Journal articles on the topic 'Human cell culture - Effect of chemicals on'
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1
KASAMAKI, Akiko, and Shozo URASAWA. "The effect of food chemicals on cell aging of human diploid cells in in vitro culture." Journal of Toxicological Sciences 18, no. 3 (1993): 143–53. http://dx.doi.org/10.2131/jts.18.3_143.
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Barile, Frank A., Dale Alexander, and Alicia Sookhoo. "Potential of Human Lung Cells for Predicting Acute Cytotoxicity." Alternatives to Laboratory Animals 23, no. 4 (July 1995): 461–68. http://dx.doi.org/10.1177/026119299502300407.
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— Human fetal lung fibroblasts (HFL1) were studied in culture to evaluate their potential as a screen for cytotoxicity. The cytotoxic concentrations determined in vitro were compared with established human and animal toxicity data. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 hours, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxicity data with rodent lethal concentrations and human lethal concentrations obtained from the testing of 50 chemicals in human lung cells, suggests that the experimental IC50 values are as accurate as predictors of human toxicity as the equivalent toxic blood concentrations derived from rodent LD50 tests. In addition, evaluation of the first 15 chemicals reveals no significant differences between results from continuous cell lines of human and rodent origin. Together with a related battery of tests, cell culture procedures have the potential to supplement or replace current animal protocols in screening chemicals for human toxicity.
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Mannelli, C., F. Ietta, C. Carotenuto, R. Romagnoli, A. Z. Szostek, T. Wasniewski, D. J. Skarzynski, and Luana Paulesu. "Bisphenol A Altersβ-hCG and MIF Release by Human Placenta: AnIn VitroStudy to Understand the Role of Endometrial Cells." Mediators of Inflammation 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/635364.
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A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end,in vitrodecidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24–48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin (β-hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF andβ-hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance ofin vitromodels including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.
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Hopkinson, Desirée, Rae Bourne, and Frank A. Barile. "In Vitro Cytotoxicity Testing: 24-hour and 72-hour Studies with Cultured Lung Cells." Alternatives to Laboratory Animals 21, no. 2 (April 1993): 167–72. http://dx.doi.org/10.1177/026119299302100208.
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This study was designed to evaluate the potential of an in vitro cell culture method for its ability to determine cytotoxicity and to compare the cytotoxic concentrations with established LD50 values for the same chemicals. Rat lung epithelial cells (L2) were incubated in the absence or presence of increasing concentrations of the test chemical for 24 hours, and the inhibition of incorporation of radio-labelled amino acids into newly synthesised proteins was used as a marker for toxicity. In addition, cultured cells were exposed to the test chemicals for 72 hours, and cell proliferation experiments were performed as parallel measures of toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. The biological significance of the results of testing 20 chemicals shows that the experimental IC50 values are as accurate as predictors of human toxicity as are equivalent toxic blood concentrations derived from rodent LD50s. Results obtained from 72-hour growth studies reveal a greater sensitivity to cytotoxicity than from the 24-hour protein synthesis experiments. Statistically, however, the differences between the two protocols are inconclusive. It is anticipated that these procedures, together with a related battery of tests, may supplement or replace animal protocols currently used for human risk assessment.
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Shibata, Michio, Takanari Tsuda, Hiroshi Itagaki, Shinobu Kato, Toshiaki Kobayashi, Hideyuki Ichikawa, and Yoshihiro Morikawa. "Interleukin-1α and Interleukin-8 Release by Human Keratinocyte Cell Culture Treated with Surfactants." Alternatives to Laboratory Animals 25, no. 2 (April 1997): 161–71. http://dx.doi.org/10.1177/026119299702500209.
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The effects of four cosmetic surfactants on interleukin (IL)-1α and IL-8 release from human keratinocytes were studied to investigate the feasibility of using these effects for the prediction of the irritation potential of chemicals. After exposure of cells to surfactants, the amounts of IL-1α and IL-8 released into culture medium were measured by ELISA. Cytotoxicity was evaluated by using the neutral red uptake (NRU) cytotoxicity assay. Cytokine release was increased 7–15 times by sodium lauryl sulphate (SLS), laurtrimonium chloride, cocamidopropyl betaine (CPB) and Oleth-5 at cytotoxic concentrations. IL-8 release was increased 3–4 times by SLS, CPB and Oleth-5 at subcytotoxic concentrations. After exposure to SLS, IL-1α was released within 1 hour, suggesting that IL-1α release is associated with membrane damage, whereas IL-8 release continued for 24 hours, suggesting that IL-8 was produced within the cells. Cytotoxicity tests and IL-8 release assays were also performed on seven other surfactants. The results show that moderate irritants CPB and PEG-4 dioleate, which have weak cytotoxic effects, significantly increased IL-8 release from human keratinocytes. It is suggested that measurement of IL-8 release is useful for predicting the irritation potential of chemicals which cannot be detected by using the NRU cytotoxicity assay.
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Dierickx, Paul J., Claudia Smit, and Ellen M. Scheers. "Cytotoxicity of the MEIC Reference Chemicals in Antioxidant-enriched Rat Hepatoma-derived Fa32 cells." Alternatives to Laboratory Animals 29, no. 3 (May 2001): 217–23. http://dx.doi.org/10.1177/026119290102900304.
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Since vitamin E increases the antioxidant status of cells, its influence on cytotoxicity was investigated. The neutral red uptake (NRU) inhibition effects of 39 MEIC reference chemicals were measured after treatment of rat hepatoma-derived Fa32 cells in the presence of vitamin E for 30 minutes. The results were quantified in terms of the NI50, the concentration of test compound required to reduce the NRU by 50%. Sodium chloride was the only chemical that was more toxic in the presence of vitamin E. This effect was related to the concentration of vitamin E in the cell culture medium. A vitamin E dose-related response was also observed for the decreased toxicity of paracetamol and caffeine. Glutathione levels were slightly increased in the presence of vitamin E, which could contribute to the protective effect of vitamin E. Of the remaining chemicals, 50% were less toxic in the presence of vitamin E, but the correlation with the acute human toxicity data of the MEIC study was not improved. The results imply that reactive oxygen species interfere with the toxicity of a high proportion of toxic chemicals. The assay described provides a quick and easy method for checking whether reactive oxygen species contribute to the toxicity of a chemical.
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Saavedra, Lorena, Kathleen Wallace, Theresa F. Freudenrich, Moritz Mall, William R. Mundy, Jorge Davila, Timothy J. Shafer, Marius Wernig, and Daniel Haag. "Comparison of Acute Effects of Neurotoxic Compounds on Network Activity in Human and Rodent Neural Cultures." Toxicological Sciences 180, no. 2 (February 4, 2021): 295–312. http://dx.doi.org/10.1093/toxsci/kfab008.
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Abstract Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking. The increasing number of differentiation protocols for generating neurons from human-induced pluripotent stem cells (hiPSCs) holds great potential to overcome the unavailability of human primary tissue and expedite cell-based assays. Yet, the variability in neuronal activity, prolonged ontogeny and rather immature stage of most neuronal cells derived by standard differentiation techniques greatly limit their utility for screening neurotoxic effects on human neural networks. Here, we used excitatory and inhibitory neurons, separately generated by direct reprogramming from hiPSCs, together with primary human astrocytes to establish highly functional cultures with defined cell ratios. Such neuron/glia cocultures exhibited pronounced neuronal activity and robust formation of synchronized network activity on multielectrode arrays, albeit with noticeable delay compared with primary rat cortical cultures. We further investigated acute changes of network activity in human neuron/glia cocultures and rat primary cortical cultures in response to compounds with known adverse neuroactive effects, including gamma amino butyric acid receptor antagonists and multiple pesticides. Importantly, we observed largely corresponding concentration-dependent effects on multiple neural network activity metrics using both neural culture types. These results demonstrate the utility of directly converted neuronal cells from hiPSCs for functional neurotoxicity screening of environmental chemicals.
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Walum, Erik, Elisabeth Hansson, and Alan L. Harvey. "In Vitro Testing of Neurotoxicity." Alternatives to Laboratory Animals 18, no. 1_part_1 (November 1990): 153–79. http://dx.doi.org/10.1177/026119299001800118.1.
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Many of the toxic compounds that are at large in the environment represent a risk to our neuronal functions. Chemicals may have a direct or indirect effect on the nervous system and they may interfere with general biochemical properties or specific neuronal structures and processes. In this review, a brief presentation of the major neurotoxicological targets is given, together with a discussion of some aspects of the use of different in vitro models for screening purposes and mechanistic studies. It is believed that in vitro methods offer special opportunities for the development of new neurotoxicological assays, and that this development will mainly involve cultured model systems. Therefore, a presentation of nerve and glia tissue culture methods is given, followed by an overview of how information on the action of mercury and mercurials, excitotoxins and acrylamide has been obtained through the use of cultured cell models. It is concluded that the developmental potential in cell neurotoxicology lies within the areas of separation and identification of cells representative for different structures in the nervous system, co-cultivation of different cell types, in vivo/in vitro (ex vivo) procedures, chemically defined media, metabolic competent cultures of human cells and improved physiological conditions for cultivation and exposure.
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Ponsoda, Xavier, Cristina Núñez, José Vicente Castell, and Maria José Gómez-Lechón. "Evaluation of the Cytotoxic Effects of MEIC Chemicals 31–50 on Primary Culture of Rat Hepatocytes and Hepatic and Non-hepatic Cell Lines." Alternatives to Laboratory Animals 25, no. 4 (July 1997): 423–36. http://dx.doi.org/10.1177/026119299702500405.
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The cytotoxicities of 20 chemicals (numbers 31–50) from the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme were assessed with a primary culture of rat hepatocytes and with two hepatic cell lines (Hep G2 and FaO) and one non-hepatic cell line (3T3). The cytotoxicities of the chemicals were evaluated by using the MTT test after the cells had been exposed to the chemicals for 24 hours. For a better evaluation of results, dose–response curves were mathematically linearised and cytotoxicity was expressed as IC50 values and IC10 values (the concentration causing 50% and 10% loss of cell viability, respectively). We found that all the compounds showed similar acute basal cytotoxicity in all four cellular systems (regardless of whether the cells were, or were not, metabolically competent or were or were not of human origin). When these results were used to predicit human toxicity in terms of a mathematical parameter (prediction error [PE]), we found that all four systems gave similar predictions of human toxicity. The best cytotoxicity parameter included in the PE calculation was the IC50/10, because of an underestimation of human toxicity by in vitro systems. However, when PEs were calculated for rodent toxicity, better results were obtained. Data from the literature obtained by using other experimental models for predicting human toxicity were analysed according to the same criteria. We conclude that cellular systems are better predictive tools for human toxicity than are prokaryotic cells or whole-organism models.
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Nishino, Taito, Takumi Mikashima, Makiko Yui, Yasuyuki Asai, Hiromitsu Nakauchi, and Atsushi Iwama. "A Chemical Approach for Ex Vivo Expansion of Human Hematopoietic Stem Cells." Blood 118, no. 21 (November 18, 2011): 481. http://dx.doi.org/10.1182/blood.v118.21.481.481.
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Abstract Abstract 481 Hematopoietic stem cells (HSCs) have been applied to treatment of a wide variety of malignant and non-malignant blood disorders. Because of low risk of graft versus-host disease, use of human cord blood (hCB) as a source of HSCs continues to increase. However, wide application of hCB is limited by the relatively low number of HSCs obtained from a single CB unit. Numerous attempts have been made to expand hCB HSCs in cultures to acquire a sufficient number of transplantable HSCs. Chemical approaches have especially showed promise for ex vivo expansion of HSCs. As an example, Boitano et al. reported that SR1, a chemically synthesized purine derivative, induced hCB HSC expansion by antagonizing the aryl hydrocarbon receptor. In this study, we initially screened several tens of thousands of small-molecule compounds for the capability to induce STAT5 activation but obtained active compounds showed no positive effect on proliferation of hCB CD34+ hematopoietic stem and progenitor cells (HSPCs). However, a derivative of the STAT5 activating compounds proved to increase the population of CD34+CD38– cells when added to hCB CD34+ cell culture. Thus, we optimized the chemical structure of the compound and identified MISK303 as the most potent compound. We next evaluated the effects of MISK303 on the ex vivo expansion of hCB CD34+ cells in detail; hCB CD34+ cells were cultured in serum-free medium supplemented with MISK303 in addition to rhTPO and rhSCF for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assays. Although the total and CD34+ cell number when cultured with MISK303 was comparable to that cultured with DMSO (vehicle), the cultures with MISK303 exhibited a >2-fold increase in the number of CD34+CD38– cells and contained 1.7-fold more high proliferative potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to those with DMSO. Correspondingly, in the NOD/SCID repopulation assay, hCB CD34+ cells cultured with MISK303 for 7 days displayed up to 2-fold higher levels of engraftment compared to control cultures and uncultured HSPCs. These data suggest that MISK303 promotes the net expansion of hematopoietic stem and progenitor cells. Furthermore, combination of SR1 and MISK303 had additive effects; cell cultures with SR1 and MISK303 showed a >4-fold increase in the number of CD34+CD38– cells. In accordance with the data, MISK303 had no antagonizing activity against the aryl hydrocarbon receptor. We also observed that a chemically-synthesized TPO receptor (c-mpl) agonist could be substituted with rhTPO and provided better expansion of CD34+CD38– cells in combination with SR1 and MISK303. To investigate the molecular mechanism by which MISK303 increases HSPCs, we first conducted gene expression profiling of CD34+ cells cultured with MK303 by DNA microarray analysis. Treatment of CD34+ cells with MISK303 for 24 hours led to up-regulation of 343-genes (>2-fold) and down-regulation of 112 genes (<0.5-fold). We also profiled effect of MISK303 over 311 protein kinases and found no inhibitory activity. We are now validating the expression of differentially expressed genes identified by DNA microarray analysis. In conclusion, we have conducted high-throughput screening and identified MK303, a novel chemically synthesized small-molecule compound, which induces the expansion of human HSCs ex vivo. The approach using MISK303 will facilitate the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures: No relevant conflicts of interest to declare.
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Zauli, Giorgio, Marco Vitale, Elisabetta Falcieri, Davide Gibellini, Alessandra Bassini, Claudio Celeghini, Marta Columbaro, and Silvano Capitani. "In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes." Blood 90, no. 6 (September 15, 1997): 2234–43. http://dx.doi.org/10.1182/blood.v90.6.2234.
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Abstract To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.
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Zauli, Giorgio, Marco Vitale, Elisabetta Falcieri, Davide Gibellini, Alessandra Bassini, Claudio Celeghini, Marta Columbaro, and Silvano Capitani. "In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes." Blood 90, no. 6 (September 15, 1997): 2234–43. http://dx.doi.org/10.1182/blood.v90.6.2234.2234_2234_2243.
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To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.
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Vandenberg, Laura N., Theo Colborn, Tyrone B. Hayes, Jerrold J. Heindel, David R. Jacobs, Duk-Hee Lee, Toshi Shioda, et al. "Hormones and Endocrine-Disrupting Chemicals: Low-Dose Effects and Nonmonotonic Dose Responses." Endocrine Reviews 33, no. 3 (March 14, 2012): 378–455. http://dx.doi.org/10.1210/er.2011-1050.
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For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of “the dose makes the poison,” because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.
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Özgür, Mustafa, Şemsi Gül Yılmaz, Ash Uçar, and Serkan Yılmaz. "Cytotoxic Effects of Bisphenol A as an Endocrine Disruptor on Human Lymphocytes." Iranian Journal of Toxicology 15, no. 2 (April 1, 2021): 115–20. http://dx.doi.org/10.32598/ijt.15.2.753.1.
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Background: Endocrine compounds, such as Bisphenol A (BPA), stimulate or inhibit the activities of hormones, nuclear receptors in the central nervous system, liver and other organs. They may be disposed of in the environment inadverdently around industrial sites. The aim of this study was to evaluate the cytotoxic effects of BPA on human lymphocytes in culture at varying concentrations. Methods: 0.1 mL heparinized 0.2 mL peripheral blood taken from a healthy male and a female were plated in culture media under sterile conditions. To prepare the reference dose at a concentration of 0.05 mg/mL, 0.027g BPA was dissolved in 1 L dimethyl sulfoxide and the highest dose of 50 μg/mL BPA solution was prepared. After separating the stock solution, 50 μg/mL BPA was diluted to prepare 20, 10 or 5 μg/mL doses. Results: After 24 h of incubation, abnormal cell±Standart Error (%)[AC±SE (%)] 1.10±1.0, chromosomal aberration/cell±Standart Error (CA/cell±SE) 0.025±0.01 was determined in control group, and AC±SE (%) 2.00±0.98 in control group. After 48 h of incubation 0.98, CA/cell±SE was found to be 0.020±0.01. After 24 and 48 h of incubation, AC±SE (%) and CA/cell±SE ratios were 30.00±3.24, 34.00±3.35 and 0.325±0.03, 0.430±0.04, respectively. Conclusion: The cytotoxic effect of BPA on human lymphocytes was investigated in this study at reference concentration and lower doses. Our findings support the fact that BPA substitutes may not be sufficiently safe for widespread use as industrial chemicals.
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Hughes, J. H. "Physical and chemical methods for enhancing rapid detection of viruses and other agents." Clinical Microbiology Reviews 6, no. 2 (April 1993): 150–75. http://dx.doi.org/10.1128/cmr.6.2.150.
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Viral replication events can be enhanced by physical, chemical, or heat treatment of cells. The centrifugation of cells can stimulate them to proliferate, reduce their generation times, and activate gene expression. Human endothelial cells can be activated to release cyclo-oxygenase metabolites after rocking for 5 min, and mechanical stress can stimulate endothelial cells to proliferate. Centrifugation of virus-infected cultures can increase cytopathic effects (CPE), enhance the number of infected cells, increase viral yields, and reduce viral detection times and may increase viral isolation rates. The rolling of virus-infected cells also has an effect similar to that of centrifugation. The continuous rolling of virus-infected cultures at < or = 2.0 rpm can enhance enterovirus, rhinovirus, reovirus, rotavirus, paramyxovirus, herpesvirus, and vaccinia virus CPE or yields or both. For some viruses, the continuous rolling of infected cell cultures at 96 rpm (1.9 x g) is superior to rolling at 2.0 rpm for viral replication or CPE production. In addition to centrifugation and rolling, the treatment of cells with chemicals or heat can also enhance viral yields or CPE. For example, the treatment of virus-infected cells with dimethyl sulfoxide can enhance viral transformation, increase plaque numbers and plaque size, increase the number of cells producing antigens, and increase viral yields. The infectivity of fowl plague virus is increased by 80-fold when 4% dimethyl sulfoxide is added to culture medium immediately after infection. The heat shocking of virus-infected cells also has been shown to have a stimulatory effect on the replication events of cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus. The effects of motion, chemicals, or heat treatments on viral replication are not well understood. These treatments apparently activate cells to make them more permissive to viral infection and viral replication. Perhaps heat shock proteins or stress proteins are a common factor for this enhancement phenomenon. The utility of these treatments alone or in combination with other methods for enhancing viral isolation and replication in a diagnostic setting needs further investigation.
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Wang, Linlin, Brent Wood, Greg Levin, and C. Anthony Blau. "Expansion of Genetically Modified Primary Human Hematopoietic Cells Using a Cell Growth Switch." Blood 106, no. 11 (November 16, 2005): 3044. http://dx.doi.org/10.1182/blood.v106.11.3044.3044.
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Abstract Methods for regulating the growth of transplanted cells have many applications in gene and cell therapy. One such method uses conditional signaling molecules that are activated by artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CID-triggered Fibroblast Growth Factor Receptor 1 (F36VFGFR1) signal in vitro and in vivo. In vitro, CD34+ cord blood cells were transduced with a lentivirus vector incorporating a chicken b-actin promoter/cytomegalovirus enhancer (CAG) that also allows for expression in human embryonic stem cells, but which is not expressed in human erythroid cells. Cells cultured in the presence of the CID expanded 40-fold relative to cells cultured in the absence of CID. The cell types evident in culture were predominantly granulocytes/monocytes but B-lymphocytes were also observed. The absence of an effect on erythroid cells is most likely attributable to the lack of transgene expression by the CAG element in erythroid cells. Contrary to our results in murine system using F36VFGFR1, cell expansion was transient, peaking at week 3 of culture. Activation of F36VFGFR1 was associated with a transient increase in CFU-GM. Ex vivo studies using a lentivirus vector in which F36VFGFR1 expression is regulated by an MSCV promoter, allowing for expression in all hematopoietic lineages, have been initiated. Preliminary results from immune deficient mice transplanted with cord blood CD34+ cells transduced with the MSCV based vector lentivirus indicate that F36VFGFR1 activation can support the CID-dependent expansion of a much wider repertoire of hematopoietic lineages, including granulocytes/monocytes, erythroid cells and lymphocytes, compared to our previous studies using a conditional derivative of the thrombopoietin receptor (F36VMpl). These findings establish CID-activated receptors as growth factors for genetically modified human hematopoietic cells.
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Brière, Normand, Joseph Ferrari, and Pierre Chailler. "Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture." Biochemistry and Cell Biology 69, no. 4 (April 1, 1991): 256–62. http://dx.doi.org/10.1139/o91-039.
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A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15–21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 °C, 95% air – 5% CO2). The addition of transferrin (5 μg/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium. The potentiation of insulin influence possibly results from an increase in the number of transferrin receptors. The above additions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors involved in human metanephrogenesis.Key words: insulin, transferrin, culture, nephrogenesis.
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Karseno, Kazuo Harada, and Kazumasa Hirata. "Effect of medium and light quality on pink pigment production of cyanobacteria Oscillatoria sp. BTCC/A0004." E3S Web of Conferences 47 (2018): 03002. http://dx.doi.org/10.1051/e3sconf/20184703002.
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Cyanobacteria are well known as promising source of valuable chemicals for human usage. Especially, cyanobacteria in tropical area are very wide in diversity and they are potent producers of unique metabolites which exhibit interesting bioactivities. Oscillatoria sp. BTCC/A0004 produce pink pigments extracellularly (OsPP). The effects of various environmental factors on the production of cyanobacteria metabolites were well documented. In this research, the effect of medium and light quality on cell growth and OsPP production were investigated. In case, three different culture media, named No 18, C, and modified C media, in which nutrient compositions are different, and light quality (white, blue, green, pink) were tested. The highest cell growth and OsPP production were obtained in modified C medium. The nitrogen concentration in modified C medium is higher (5 g/L) than in No 18 medium (1.5 g/L) or C medium (1 g/L). In addition, cell growth and OsPP production were significantly stimulated by pink light radiation.
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Schmidt, Craig M., Chun N. Cheng, Angelo Marino, Roula Konsoula, and Frank A Barile. "Hormesis effect of trace metals on cultured normal and immortal human mammary cells." Toxicology and Industrial Health 20, no. 1-5 (February 2004): 57–68. http://dx.doi.org/10.1191/0748233704th192oa.
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An in vitro study was conducted to determine the effects of variable concentrations of trace metals on human cultured mammary cells. Monolayers of human mortal (MCF-12A) and immortal (MDAMB231) mammary epithelial cells were incubated in the absence or presence of increasing concentrations of arsenic (As), mercury (Hg) and copper (Cu) for 24-h, 72-h, 4-d, and 7-d. The MTT assay was used to assess viability for all time periods and cell proliferation was monitored for 4-d and 7-d studies. Monolayers were also labeled with rhodamine-110 (R-6501), Sytox green®, and Celltiter blueTM fluorescent dyes as indicators for intracellular esterase activity, nucleic acid staining, and cell reduction/viability, respectively. Total incubation time with chemical plus dyes was 24 h. For 24-h and 72-h studies, cells were seeded in 96-well plates, after which confluent monolayers were exposed to increasing concentrations of chemicals. For 4-d and 7-d studies, cells were seeded in 12-well plates at 1/3 confluent density (day 0) and exposed to increasing concentrations of metals on day 1. All cells were counted on days 4 and 7. In addition, test medium was removed from select groups of cultures on day 4, replaced with fresh medium in the absence of chemical (recovery studies), and assays were performed on day 7 as above. The data suggest that there is a consistent protective and/or stimulating effect of metals at the lowest concentrations in MCF-12A cells that is not observed in immortal MDA-MB231 cells. In fact, cell viability of MCF-12A cells is stimulated by otherwise equivalent inhibitory concentrations of As, Cu, and Hg on MDA-MB231 cells at 24-h. Whereas As and Hg suppress proliferation and viability in both cell lines after 4-d and 7-d of exposure, Cu enhances cell proliferation and viability of MCF-12A cells. MDA-MB231, however, recover better after 4-days of toxic insult. In addition, nutritional manipulation of media between the cell lines, or pretreatment with penicillamine, did not alter the hormesis effect displayed by MCF- 12A. Growth of these cells however was not maintained in the alternative medium. The study demonstrates that a hormesis effect from trace metals is detectable in cultured mammary cells; fluorescent indicators, however, are not as sensitive as cell proliferation or MTT in recognizing the subtle responses. Also, sensitivity of mammary cells to lower concentrations of Cu, a biologically important trace metal, may play an important role in controlling cellular processes and proliferation. The ability to detect this in vitro phenomenon implies that similar processes, occurring in vivo, may be responsible for the development, induction, or enhancement of human cancers.
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Shao, L., NL Jr Frigon, AL Young, AL Yu, LS Mathews, J. Vaughan, W. Vale, and J. Yu. "Effect of activin A on globin gene expression in purified human erythroid progenitors." Blood 79, no. 3 (February 1, 1992): 773–81. http://dx.doi.org/10.1182/blood.v79.3.773.773.
Full textAbstract:
Abstract The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).
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Shao, L., NL Jr Frigon, AL Young, AL Yu, LS Mathews, J. Vaughan, W. Vale, and J. Yu. "Effect of activin A on globin gene expression in purified human erythroid progenitors." Blood 79, no. 3 (February 1, 1992): 773–81. http://dx.doi.org/10.1182/blood.v79.3.773.bloodjournal793773.
Full textAbstract:
The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).
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Kita, Yuki, Akihiro Hamada, Ryoichi Saito, Yuki Teramoto, Ryusuke Tanaka, Keishi Takano, Kenji Nakayama, et al. "Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies." British Journal of Cancer 121, no. 12 (November 1, 2019): 1027–38. http://dx.doi.org/10.1038/s41416-019-0609-0.
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Abstract Background Since the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC. Methods We performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened. Results Disulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA–platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin. Conclusions The present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.
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Danovi, Davide, Anna Falk, Peter Humphreys, Richard Vickers, Jon Tinsley, Austin G. Smith, and Steven M. Pollard. "Imaging-based chemical screens using normal and glioma-derived neural stem cells." Biochemical Society Transactions 38, no. 4 (July 26, 2010): 1067–71. http://dx.doi.org/10.1042/bst0381067.
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The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.
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Gaddipati, Jaya P., N. V. Rajeshkumar, Jason C. Grove, Susan V. M. Maharaj, Jose A. Centeno, Radha K. Maheshwari, and Wayne B. Jonas. "Low-Dose Cadmium Exposure Reduces Human Prostate Cell Transformation in Culture and Up-Regulates Metallothionein and MT-1G mRNA." Nonlinearity in Biology, Toxicology, Medicine 1, no. 2 (April 1, 2003): 154014203914343. http://dx.doi.org/10.1080/15401420391434333.
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Chronic low-level exposure to environmental toxins, including cadmium (Cd), is a growing problem in the industrialized world. One promising strategy for protection from these toxins is the use of low-dose exposure of environmental chemicals to induce cell tolerance and recovery, a phenomenon known as “protective hormesis”. Hormetic [low-dose stimulatory] effects occur in a variety of systems and with a number of chemicals. Cd is a potent carcinogen in rodents and has also been linked to human lung and prostate cancers. In the present study, we have evaluated the protective effects of low and ultra-low dose, long-term Cd exposure in the normal human prostate cells, RWPE-1. Cells were exposed to low and ultra-low doses (0, 0 (S−36), 10−6, 10−7, 10−18, 10−21, 10−32, or 10−36 M) of Cd for 20 weeks followed by treatment with 10−5 M Cd for another 8 weeks. Continuous exposure of RWPE-1 cells to 10−5 M Cd results in malignant transformation. However, cells pretreated with low and ultra-low doses of Cd had delayed transformation compared with controls. In addition, the number of transformed cell mounds was lower in pretreated cells indicating that low and ultra-low dose exposure had protective effects against high-dose Cd induced carcinogenesis. The expression of metallothionein (MT), the primary Cd detoxification protein, was induced by low-dose exposure to Cd and maintained during the 20 weeks. In addition, MT-1G mRNA was up-regulated 2- to 3-fold by low-dose and ultralow-dose Cd exposures and may be the mechanism of protective hormesis in this model. MT-1G mRNA might also serve as a biological indicator of very low-dose environmental Cd exposure.
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Facioni, Maria Sole, Joana Soares, Barabara Adinolfi, Sara Gomes, Liliana Raimundo, Adele Contini, Barbara Ruffoni, et al. "Biological Effects of Saponin Fractions from Astragalus verrucosus in Tumor and Non-tumor Human cells." Natural Product Communications 13, no. 9 (September 2018): 1934578X1801300. http://dx.doi.org/10.1177/1934578x1801300903.
Full textAbstract:
Among natural chemicals used as cancer chemo-preventive and/or chemotherapeutic agents, saponins represent one of the most promising and interesting family of compounds. In this work, we aimed to elucidate the biological effects on human cells of six saponin fractions (SFs) obtained from in vitro cultures of Astragalus verrucosus Moris, a poorly characterized species. Interestingly, SF (3) showed a strongly inhibitory effect on the proliferation of human colon adenocarcinoma cell line (HCT116) via activation of a p53-dependent apoptotic pathway. In addition, SF (3) and the other SFs did not display genotoxic activity in human peripheral lymphocytes.
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Iwasa, F., S. Sassa, and A. Kappas. "δ-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals." Biochemical Journal 262, no. 3 (September 15, 1989): 807–13. http://dx.doi.org/10.1042/bj2620807.
Full textAbstract:
delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.
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Li, Qiongfang, Bo Zhang, Naresh Kasoju, Jinmin Ma, Aidong Yang, Zhanfeng Cui, Hui Wang, and Hua Ye. "Differential and Interactive Effects of Substrate Topography and Chemistry on Human Mesenchymal Stem Cell Gene Expression." International Journal of Molecular Sciences 19, no. 8 (August 9, 2018): 2344. http://dx.doi.org/10.3390/ijms19082344.
Full textAbstract:
Variations in substrate chemistry and the micro-structure were shown to have a significant effect on the biology of human mesenchymal stromal cells (hMSCs). This occurs when differences in the surface properties indirectly modulate pathways within numerous signaling networks that control cell fate. To understand how the surface features affect hMSC gene expression, we performed RNA-sequencing analysis of bone marrow-derived hMSCs cultured on tissue culture-treated polystyrene (TCP) and poly(l-lactide) (PLLA) based substrates of differing topography (Fl: flat and Fs: fibrous) and chemistry (Pr: pristine and Am: aminated). Whilst 80% of gene expression remained similar for cells cultured on test substrates, the analysis of differentially expressed genes (DEGs) revealed that surface topography significantly altered gene expression more than surface chemistry. The Fl and Fs topologies introduced opposite directional alternations in gene expression when compared to TCP control. In addition, the effect of chemical treatment interacted with that of topography in a synergistic manner with the Pr samples promoting more DEGs than Am samples in all gene ontology function groups. These findings not only highlight the significance of the culture surface on regulating the overall gene expression profile but also provide novel insights into cell-material interactions that could help further design the next-generation biomaterials to facilitate hMSC applications. At the same time, further studies are required to investigate whether or not the observations noted correlate with subsequent protein expression and functionality of cells.
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Nishino, Taito, and Atsushi Iwama. "Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol." Blood 116, no. 21 (November 19, 2010): 1174. http://dx.doi.org/10.1182/blood.v116.21.1174.1174.
Full textAbstract:
Abstract Abstract 1174 Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) have recently been explored to optimize autologous and allogeneic HSPC transplantation and shown to be effective in the field of stem cell biology. However, to our knowledge, identification of culture conditions that allow HSPCs expansion and long-term hematopoietic reconstitution have remained incomplete, and clinical methods to expand human HSPCs have yet to be realized. In this study, we assumed that some small molecule compounds may preferentially activate signals that are required for optimal HSPC expansion and facilitate self-renewal of hematopoietic stem cells (HSCs). Thus, we evaluated the effects of several biologically active compounds on the ex vivo expansion of CD34+ hematopoietic stem and progenitor cells from human cord blood (hCB) and identified Garcinol, a plant-derived natural product as a novel modulator of HSPC proliferation. We cultured hCB CD34+ cells in serum-free medium supplemented with human thrombopoietin, human stem cell factor and Garcinol for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with Garcinol was similar to those cultured without Garcinol, the cultures with Garcinol showed >2-fold increase in the number of CD34+CD38- hematopoietic stem and progenitor cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to control cultures. Correspondingly, SCID-repopulating cells (SRCs) were increased 2-fold during a 7-day culture with Garcinol compared to cultures without Garcinol. These findings suggest that Garcinol efficiently promotes the net expansion of HPSCs. To investigate the structure-activity relationship of Garcinol, we synthesized the chemical derivatives of Garcinol and evaluated the effect of Garcinol and its derivatives, Isogarcinol and O, O'-dimethylisogarcinol, on the proliferation of CD34+CD38- cells. Although Isogarcinol exhibited almost the same activity as Garcinol, O, O'-dimethyl isogarcinol was scarcely effective in the CD34+CD38- cell proliferation. Correspondingly, O, O'-dimethylisogarcinol had no effect on numbers of HPP-CFCs. These results indicate that dihydroxybenzoyl moiety is crucial for the positive effect of Gacinol on HSPCs.Garcinol has been reported to be a potent inhibitor of histone acetyltransferases (HAT). Thus, we estimated the HAT activity in cells treated with Garcinol and its derivatives. Garcinol and Isogarcinol inhibited HAT activity while O, O'-dimethylisogarcinol showed much less HAT inhibitory activity as compared to Garcinol and Isogarcinol, which suggested that HAT inhibitory activity of Garcinol is correlate with the expansion of HPSCs. We are now investigating gene expression profiling in cells cultured with Garcinol using DNA microarray analysis and Q-PCR. In conclusion, we have identified Garcinol, a plant-derived small-molecule compound, which exhibits inhibitory effect on HAT activity, as a novel stimulator of HSPC expansion. The results reported here indicate that Garcinol would be applied as a useful tool for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures: No relevant conflicts of interest to declare.
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Nassar, Mahmoud Ibrahim, A. Ahmed, A. Dina, and A. Abdullah. "The Effect of Recombinant Human Bone Morphogenetic Protein-7 on the Osteoblast-like Cells Cultured on Implant." Open Access Macedonian Journal of Medical Sciences 8, no. D (October 31, 2020): 224–28. http://dx.doi.org/10.3889/oamjms.2020.4908.
Full textAbstract:
AIM: The aim of the study was to study the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the osteoblast-like cells cultured on implant.
MATERIALS AND METHODS: The osteoblast-like osteo-1 cell line was used in this experiment and derived from the parietal bone tissue of newborn albino rats. The cells were incubated in a humid atmosphere of 95% air and 5% carbon dioxide at 37°C. The medium was changed every 2 days. Four groups were conducted as follows osteoclast-like cell (machined), implanted osteoclast-like cell on titanium (Ti) (modified), osteoclast-like cell supplemented with BMP-7 (Machined + BMP-7), and implanted osteoclast-like cell on Ti and supplemented with BMP-7 (Modified + BMP-7).
RESULTS: Cell proliferation was influenced by rhBMP-7, as demonstrated by a significant increase in collagen content after 7 and 21 days of culture (p = 0.005) and a significant increase in alkaline phosphatase (ALP) activity after 7 days (p < 0.001). The addition of rhBMP-7 influenced ALP activity, and a significant increase was observed after 21 days (p < 0.001).
CONCLUSION: Within the limitations of the study, we concluded that the presence of rhBMP-7 did not influence cell adhesion to chemically modified Ti surfaces but provided an additional stimulus during the differentiation of rat osteo-1 cells cultured on this type of surface.
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Hopkinson, Desirée, Peter Scheiner, and Frank A. Barile. "In Vitro Cytotoxicity Testing of Potentially Active Anti-HIV Drugs with Cultured Cells." Alternatives to Laboratory Animals 24, no. 3 (June 1996): 413–18. http://dx.doi.org/10.1177/026119299602400316.
Full textAbstract:
This study compared the ability of two continuous cell lines to predict the cytotoxicity of potentially active anti-HIV drugs. Human fetal lung fibroblasts (HFL1) and CD4+ T-lymphocytes (CEM-IW) were incubated in the absence or presence of increasing concentrations of 12 antiviral compounds. These six-membered unsaturated nucleoside analogues were stereospecifically synthesised in our laboratories, and were evaluated for cytotoxicity as well as for antiviral activity. Cells were incubated for six days and mitochondrial activity (XTT and MTT assays) was used to assess cytotoxicity. IC50 values were derived from concentration–effect curves after linear regression analysis. Comparison of the two sets of cytotoxicity data suggests that the experimental IC50 values from HFL1 cells correlate well with the values obtained in lymphocyte studies performed at the National Cancer Institute laboratories (r value = 0.93). For the 12 antiviral chemicals, and those we have tested previously, these methods probably detect basal cytotoxicity, i.e. the toxicity of a chemical to basic cellular functions and structures common to all mammalian specialised cells. However, as with any testing procedure, some chemicals may elude the cytotoxicity screen, as a result of false negatives due to solubility, miscibility and organ-specific effects, and could be mislabelled as having low toxic potential. It is therefore conceivable that tests involving continuous differentiated cell lines of various origins could be developed to cover a large percentage of toxic effects, thereby reducing the need to introduce many laborious assay systems with freshly-isolated primary cultures.
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Bogdanovic, Visnja, and Mihajlo Spasic. "Redox regulation of cell cycle through nitric oxide." Chemical Industry 62, no. 5 (2008): 301–4. http://dx.doi.org/10.2298/hemind0805301b.
Full textAbstract:
This paper investigates the effects of sodium nitroprusside as NO donor on two cell lines in culture: transformed cells of mice fibroblasts (L929) and malignant cells of human eritroleukemia (K562). Low concentrations of NO have stimulative effect, while high concentrations have inhibitive effects on proliferation of K562 cells in a dose-dependent manner. In our experiments, by using sodium nitroprusside (SNP) as NO donor and two kinds of superoxide dismutase, Cu,Zn-SOD and Mn-SOD, we created conditions to generate several kinds of signal molecules and investigated reaction of transformed (L929) and malignant (K562) cells to dose. Results of experiments are showing that chosen parameters (amount of free thiol groups and glutathione) may be relevant in monitoring the effect of exogenous nitrate oxide and its redox descendants in different, both transformed and malignant cell lines.
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Chen, Baosheng, Methodius G. Tuuli, Mark S. Longtine, Joong Sik Shin, Russell Lawrence, Terrie Inder, and D. Michael Nelson. "Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts." American Journal of Physiology-Endocrinology and Metabolism 302, no. 9 (May 1, 2012): E1142—E1152. http://dx.doi.org/10.1152/ajpendo.00003.2012.
Full textAbstract:
The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35∼38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus.
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Argentati, Chiara, Francesco Morena, Chiara Fontana, Ilaria Tortorella, Carla Emiliani, Loredana Latterini, Giulia Zampini, and Sabata Martino. "Functionalized Silica Star-Shaped Nanoparticles and Human Mesenchymal Stem Cells: An In Vitro Model." Nanomaterials 11, no. 3 (March 18, 2021): 779. http://dx.doi.org/10.3390/nano11030779.
Full textAbstract:
The biomedical translational applications of functionalized nanoparticles require comprehensive studies on their effect on human stem cells. Here, we have tested neat star-shaped mesoporous silica nanoparticles (s-MSN) and their chemically functionalized derivates; we examined nanoparticles (NPs) with similar dimensions but different surface chemistry, due to the amino groups grafted on silica nanoparticles (s-MSN-NH2), and gold nanoseeds chemically adsorbed on silica nanoparticles (s-MSN-Au). The different samples were dropped on glass coverslips to obtain a homogeneous deposition differing only for NPs’ chemical functionalization and suitable for long-term culture of human Bone Marrow–Mesenchymal stem cells (hBM-MSCs) and Adipose stem cells (hASCs). Our model allowed us to demonstrate that hBM-MSCs and hASCs have comparable growth curves, viability, and canonical Vinculin Focal adhesion spots on functionalized s-MSN-NH2 and s-MSN-Au as on neat s-MSN and control systems, but also to show morphological changes on all NP types compared to the control counterparts. The new shape was stem-cell-specific and was maintained on all types of NPs. Compared to the other NPs, s-MSN-Au exerted a small genotoxic effect on both stem cell types, which, however, did not affect the stem cell behavior, likely due to a peculiar stem cell metabolic restoration response.
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Rathjen, Joy, Christine Yeo, Charlotte Yap, Boon Siang Nicholas Tan, Peter D. Rathjen, and David K. Gardner. "Culture environment regulates amino acid turnover and glucose utilisation in human ES cells." Reproduction, Fertility and Development 26, no. 5 (2014): 703. http://dx.doi.org/10.1071/rd12276.
Full textAbstract:
Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also increased glucose uptake and changed amino acid turnover by the cells. These changes were distinct from those induced by serum and occurred in the absence of detectable differentiation. Induction of differentiation by bone morphogenetic protein 4 (BMP4), in contrast, did not alter metabolite turnover. Deviations from metabolite turnover by ES cells in fully defined medium demonstrated that culture environment can alter metabolite use. The challenge remains to understand the impact of metabolic changes on long-term cell maintenance and the functionality of derived cell populations.
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Richard, Robert E., Brent Wood, Hui Zeng, Liqing Jin, Thalia Papayannopoulou, and C. Anthony Blau. "Expansion of genetically modified primary human hemopoietic cells using chemical inducers of dimerization." Blood 95, no. 2 (January 15, 2000): 430–36. http://dx.doi.org/10.1182/blood.v95.2.430.
Full textAbstract:
The inability to deliver a therapeutic gene to a sufficient percentage of hematopoietic stem cells is the major obstacle to using gene therapy to treat blood disorders. Providing genetically corrected stem cells with a reversible growth advantage could solve this problem. To this end we have employed small synthetic molecules that can reversibly dimerize and activate fusion proteins which contain a growth factor receptor signaling domain. We have shown that the thrombopoietin receptor (mpl) signaling domain can be used in this system to expand transduced multipotential progenitor cells from mouse bone marrow. In the present study we tested a similar retroviral vector in human CD34-selected cord blood cells. Following transduction, cells cultured in the presence of the dimerizing molecule AP1903 expanded 13.8- to 186-fold relative to cells cultured in the absence of AP1903. The cell type that emerged in suspension culture was erythroid. Contrary to our results in the murine system, cell expansion was transient. Activation of mpl caused the disappearance of BFU-E followed by a transient increase in CFU-E. In contrast, mpl activation had no discernable effect on transduced myeloid progenitor cells. AP1903-mediated expansion was restricted to transduced cells, as demonstrated by immunohistochemical staining. These findings indicate that synthetic dimerizing molecules can be used to expand primary human hematopoietic cells.
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Tonks, Alex, Lorna Pearn, Amanda Tonks, Ken I. Mills, Alan K. Burnett, and Richard L. Darley. "Expression of RUNX1-RUNX1T1 Alone Has No Effect on the Intrinsic Susceptibility to Cytotoxic Chemicals." Blood 108, no. 11 (November 16, 2006): 2602. http://dx.doi.org/10.1182/blood.v108.11.2602.2602.
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Abstract The RUNX1 gene (aka AML1 on chromosome 21) encodes the alpha component of the Core Binding Factor (CBF) complex. This heterodimeric transcription factor is central to haematopoietic development and has been shown to be involved in the regulation of a number of haematopoietic-specific genes including IL-3, MPO and GM-CSF. RUNX1 is one of the most frequently disrupted genes in acute myeloid leukaemia (AML); and is particularly associated with chromosomal translocations. The t(8;21) encodes the RUNX1-RUNX1T1 (aka AML1-ETO) fusion protein which promotes self-renewal of haematopoietic cells and also inhibits their subsequent differentiation. Leukaemias expressing this abnormality are generally associated with a good prognosis in terms of complete remission, relapse risk and overall survival compared with other subtypes and tends to respond favourably to chemotherapeutic agents. However it is not currently known why patients expressing the t(8;21) have a good prognosis. It has been suggested that in AML patients expressing RUNX1-RUNX1T1, the fusion gene may promote the expression of p-glycoprotein (encoded by MDR-1) in mediating drug resistance. We therefore tested this hypothesis directly by expressing the RUNX1-RUNX1T1 fusion as a single abnormality in human haematopoietic cell subsets and performed Affymetrix microarray analysis to determine whether this fusion had any effect on the transcription of MDR genes. Using this approach we generated independent replicate sets of data from control and RUNX1-RUNX1T1 matched CD34+ cultures as well as matched sets constituting granulocytic (CD14lo, CD36lo, CD15hi) and monocytic (CD14hi) unilineage populations (isolated from day 6 cultures by immunomagnetic sorting). cRNA was prepared from each sample and hybridised to Affymetrix human 133A oligonucleotide arrays which allowed the simultaneous analysis of 6 MDR family gene members. In each of these populations, the expression of MDR genes was not significantly different from controls. We could therefore find no evidence that RUNX1-RUNX1T1 expression directly influences MDR gene expression as a single abnormality. We next addressed the issue of whether the t(8;21) abnormality directly influences the susceptibility to chemotherapeutic agents. We therefore assessed the sensitivity of CD34+ cells expressing RUNX1-RUNX1T1 to a number drugs commonly used to treat AML (Daunorubicin, Cytarabine, Fludarabine, Idarubicin or Etoposide) in comparison with matched controls. Remarkably, none of these agents differentially affected the growth of RUNX1-RUNX1T1 transduced cells. Since treatment of AML commonly involves multiple drugs, we also determined the effect of combining two or more of these chemotherapeutic agents. Again, we observed little difference in the in vitro growth response of RUNX1-RUNX1T1 expressing cells compared to controls. Taken together, these data suggest that expression of RUNX1-RUNX1T1 itself has no effect on the intrinsic susceptibility to cytotoxic chemicals. This raises the alternative hypothesis that RUNX1-RUNX1T1 moderates the influence of secondary abnormalities which are required for RUNX1-RUNX1T1 expressing cells to undergo leukaemic transformation.
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Koss-Mikołajczyk, Izabela, Monika Baranowska, Jacek Namieśnik, and Agnieszka Bartoszek. "Determination of antioxidantactivity of phytochemicals in cellular models by fluorescence/luminescence methods." Postępy Higieny i Medycyny Doświadczalnej 71, no. 1 (July 30, 2017): 0. http://dx.doi.org/10.5604/01.3001.0010.3841.
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As soon as the role of Reactive Oxygen Species (ROS) in so-called civilization diseases, which include non-infectious chronic diseases such as cancer, diabetes or high blood pressure has been discovered, and the possibility of employing antioxidants as a remedy for these diseases have been proposed, scientists developed a broad spectrum of methods to determine antioxidant activity of pure chemicals and plant extracts, as well as dietary supplements. Most of these methods are based on simple redox reactions between antioxidant and ROS (for example ABTS, DPPH, or FRAP tests). However, chemical methods of assessing antioxidant activity are rarely biologically relevant. They do not mirror the real effect of antioxidants in living organisms, because they are used in non-physiological conditions of temperature and pH; neither they take metabolism nor intracellular transport under consideration. The perfect model for assessment of antioxidant activity in living organisms would be human or animal model, but such determinations are very complicated and often ambiguous. The current best alternative to chemical and human tests are assays employing cell culture models being less expensive than human tests, yet still reflecting biological systems more convincingly than chemical assays. Cellular antioxidant assays are performed under physiological pH and temperature, but most importantly, they take metabolism and intracellular transport under consideration. In this review, we present cellular tests used to determine antioxidant activity that are based on luminescence and fluorescence methods.
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Suzuki, Takahiro, Yasuhisa Yokoyama, Keiki Kumano, Minoko Takanashi, Shiro Kozuma, Tsuyoshi Takato, Tatsutoshi Nakahata, et al. "Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein." Blood 108, no. 11 (November 16, 2006): 1332. http://dx.doi.org/10.1182/blood.v108.11.1332.1332.
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Abstract [Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133. [Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods. [Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.
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Shirinsky, V. P., A. S. Antonov, K. G. Birukov, A. V. Sobolevsky, Y. A. Romanov, N. V. Kabaeva, G. N. Antonova, and V. N. Smirnov. "Mechano-chemical control of human endothelium orientation and size." Journal of Cell Biology 109, no. 1 (July 1, 1989): 331–39. http://dx.doi.org/10.1083/jcb.109.1.331.
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Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.
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Kim, Kevin J., Jamie Wang, Xiaohong Xu, Sharon Wu, Wei Zhang, Zhen Qin, Fenglan Wu, et al. "A Chemical Genomics Screen to Discover Genes That Modulate Neural Stem Cell Differentiation." Journal of Biomolecular Screening 17, no. 2 (September 23, 2011): 129–39. http://dx.doi.org/10.1177/1087057111422379.
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The authors designed a chemical genomics screen with the aim of understanding genes and pathways that modulate neural stem/precursor cell differentiation. Multipotent mouse neural precursor cells isolated from cortices of embryonic day 12 (E12) embryos were subjected to spontaneous differentiation triggered by growth factor withdrawal. A quantitative whole-well immunofluorescence assay was set up to screen tool compound sets to identify small molecules with potent, dose-dependent, and reproducible effects on increasing neural stem cell differentiation toward neuronal lineage. Among the pro-neuronal compounds, kinase inhibitors were shown to exert pro-neuronal effect via a signaling pathway associated with the kinase. The global effect of hit compounds on modulating neuronal differentiation was confirmed by an in vivo mouse study and human neural stem cells culture. This study demonstrates that a phenotypic assay using cell type–specific antibody markers can be used for a large-scale compound screen to discover targets and pathways with impacts on differentiation of lineage-restricted precursor cells toward specific lineages.
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Morley, P., D. T. Armstrong, and R. E. Gore-Langton. "Ganglioside inhibition of attachment and differentiation of cultured rat granulosa cells: interactions with fibronectin." Journal of Cell Science 88, no. 2 (September 1, 1987): 205–17. http://dx.doi.org/10.1242/jcs.88.2.205.
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The involvement of fibronectin in the attachment and differentiation of rat granulosa cells, cultured in a chemically defined medium, was investigated using the inhibitory properties of mixed brain gangliosides (MBGs) and highly purified disialoganglioside, GD1a. MBGs inhibited cell attachment to plastic culture surfaces in a concentration-dependent manner, with 0.1 mmol l-1 causing significantly decreased attachment between 0.5 and 24 h of incubation. Inhibition of attachment to a fibronectin-coated substratum was even greater. The inhibitory effect of MBGs was not caused by binding to the cell surface, but instead the inhibitory factor(s) were adsorbed on a surface of immobilized human plasma fibronectin, thereby preventing cell attachment to this surface. The inhibitory action of MBGs was also neutralized by the addition of soluble fibronectin. Furthermore, at least one component of MBGs, detected chemically following thin-layer chromatography, was directly shown to bind to human fibronectin. MBGs inhibited to varying degrees the follicle-stimulating hormone(FSH)-dependent responses: augmentation of cellular protein content, production of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and progestins (progesterone + 20 alpha-hydroxypregn-4-en-3-one + pregnenolone), and induction of aromatase activity. These inhibitory activities of MBGs could not be eliminated by adsorption on immobilized fibronectin or reversed by addition of soluble fibronectin, thus distinguishing these actions from the early inhibition of cell attachment. FSH-dependent responses were also inhibited by GD1a, while responses to stimulation by dibutyryl cyclic AMP plus 3-isobutyl-1-methyl xanthine were less affected by this ganglioside. These results suggest that gangliosides inhibit attachment of granulosa cells in culture by binding to fibronectin, whereas the inhibition of FSH-dependent differentiation occurs by other modes of action that are unrelated to the effects on cell adhesion.
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Lambertini, Elisabetta, Letizia Penolazzi, Giulia Pellielo, Caterina Pipino, Assunta Pandolfi, Serena Fiorito, Francesco Epifano, Salvatore Genovese, and Roberta Piva. "Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures." Nutrients 13, no. 8 (July 30, 2021): 2633. http://dx.doi.org/10.3390/nu13082633.
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Traditional medicines rely mainly on use of plant extracts to mitigate or treat a wide range of disorders, including those that affect skeletal homeostasis. In this study, we investigated for the first time the potential pro-osteogenic effects of hexane, acetone and methanol extracts of the leaves of Cucurbita moschata, a very popular pumpkin cultivar in Western countries. We found that in Cucurbita moschata leaves, there are acetone-extractable substances—in particular, fatty acids such as 13-OH-9Z,11E,15E-octadecatrienoic acid (PU-13OH-FA), which is capable of both stimulating the function of human primary osteoblasts, which are responsible for bone formation, and inhibiting the differentiation of human osteoclasts, which are responsible for bone resorption. This dual effect was monitored by analyzing Runx2 expression, deposition of mineralized matrix, ALP activity, TRAP and actin ring staining respectively. This study suggests that bioactive chemicals from Cucurbita moschata leaves are potentially suitable as therapeutics for managing metabolic bone disorders such as osteoporosis and rheumatoid arthritis, and promoting tissue healing and functional recovery after bone fractures. The data we obtained increase knowledge on the biological activities of Cucurbita moschata, and in particular underline the potential benefits of consuming leaves which are a part of the plant currently little considered in the Western world.
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Lukaszewska-Kuska, Magdalena, Malgorzata Idzior-Haufa, and Barbara Dorocka-Bobkowska. "Evaluation of human osteoblast metabolic activity in modified titanium-conditioned medium." Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine 234, no. 6 (March 13, 2020): 603–11. http://dx.doi.org/10.1177/0954411920911281.
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To evaluate human osteoblast metabolic activity cultured in medium conditioned with commercially pure titanium after surface treatments with alumina or ceramic grit-blasting followed by acid etching. Commercially available, pure Grade 4 titanium disks were used and subjected to seven different surface modifications: (1) machined (MA)—used as the control group; (2) blasted with Al2O3 (Al2O3); (3) blasted with sintered ceramic (HAS); (4) blasted with non-sintered ceramics (HA); (5) blasted with Al2O3 and etched with HCl/H2SO4 (Al2O3 DE); (6) blasted with sintered ceramic and etched with HCl/H2SO4 (HAS DE), and (7) blasted with non-sintered ceramic and etched with HCl/H2SO4 (HA DE). A samples roughness evaluation test was carried out with an interference microscope, and energy-dispersive X-ray spectroscopy was performed to evaluate the presence of aluminum, phosphorus, and calcium deposited during the titanium surface treatment along with carbon contaminants acquired by the surface during processing. A culture medium conditioned with the respective samples was prepared in five dilutions, and its effect on human osteoblast cell viability was evaluated using the relative viability of cells. Human osteoblast metabolic activity was found to be the most intensive for the Al2O3 DE sample. The lowest activity was observed for the HAS DE. The material’s cytocompatibility depended on both the surface roughness and its chemical composition. Etching had a dual effect on cell activity, depending on the chemical composition of the titanium surface after blasting.
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De Leon, Sorel E., Lana Cleuren, Zay Yar Oo, Paul R. Stoddart, and Sally L. McArthur. "Extending In-Plane Impedance Measurements from 2D to 3D Cultures: Design Considerations." Bioengineering 8, no. 1 (January 13, 2021): 11. http://dx.doi.org/10.3390/bioengineering8010011.
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Three-dimensional (3D) cell cultures have recently emerged as tools for biologically modelling the human body. As 3D models make their way into laboratories there is a need to develop characterisation techniques that are sensitive enough to monitor the cells in real time and without the need for chemical labels. Impedance spectroscopy has been shown to address both of these challenges, but there has been little research into the full impedance spectrum and how the different components of the system affect the impedance signal. Here we investigate the impedance of human fibroblast cells in 2D and 3D collagen gel cultures across a broad range of frequencies (10 Hz to 5 MHz) using a commercial well with in-plane electrodes. At low frequencies in both 2D and 3D models it was observed that protein adsorption influences the magnitude of the impedance for the cell-free samples. This effect was eliminated once cells were introduced to the systems. Cell proliferation could be monitored in 2D at intermediate frequencies (30 kHz). However, the in-plane electrodes were unable to detect any changes in the impedance at any frequency when the cells were cultured in the 3D collagen gel. The results suggest that in designing impedance measurement devices, both the nature and distribution of the cells within the 3D culture as well as the architecture of the electrodes are key variables.
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Kongkiatkamon, Sunisa, Yihong Guan, Anand Tiwari, Phillip J. Maciejewski, Dale Grabowski, Sergei Vatolin, Vera Adema, et al. "Reversible TET Inhibitor As a Hematopoietic Stem Cell Booster: A Novel Strategy to Improve Stem Cell Function." Blood 134, Supplement_1 (November 13, 2019): 2472. http://dx.doi.org/10.1182/blood-2019-129056.
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Hematopoietic stem cells (HSCs) are responsible for the adaptation capacity in times of need but are also subjected to disease processes, natural or iatrogenic damage, and age-related attrition. The latter can lead to deficient production, decreased compensatory capacity and degenerative diseases such as MDS. In childhood, hereditary BMF predominates but increasingly with age, acquired idiopathic AA is a leading cause of HSC failure. Throughout life iatrogenic cases and cumulative exposures to environmental toxicities may lead to failure of the HSC compartment. Pharmacologic HSC boosters capable of expanding HSCs would have a wide range of clinical applications in acquired and inherited BMF states, including reconstitution of exhausted hematopoiesis after chemotherapy, aging, or HSC grafting. Currently, hematopoietic growth factors (HGF) are used but lead to progenitor rather than HSC expansion despite their success in clinical application. Depletion of TET2 in murine models leads to impairment of cellular differentiation and increases the proportion of HSCs and progenitors suggesting that TET2 is a key regulator of hematopoietic homeostasis and HSC self-renewal. Alterations of TET2 via somatic mutations and/ or deletion are frequent in MDS whereby HSC and progenitor expansion may be a key component of neoplastic evolution. We hypothesized that chemical agents reversibly inhibiting TET2 activity might phenocopy HSC expansion due to mutations and be applied as HSC boosters. Using structure guided approaches, we designed, synthesized and subsequently optimized bioavailable TET inhibitors (TETi). Among them, the one of the most effective TETi, named TETi76, showed dose-dependent inhibitory activity against TET dioxygenases in in vitro cell-free and cell culture systems with 5hmC production as a read out. We developed esterified forms of TETi76 and used in our cellular experimental models. Esterified TETi is bioavailable and non-toxic to normal bone marrow (BM) cells in therapeutically effective doses. Treatment of TETi76 resulted in a 44+15% and 42+17% increase in the clonogenic potential of human and murine BM cells consistent with the proliferative advantage gained by loss of TET activity in HSCs. We then performed serial replating experiments to determine the effect of TET inhibition on immature hematopoietic progenitor cells. Over 3 consecutive passages, TETi76 treatment prolonged the durability and capacity of human HSCs to maintain colony-forming cells (155+24 vs.107+14 colonies per 1x105 P1 cells). As long-term culture initiating cells (LTC-IC) are the best in vitro surrogates of HSCs, we also investigated the effect of TETi76 in LTC-IC cultures (n=3). The weekly addition of 1μM-TETi76 resulted in nearly 2-fold increase in LTC-IC numbers at the end of the culture (116±27 vs. 64 ±26 colonies per 2x106 P1 cells, p=.011). Expansion of grafts e.g., in the setting of umbilical cord HSC transplant (UCHSC) could be an important medical area of application of TETi76. We performed suspension cultures (n=3) with an optimal cocktail of hematopoietic growth factors (HGF) in the presence or absence of TETi76 (1μM). In control cultures, total cellular output peaked on day 14, but in the presence of TETi, growth continued beyond day 28. Cumulatively, total cellular output per 106 input was 27+2.61 x106cells/mL in control cultures and 32+0.642 x106 cells/mL in TETi treated cultures on day 28. CD34+ cell output was significantly higher in cultures treated with TETi vs. vehicle (2.5x105vs. 0.9X106)CD34+ cells per 1x104 CD34+ cell input). Similar effects were observed in murine BM suspension cultures. BM cells from C57BL/6 mice (n=3) were supplemented with HGF± TETi76 (1μM). TETi treatment led to a 5-fold HSC expansion compared to vehicle treated cells at 20 days of culture. The effect of TETi76 was reversed by treatment with ascorbic acid (50 μM), a known TET activator. Cumulatively our in vitro results suggest that the presence of TETi prevents exhaustion of immature cells, observed with growth factor driven expansion. In summary, our study indicates that novel agents modulating TET activity prevent exhaustion of HSC and may help expand the HSC in vitro. In vivo experiments examining the effects of TETi on hematopoietic recovery following radiation-induced aplasia, and competitive transplant experiments of grafts exposed in vivo and in vitro to TETi are underway. Disclosures Sekeres: Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.
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Palomino, Olga, Ana García-Aguilar, Adrián González, Carlos Guillén, Manuel Benito, and Luis Goya. "Biological Actions and Molecular Mechanisms of Sambucus nigra L. in Neurodegeneration: A Cell Culture Approach." Molecules 26, no. 16 (August 10, 2021): 4829. http://dx.doi.org/10.3390/molecules26164829.
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Sambucus nigra flowers (elderflower) have been widely used in traditional medicine for the relief of early symptoms of common cold. Its chemical composition mainly consists of polyphenolic compounds such as flavonoids, hydroxycinnamic acids, and triterpenes. Although the antioxidant properties of polyphenols are well known, the aim of this study is to assess the antioxidant and protective potentials of Sambucus nigra flowers in the human neuroblastoma (SH-SY5Y) cell line using different in vitro approaches. The antioxidant capacity is first evaluated by the oxygen radical absorbance capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cell viability is assessed by the crystal violet method; furthermore, the intracellular ROS formation (DCFH-DA method) is determined, together with the effect on the cell antioxidant defenses: reduced glutathione (GSH) and antioxidant enzyme activities (GPx, GR). On the other hand, mTORC1 hyperactivation and autophagy blockage have been associated with an increase in the formation of protein aggregates, this promoting the transference and expansion of neurodegenerative diseases. Then, the ability of Sambucus nigra flowers in the regulation of mTORC1 signaling activity and the reduction in oxidative stress through the activation of autophagy/mitophagy flux is also examined. In this regard, search for different molecules with a potential inhibitory effect on mTORC1 activation could have multiple positive effects either in the molecular pathogenic events and/or in the progression of several diseases including neurodegenerative ones.
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Chana, Ravinder S., W. Dale Treleaven, Robert J. Cushley, and Urs P. Steinbrecher. "Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins." Biochemistry and Cell Biology 68, no. 1 (January 1, 1990): 180–88. http://dx.doi.org/10.1139/o90-024.
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Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6–20 mol%) and into VLDL (7–10 mol%). Electron microscopy, light scattering, and 31P nuclear magnetic resonance indicated that particle size remained unchanged. Gel exclusion chromatography and chemical analysis showed no difference in hydrodynamic properties and only slight alteration to particle component ratios. Biological activity of labelled VLDL was measured from the rate of cholesterol esterification by cultured J774A.1 cells. Effect of labelling LDL was evaluated by monitoring LDL uptake and degradation by cultured human skin fibroblasts. In all cases the lipoproteins containing labels were indistinguishable from their native counterparts.Key words: deuterium incorporation, cell culture studies, light scattering, lipoproteins, electron microscopy.
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Engin, Ayse Basak, Evren Doruk Engin, and Bensu Karahalil. "Effect of N-acetyl cysteine, Neopterin and Dexamethasone on the Viability of Titanium dioxide Nanoparticles Exposed Cell Lines." Pteridines 23, no. 1 (February 2012): 111–22. http://dx.doi.org/10.1515/pteridines.2012.23.1.111.
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Abstract Titanium is extensively used for a wide range of implanted medical devices due to its advantageous combination of physico-chemical and biological properties. Nano-size titanium dioxide (TiO2) is also used in a variety of consumer products. Such widespread use and its potential entry through various routes of the body suggest that TiO2 could pose an exposure risk to humans. Nano-size particles (NP) enter systemic circulation, accumulate and damage tissues that are especially sensitive to oxidative stress. We hypothesized that TiO2 NPs can exert diverse cytotoxic effects on various human cell type especially neural cell lines. In order to test our hypothesis, putative cytotoxic effects of oxidative stress due to TiO2 NPs exposure on IM9, U937 and SHSY5Y (human neuroblastoma cells) were investigated in N-acetyl cysteine (NAC), neopterin and dexamethasone pre-treated cell cultures. IM9, U937 and SHSY5Y cells were exposed to ten different concentrations of 25 and 10 nm diameter TiO2 NPs in three different time periods before and after treatment with NAC, neopterin and dexamethasone. To determine toxicity levels of NPs, cell viability was estimated by MTT test. Concentration of cells was assessed by counting trypan blue stained cells with a heamo-cytometer. Toxicity of 25nm TiO2 particles was significantly increased (p<0.05) by adding fetal bovine serum (FBS) in SHYS5Y and U937 cell lines culture medium. Concentrationdependent toxicity of 10 nm TiO2 NP was weakly increased by adding FBS to SHYS5Y, IM9 and U937 cell culture media. NAC pre-treatment provided significant protection for only SHYS5Y cell exposed to 25 nm and 10 nm of TiO2 NPs after a 24-hour incubation period. Neopterin pre-treated SHYS5Y cells displayed significant increases in viability after 24-hour exposure to 10 nm and 25 nm TiO2 NPs. While exposure of dexamethasone pre-treated U937 cells to 25 nm of TiO2 NPs induced a significant increase in cell survival at only 100 mg/ml particle concentration, increase in viability of SHYS5Y cells were observed at all concentrations against 10 nm particle challenge. Our study demonstrated that exposure of SHYS5Y to TiO2 NPs for 24 hours regularly induced reduction of cell viability. We also found similar dose-related effects of TiO2 NPs in reducing cell survival in IM9 cells. This study clearly indicated that FBS is an effective dispersing agent for TiO2 NPs and increased TiO2 toxicity in all cell lines. NAC and neopterin significantly protected the SHYS5Y cell against the putative cytotoxic effects of TiO2 NPs.
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Ossowski, L., and D. Belin. "Effect of dimethyl sulfoxide on human carcinoma cells, inhibition of plasminogen activator synthesis, change in cell morphology, and alteration of response to cholera toxin." Molecular and Cellular Biology 5, no. 12 (December 1985): 3552–59. http://dx.doi.org/10.1128/mcb.5.12.3552-3559.1985.
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Human carcinoma HEp-3 lost its tumorigenic and metastatic potential upon prolonged culture in vitro. This change was accompanied by a reduced production of plasminogen activator (PA) of the urokinase type (uPA), which is secreted by HEp-3 cells, a change in response to effectors that modulate uPA production, and an alteration of cell morphology. Similar but more rapid changes occurred when malignant HEp-3 cells were exposed to dimethyl sulfoxide (DMSO). uPA activity in the culture medium dropped below 50% of the control level within 6 h after the addition of DMSO and became undetectable after 24 h of treatment. This drop in uPA activity was not caused by an increased production of PA inhibitors. The cell-associated uPA decreased to 25 to 30% of the control level within 6 h of DMSO treatment and remained at this level for at least 96 h; the reduced uPA production was partially accounted for by a rapid decrease in the functional and chemical concentration of uPA mRNA. In contrast, the concentrations of most of the abundant mRNA species did not appear to be significantly affected, and cell growth was only slightly inhibited in the presence of DMSO. Malignant HEp-3 cells treated with DMSO responded to cholera toxin with an enhanced production of uPA, and their morphology became indistinguishable from that of nonmalignant HEp-3 cells grown in vitro for prolonged periods of time. All of the above changes were fully and rapidly reversible. The inhibitory effect of DMSO on PA production appears to be specific for uPA of human cell lines.
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Graikou, Konstantia, Harilaos Damianakos, Christos Ganos, Katarzyna Sykłowska-Baranek, Małgorzata Jeziorek, Agnieszka Pietrosiuk, Christos Roussakis, and Ioanna Chinou. "Chemical Profile and Screening of Bioactive Metabolites of Rindera graeca (A. DC.) Bois. & Heldr. (Boraginaceae) In Vitro Cultures." Plants 10, no. 5 (April 21, 2021): 834. http://dx.doi.org/10.3390/plants10050834.
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Rindera graeca is a rare endemic plant where in vitro culture has been used in order to investigate bioactive metabolites. Phytochemical study of the in vitro shoots and hairy roots led to the isolation of seven phenolic derivatives and the unusual furano-naphthoquinone rinderol. R. graeca was also analyzed for its pyrrolizidine alkaloids content by LC-MS, and it was found to contain echinatine together with echinatine and rinderine N-oxides. Rinderol, isolated only from in vitro hairy root culture for the first time in the genus, revealed promising bioactivities. It was evaluated in vitro against a panel of microorganisms, showing very strong activity specifically against Gram-positive bacteria (MIC values 0.98 × 10−2–1.18 µg/mL) as well as very interesting antiproliferative effect against the human non-small-cell bronchopulmonary carcinoma cell line NSCLC-N6-L16 and the epidermoid lung cancer cell line A549. These findings were compared with the chemical profile of the plant from nature, while this study is the first to report on the effects of R. graeca extracts obtained from in vitro culture, providing a valuable contribution to the scientific community towards this sustainable method of production of potential bioactive molecules.