303 THE MODIFIED CULTURE SYSTEM OF PORCINE IN VITRO PRODUCTION IMPROVES COLONIZATION OF PUTATIVE EMBRYONIC STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 299
Author(s):  
S. A. Cheong ◽  
S. S. Kwak ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
S. H. Hyun
Keyword(s):  
Control System ◽  
Cell Line ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Attachment Rate ◽  
Blastocyst Quality ◽  
Cell Line Derivation ◽  
Colonization Rates

The establishment of porcine embryonic stem cells (pESC) using in vitro-produced blastocysts derived from IVF, parthenogenesis (PA), and somatic cell nuclear transfer (SCNT) have a great potential. However, porcine in vitro-produced blastocysts show a lower quality than do in vivo-derived blastocysts. In this study, to improve in vitro-derived blastocyst quality, and then to establish pESC, we treated IVF embryos and PA embryos with resveratrol (RES), porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), and β-mercaptoethanol (β-ME). The control system was produced using M199 medium in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The modified group was produced using M199 with 2 µM RES in IVM and PZM5 with 10 ng mL–1 pGM-CSF, 2 µM RES, and 10 µM β-ME in IVC. Data were analysed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test. In total, 1210 PA, 612 IVF, and 5 SCNT embryos were evaluated (for SCNT, we examined only the control system). We observed that overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P < 0.05) in the modified system (54.5%) compared with the control system (43.4%) in PA and hatched blastocysts rates at Day 6 and 7 were also increased significantly. Total blastocyst cell numbers were significantly higher (P < 0.05) in the modified system (55.1) compared with the control system (45.6). Hatching rate of Day 7 IVF blastocysts was also significantly increased. After seeding porcine blastocysts, the attachment rates were higher in the modified system (32.2% in PA and 36.2% in IVF; not examined in SCNT) than the control system (19.5% in PA, 26.6% in IVF, and 40.0% in SCNT). In addition, colonization rates and cell line derivation rates were higher in the modified system than in the control system. Colonization rates of modified system were 13.1% for PA and 17.75% for IVF embryos, whereas the control system generated 2.4% for PA, 10.8% for IVF, and 20.0% for SCNT. We also investigated the correlation between blastocyst state and attachment rate. The highest attachment rate is in hatched blastocyst (78.35 ± 15.74%). Therefore, the modified system increased quality of porcine blastocysts in vitro produced, and subsequently increased attachment rates. The cell line derivation rates were 2.4% (PA), 4.2% (IVF), and 20.0% (SCNT) in control group. In modified system, they were 7.2% (PA) and 10.0% (IVF). We established 3 cell lines from PA blastocysts (1 cell line in control system and 2 cell lines in modified system) and 1 cell line from SCNT-control system. All cell lines showed alkaline phosphatase activity and expressed pluripotent markers. In conclusion, the modified system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of in vitro-produced porcine blastocysts, and subsequently increased derivation rates of putative pESC. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#2012004885).

205 DERIVATION OF CAT EMBRYONIC STEM-LIKE CELLS FROM IN VITRO-PRODUCED BLASTOCYSTS AND THEIR SUPPORT BY INTRASPECIFIC VS. INTERSPECIFIC FEEDER CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 210 ◽  
Author(s):  
M. A. Serrano ◽  
M. C. Gómez ◽  
M. Lopez ◽  
C. L. Dumas ◽  
K. E. Smith ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Mitomycin C ◽  
Cell Lines ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Nuclear Reprogramming ◽  
Important Species ◽  
Feeder Layers

Interspecific nuclear transfer has been successfully demonstrated in nondomestic cats (Gomez et al. 2004 Cloning Stem Cells 6, 247); however, the efficiency remains low and may be attributable to nuclear reprogramming errors. Embryonic stem cells (ESC) may complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing cloning success (Jaenisch et al. 2002 Cloning Stem Cells 4, 389). The objectives of this study were to: (1) compare efficiency of immunosurgery vs. mechanical separation for isolating the inner cell mass (ICM) of in vitro-derived cat blastocysts; and (2) determine the influence of mouse (MEF: CF-1) and cat (CEF) embryonic fibroblast feeder layers on ICM attachment and growth of ES-like cells. After ICMs were isolated from in vitro-derived blastocysts (n = 142) by immunosurgery or mechanically, they were plated either on mitotically inactivated CEF (40 �L/mL Mitomycin-C; 5 h) or MEF (30 �L/mL Mitomycin-C; 2.5 h). Cells were cultured in DMEM-F12, 1 mM L-Glutamine, 0.1 mM 2-mercaptoethanol, 1.25% nonessential amino acids, 15% knock-out replacement serum, 5% fetal bovine serum, 40 ng/mL leukemia inhibitory factor, 5 ng/mL basic fibroblast growth factor, 100 IU penicillin, 100 �g/mL streptomycin, and 25 �L/mL amphotericin-B in a humidified atmosphere of 5% CO2 in air at 38�C. Our results indicated that ICM isolation and attachment were not affected by either the method of isolation (immunosurgery: 75.8 � 6.9% vs. mechanical: 89.5 � 6.4%) or the feeders (MEF: 74.6 � 6.7% vs. CEF: 90.7 � 6.6%). However, the incidence of ES-like cell colony formation was significantly affected by the feeder layer (CEF: 55.4 � 7.2% vs. MEF: 12.7 � 7.2%; P < 0.001). A total of 32 ES-like cell lines were derived on CEF (n = 26) and MEF (n = 6), of which 50% were alkaline phosphatase (AP)-positive. One ES-like cell line derived on MEF spontaneously differentiated into myocardiocytes after 14 days in culture. Three ES-like cell lines derived on CEF were immunostained for ESC-markers Oct-4, SSEA-1, and SSEA-4, and for AP. Positive results for all markers were observed in a few colonies of each line, with colonies from one cell line appearing on Day 23 and remaining in culture for 102 days (12 passages). Colonies from the other two cell lines appeared on Day 17 and remained in culture for 78 days (9 passages). Colonies derived on MEF appeared on average at 17.9 days and remained in culture an additional 15 to 61 days without further characterization. The present results describe the first isolation of cat ES-like cells. We have demonstrated an important species-specific relationship between feeder layers and the derivation of cat ESCs. Further studies are in progress to improve culture conditions for the derivation and expansion of stable cat ESC lines.

scholarly journals ID: 1065 Establishment of parthenogenetic diploid embryonic stem cells in mice

2017 ◽  
Vol 4 (S) ◽  
pp. 147
Author(s):  
Ho Thi-Kim Ngan ◽  
Nguyen Van Thuan ◽  
Hong-Thuy Bui
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Strontium Chloride ◽  
Blastocyst Quality ◽  
Parthenogenetic Embryos

Parthenogenesis is a process in which zygotes are produced without sperm presence. Due to lack of paternal genes, parthenogenetic embryos cannot develop to full-term; however, these embryos show a great potential to generate histocompatible stem cells (parthenogenetic embryonic stem – pES cells) for transplantation. In this research, parthenogenetic activation in the mouse was carried out using strontium chloride (SrCl2) combined with cytochalasin B (CB). The rate of embryo development, blastocyst quality and expression of acetylation of histone H4 lysine 12 (H4K12Ac) were investigated, while parthenogenetic blastocysts were used to establish pES cells. The results showed that rate of in vitro blastulation of parthenogenetic embryos was lower than that of fertilized ones (45.1% vs 98.0%, respectively). In addition, blastocysts developed from parthenogenetic embryos also expressed lower quality, which was demonstrated by lower total cell number. Moreover, H4K12Ac expression significantly decreased in the inner cell mass (ICM) of parthenogenetic blastocysts compared to fertilized ones, indicating a possible reason for lower blastocyst quality. Following embryo collection and activation, two ES cell lines – fertilized (fES) and pES cell lines have been successfully established and maintained long term in vitro. To sum up, differences in blastocyst quality and H4K12Ac expression in ICM cells of blastocyst may contribute to aberrant developmental and embryonic stem cell formation in parthenogenetic embryos.

scholarly journals New models of hepatitis E virus replication in human and porcine hepatocyte cell lines

2013 ◽  
Vol 94 (3) ◽  
pp. 549-558 ◽  
Author(s):  
Sophie Rogée ◽  
Neil Talbot ◽  
Thomas Caperna ◽  
Jérôme Bouquet ◽  
Elodie Barnaud ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hepatitis E Virus ◽  
Embryonic Stem ◽  
Hepatitis E ◽  
Species Barrier ◽  
Barrier Crossing ◽  
New Models ◽  
Culture Models

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

ANTITUMOR EFFECT OF SUNITINIB AND BORTEZOMIB ON BREAST CANCER CELL LINE IN VITRO

2017 ◽  
Vol 63 (1) ◽  
pp. 141-145
Author(s):  
Yuliya Khochenkova ◽  
Eliso Solomko ◽  
Oksana Ryabaya ◽  
Yevgeniya Stepanova ◽  
Dmitriy Khochenkov
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Antitumor Effect ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Actual Problem

The discovery for effective combinations of anticancer drugs for treatment for breast cancer is the actual problem in the experimental chemotherapy. In this paper we conducted a study of antitumor effect of the combination of sunitinib and bortezomib against MDA-MB-231 and SKBR-3 breast cancer cell lines in vitro. We found that bortezomib in non-toxic concentrations can potentiate the antitumor activity of sunitinib. MDA-MB-231 cell line has showed great sensitivity to the combination of bortezomib and sunitinib in vitro. Bortezomib and sunitinib caused reduced expression of receptor tyrosine kinases VEGFR1, VEGFR2, PDGFRa, PDGFRß and c-Kit on HER2- and HER2+ breast cancer cell lines

Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

2020 ◽  
Vol 21 (1) ◽  
pp. 42-60
Author(s):  
Farah Nawaz ◽  
Ozair Alam ◽  
Ahmad Perwez ◽  
Moshahid A. Rizvi ◽  
Mohd. Javed Naim ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Kinase Assay ◽  
Cervix Uteri ◽  
Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

Survival and Function of Fibroblasts Stored as Stock Cultures at a Non-freezing Temperature

1993 ◽  
Vol 21 (2) ◽  
pp. 206-209
Author(s):  
Anders H. G. Andrén ◽  
Anders P. Wieslander
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Freezing Temperature ◽  
Mouse Fibroblast ◽  
Toxic Response ◽  
Normal Manner ◽  
And Function

Cytotoxicity, measured as inhibition of cell growth of cultured cell lines, is a widely used method for testing the safety of biomaterials and chemicals. One major technical disadvantage with this method is the continuous routine maintenance of the cell lines. We decided to investigate the possibility of storing stock cultures of fibroblasts (L-929) in an ordinary refrigerator as a means of reducing the routine workload. Stock cultures of the mouse fibroblast cell line L-929 were prepared in plastic vials with Eagle's minimum essential medium. The vials were stored in a refrigerator at 4–10°C for periods of 7–31 days. The condition of the cells after storage was determined as cell viability, cell growth and the toxic response to acrylamide, measured as cell growth inhibition. We found that the L-929 cell line can be stored for 2–3, weeks with a viabilty > 90% and a cell growth of about 95%, compared to L-929 cells grown and subcultured in the normal manner. The results also show that the toxic response to acrylamide, using refrigerator stored L-929 cells, corresponds to that of control L-929 cells. We concluded that it is possible to store L-929 cells in a refrigerator for periods of up to 3 weeks and still use the cells for in vitro cytotoxic assays.

Derivation of goat embryonic stem cell-like cell lines from in vitro produced parthenogenetic blastocysts

2013 ◽  
Vol 113 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Arun Kumar De ◽  
Shweta Garg ◽  
Dinesh Kumar Singhal ◽  
Hrudananda Malik ◽  
Ayan Mukherjee ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Embryonic Stem

scholarly journals Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

1983 ◽  
Vol 96 (1) ◽  
pp. 37-50 ◽  
Author(s):  
E Schmid ◽  
DL Schiller ◽  
C Grund ◽  
J Stadler ◽  
WW Franke
Keyword(s):  
Mammary Gland ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Striking Difference ◽  
Intermediate Filament Proteins ◽  
Cell Clones ◽  
Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

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