses a dominant transacting factor(s) that represses {beta}-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary {beta}-cells as well as MIN6, but wasmore » repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in {beta}-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the {beta}-cell differentiated state.« less

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential.

PubMed

Muraglia, Anita; Todeschi, Maria Rosa; Papait, Andrea; Poggi, Alessandro; Spanò, Raffaele; Strada, Paolo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2015-12-01

Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

NANOG regulates epithelial-mesenchymal transition and chemoresistance in ovarian cancer.

PubMed

Qin, Shan; Li, Yanfang; Cao, Xuexia; Du, Jiexian; Huang, Xianghua

2017-02-28

A key transcription factor associated with poor prognosis and resistance to chemotherapy in ovarian cancer is NANOG. However, the mechanism by which NANOG functions remains undefined. It has been suggested that epithelial-to-mesenchymal transition (EMT) also contributes to development of drug resistance in different cancers. We thus determined whether NANOG expression was associated with EMT and chemoresistance in epithelial ovarian cancer cells. NANOG expression was increased in epithelial ovarian cancer cell lines compared with its expression in normal epithelial ovarian cell lines. NANOG expression in SKOV-3 or OV2008 cells directly correlated with high expression of mesenchymal cell markers and inversely with low expression of epithelial cell marker. RNAi-mediated silencing of NANOG in SKOV-3 reversed the expression of mesenchymal cell markers and restored expression of E-cadherin. Reversibly, stable overexpression of NANOG in Moody cells increased expression of N-cadherin whereas down-regulating expression of E-cadherin, cumulatively indicating that NANOG plays an important role in maintaining the mesenchymal cell markers. Modulating NANOG expression did not have any effect on proliferation or colony formation. Susceptibility to cisplatin increased in SKOV-3 cells on down-regulating NANOG and reversible results were obtained in Moody cells post-overexpression of NANOG. NANOG silencing in SKOV-3 and OV2008 robustly attenuated in vitro migration and invasion. NANOG expression exhibited a biphasic pattern in patients with ovarian cancer and expression was directly correlated to chemoresistance retrospectively. Cumulatively, our data demonstrate that NANOG expression modulates chemosensitivity and EMT resistance in ovarian cancer. © 2017 The Author(s).

Imaging intraflagellar transport in mammalian primary cilia.

PubMed

Besschetnova, Tatiana Y; Roy, Barnali; Shah, Jagesh V

2009-01-01

The primary cilium is a specialized organelle that projects from the surface of many cell types. Unlike its motile counterpart it cannot beat but does transduce extracellular stimuli into intracellular signals and acts as a specialized subcellular compartment. The cilium is built and maintained by the transport of proteins and other biomolecules into and out of this compartment. The trafficking machinery for the cilium is referred to as IFT or intraflagellar transport. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella. Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations. Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling. 2009 Elsevier Inc. All rights reserved.

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

NASA Astrophysics Data System (ADS)

Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

2017-05-01

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

The Impact of Microgravity and Hypergravity on Endothelial Cells

PubMed Central

Maier, Jeanette A. M.

2015-01-01

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature. PMID:25654101

The impact of microgravity and hypergravity on endothelial cells.

PubMed

Maier, Jeanette A M; Cialdai, Francesca; Monici, Monica; Morbidelli, Lucia

2015-01-01

The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

Tissue engineering, stem cells and cloning: current concepts and changing trends.

PubMed

Atala, Anthony

2005-07-01

Organ damage or loss can occur from congenital disorders, cancer, trauma, infection, inflammation, iatrogenic injuries or other conditions and often necessitates reconstruction or replacement. Replacement may take the form of organ transplant. At present, there is a severe shortage of donor organs that is worsening with the aging of the population. Tissue engineering follows the principles of cell transplantation, materials science and engineering towards the development of biological substitutes that can restore and maintain normal tissue function. Therapeutic cloning involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications. This paper reviews recent advances in stem cell research and regenerative medicine, and describes the clinical applications of these technologies as novel therapies for tissue or organ loss.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

PubMed Central

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

PubMed

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Involvement of HIF-1α activation in the doxorubicin resistance of human osteosarcoma cells.

PubMed

Roncuzzi, Laura; Pancotti, Fabia; Baldini, Nicola

2014-07-01

Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1α (HIF‑1α) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1α, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1α expression. The emerging role of HIF-1α in osteosarcoma biology indicates its use as a valuable therapeutic target.

Space Photovoltaic Concentrator Using Robust Fresnel Lenses, 4-Junction Cells, Graphene Radiators, and Articulating Receivers

NASA Technical Reports Server (NTRS)

O'Neill, Mark; McDanal, A. J.; Brandhorst, Henry; Spence, Brian; Iqbal, Shawn; Sharps, Paul; McPheeters, Clay; Steinfeldt, Jeff; Piszczor, Michael; Myers, Matt

2016-01-01

At the 42nd PVSC, our team presented recent advances in our space photovoltaic concentrator technology. These advances include more robust Fresnel lenses for optical concentration, more thermally conductive graphene radiators for waste heat rejection, improved color-mixing lens technology to minimize chromatic aberration losses with 4-junction solar cells, and an articulating photovoltaic receiver enabling single-axis sun-tracking, while maintaining a sharp focal line despite large beta angles of incidence. In the past year, under a NASA Phase II SBIR program, our team has made much additional progress in the development of this new space photovoltaic concentrator technology, as described in this paper.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

PubMed

Zhang, Sen; Anjum, Rana; Squillace, Rachel; Nadworny, Sara; Zhou, Tianjun; Keats, Jeff; Ning, Yaoyu; Wardwell, Scott D; Miller, David; Song, Youngchul; Eichinger, Lindsey; Moran, Lauren; Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Wang, Yihan; Mohemmad, Qurish; Jang, Hyun Gyung; Ye, Emily; Narasimhan, Narayana; Wang, Frank; Miret, Juan; Zhu, Xiaotian; Clackson, Tim; Dalgarno, David; Shakespeare, William C; Rivera, Victor M

2016-11-15

Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK + ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK + cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK + tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK + , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR. ©2016 American Association for Cancer Research.

Ex vivo delivery of GDNF maintains motor function and prevents neuronal loss in a transgenic mouse model of Huntington's disease.

PubMed

Ebert, Allison D; Barber, Amelia E; Heins, Brittany M; Svendsen, Clive N

2010-07-01

Huntington's disease (HD) is an autosomal dominant disorder caused by expansion of polyglutamine repeats in the huntingtin gene leading to loss of striatal and cortical neurons followed by deficits in cognition and choreic movements. Growth factor delivery to the brain has shown promise in various models of neurodegenerative diseases, including HD, by reducing neuronal death and thus limiting motor impairment. Here we used mouse neural progenitor cells (mNPCs) as growth factor delivery vehicles in the N171-82Q transgenic mouse model of HD. mNPCs derived from the developing mouse striatum were isolated and infected with lentivirus expressing either glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Next, mNPCs(GDNF) or mNPCs(GFP) were transplanted bilaterally into the striatum of pre-symptomatic N171-82Q mice. We found that mNPCs(GDNF), but not mNPCs(GFP), maintained rotarod function and increased striatal neuron survival out to 3months post-transplantation. Importantly, histological analysis showed GDNF expression through the duration of the experiment. Our data show that mNPCs(GDNF) can survive transplantation, secrete GDNF for several weeks and are able to maintain motor function in this model of HD. Copyright 2010 Elsevier Inc. All rights reserved.

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis

PubMed Central

Wu, Yuhsin V.; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O’Connor, Rachael; Geha, Rula C.; Somberg, C. Joy; Antonescu, Cristina; Singer, Samuel

2012-01-01

Well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n=84), WDLS (n=79), and normal fat (n=23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS compared to both WDLS and normal fat (15.2 fold and 27.8 fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for dedifferentiated liposarcomas. PMID:22170698

Complex interactions in EML cell stimulation by stem cell factor and IL-3.

PubMed

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M

2011-03-22

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

PubMed Central

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M.

2011-01-01

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34− cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone. PMID:21383156

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion.

PubMed

Watanabe, M; Noda, M; Nakajin, S

2007-09-01

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.

Targeting human liver cancer cells with lactobionic acid-G(4)-PAMAM-FITC sorafenib loaded dendrimers.

PubMed

Iacobazzi, Rosa Maria; Porcelli, Letizia; Lopedota, Angela Assunta; Laquintana, Valentino; Lopalco, Antonio; Cutrignelli, Annalisa; Altamura, Emiliano; Di Fonte, Roberta; Azzariti, Amalia; Franco, Massimo; Denora, Nunzio

2017-08-07

Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R. Copyright © 2017 Elsevier B.V. All rights reserved.

Persistence of hAQP1 expression in human salivary gland cells following AdhAQP1 transduction is associated with a lack of methylation of hCMV promoter

PubMed Central

Zheng, C; Baum, BJ; Liu, X; Goldsmith, CM; Perez, P; Jang, S-I; Cotrim, AP; McCullagh, L; Ambudkar, IS; Alevizos, I

2017-01-01

In 2012, we reported that 5 out of 11 subjects in a clinical trial (NCT00372320) administering AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first-generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). As subject peak responses were at times much longer (7–42 days) than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. This hypothesis was supported in human salivary gland primary cultures and human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. Conversely, when we transduced mouse and rat cell lines in vitro and submandibular glands in vivo with AdhAQP1, the hCMVp was gradually methylated over time and associated with decreased hAQP1 expression and function in vitro and decreased hAQP1 expression in vivo. These data suggest that the hCMVp in AdhAQP1was probably not methylated in transduced human salivary gland cells of responding subjects, resulting in an unexpectedly longer functional expression of hAQP1. PMID:26177970

Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

PubMed Central

Tennis, Meredith A.; VanScoyk, Michelle M.; Wilson, Lora A.; Kelley, Nicole; Winn, Robert A.

2012-01-01

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC. PMID:22403725

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

PubMed

Marunaka, Y; Hagiwara, N; Tohda, H

1992-09-01

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Barriers for Deriving Transgene-Free Pig iPS Cells with Episomal Vectors.

PubMed

Du, Xuguang; Feng, Tao; Yu, Dawei; Wu, Yuanyuan; Zou, Huiying; Ma, Shuangyu; Feng, Chong; Huang, Yongye; Ouyang, Hongsheng; Hu, Xiaoxiang; Pan, Dengke; Li, Ning; Wu, Sen

2015-11-01

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets. © 2015 AlphaMed Press.

Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum

PubMed Central

Kornilov, Roman; Puhka, Maija; Mannerström, Bettina; Hiidenmaa, Hanna; Peltoniemi, Hilkka; Siljander, Pia; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy

2018-01-01

ABSTRACT Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. PMID:29410778

Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors.

PubMed

Lam, Alan Tin-Lun; Li, Jian; Toh, Jessica Pei-Wen; Sim, Eileen Jia-Hui; Chen, Allen Kuan-Liang; Chan, Jerry Kok-Yen; Choolani, Mahesh; Reuveny, Shaul; Birch, William R; Oh, Steve Kah-Weng

2017-03-01

Large numbers of human mesenchymal stromal cells (MSCs) used for a variety of applications in tissue engineering and cell therapy can be generated by scalable expansion in a bioreactor using microcarriers (MCs) systems. However, the enzymatic digestion process needed to detach cells from the growth surface can affect cell viability and potentially the potency and differentiation efficiency. Thus, the main aim of our study was to develop biocompatible and biodegradable MCs that can support high MSC yields while maintaining their differentiation capability and potency. After cell expansion, the cells that covered MCs can be directly implanted in vivo without the need for cell harvesting or use of scaffold. Poly-ε-caprolactone (PCL) is known as a biocompatible and biodegradable material. However, it cannot be used for generation of MCs because its high density (1.14 g/cm 3 ) would exclude its applicability for suspension MCs in stirred reactors. In this article, we describe expansion and potency of MSCs propagated on low-density (1.06 g/cm 3 ) porous PCL MCs coated with extracellular matrices (LPCLs) in suspended stirred reactors. Using these LPCLs, cell yields of about 4 × 10 4 cells/cm 2 and 7- to 10-fold increases were obtained using four different MSC lines (bone marrow, cord blood, fetal and Wharton's jelly). These yields were comparable with those obtained using non-degradable MCs (Cytodex 3) and higher than two-dimensional monolayer (MNL) cultures. A fed-batch process, which demonstrated faster cell expansion (4.5 × 10 4 cells/cm 2 in 5 days as compared with 7 days in batch culture) and about 70% reduction in growth media usage, was developed and scaled up from 100-mL spinner flask to 1-L controlled bioreactor. Surface marker expression, trilineage differentiation and clonogenic potential of the MSCs expanded on LPCL were not affected. Cytokine secretion kinetics, which occurred mostly during late logarithmic phase, was usually comparable with that obtained in Cytodex 3 cultures and higher than MNL cultures. In conclusion, biodegradable LPCL can be used to efficiently expand a variety of MSC lines in stirred scalable reactors in a cost-effective manner while maintaining surface markers expression, differentiation capability and high levels of cytokine secretion. This study is the first step in testing these cell-biodegradable porous MC aggregates for tissue engineering and cell therapy, such as bone and cartilage regeneration, or wound healing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

PubMed Central

Arason, Ari Jon; Jonsdottir, Hulda R.; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K.

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium. PMID:24533135

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

PubMed

Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

PubMed Central

Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

2012-01-01

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

Epidermal Homeostasis and Radiation Responses in a Multiscale Tissue Modeling Framework

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2013-01-01

The surface of skin is lined with several thin layers of epithelial cells that are maintained throughout life time by a small population of stem cells. High dose radiation exposures could injure and deplete the underlying proliferative cells and induce cutaneous radiation syndrome. In this work we propose a multiscale computational model for skin epidermal dynamics that links phenomena occurring at the subcellular, cellular, and tissue levels of organization, to simulate the experimental data of the radiation response of swine epidermis, which is closely similar to human epidermis. Incorporating experimentally measured histological and cell kinetic parameters, we obtain results of population kinetics and proliferation indexes comparable to observations in unirradiated and acutely irradiated swine experiments. At the sub-cellular level, several recently published Wnt signaling controlled cell-cycle models are applied and the roles of key components and parameters are analyzed. Based on our simulation results, we demonstrate that a moderate increase of proliferation rate for the survival proliferative cells is sufficient to fully repopulate the area denuded by high dose radiation, as long as the integrity of underlying basement membrane is maintained. Our work highlights the importance of considering proliferation kinetics as well as the spatial organization of tissues when conducting in vivo investigations of radiation responses. This integrated model allow us to test the validity of several basic biological rules at the cellular level and sub-cellular mechanisms by qualitatively comparing simulation results with published research, and enhance our understanding of the pathophysiological effects of ionizing radiation on skin.

College Students' Conceptions of Stem Cells, Stem Cell Research, and Cloning

NASA Astrophysics Data System (ADS)

Concannon, James P.; Siegel, Marcelle A.; Halverson, Kristy; Freyermuth, Sharyn

2010-04-01

In this study, we examined 96 undergraduate non-science majors' conceptions of stem cells, stem cell research, and cloning. This study was performed at a large, Midwest, research extensive university. Participants in the study were asked to answer 23 questions relating to stem cells, stem cell research, and cloning in an on-line assessment before and after instruction. Two goals of the instruction were to: (1) help students construct accurate scientific ideas, and (2) enhance their reasoning about socioscientific issues. The course structure included interactive lectures, case discussions, hands-on activities, and independent projects. Overall, students' understandings of stem cells, stem cell research, and cloning increased from pre-test to post-test. For example, on the post-test, students gained knowledge concerning the age of an organism related to the type of stem cell it possesses. However, we found that some incorrect ideas that were evident on the pre-test persisted after instruction. For example, before and after instruction several students maintained the idea that stem cells can currently be used to produce organs.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

PubMed

Bar-Nur, Ori; Gerli, Mattia F M; Di Stefano, Bruno; Almada, Albert E; Galvin, Amy; Coffey, Amy; Huebner, Aaron J; Feige, Peter; Verheul, Cassandra; Cheung, Priscilla; Payzin-Dogru, Duygu; Paisant, Sylvain; Anselmo, Anthony; Sadreyev, Ruslan I; Ott, Harald C; Tajbakhsh, Shahragim; Rudnicki, Michael A; Wagers, Amy J; Hochedlinger, Konrad

2018-05-08

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7 + cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.