Mouse Fibroblasts Are Reprogrammed to Oct-4 and Rex-1 Gene Expression and Alkaline Phosphatase Activity by Embryonic Stem Cell Extracts

2007 ◽  
Vol 9 (3) ◽  
pp. 394-406 ◽  
Author(s):  
Tui Neri ◽  
Manuela Monti ◽  
Paola Rebuzzini ◽  
Valeria Merico ◽  
Silvia Garagna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Embryonic Stem ◽  
Mouse Fibroblasts ◽  
Cell Extracts

299 A FEATURE OF SELF-RENEWAL PORCINE EMBRYONIC STEM CELL-LIKE CELL LINES ESTABLISHED BY INHIBITORS

2013 ◽  
Vol 25 (1) ◽  
pp. 297
Author(s):  
S. Haraguchi ◽  
T. Tokunaga ◽  
T. Furusawa ◽  
K. Ohkoshi ◽  
M. Nakai ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Phosphatase Activity ◽  
Cell Lines ◽  
Alkaline Phosphatase Activity ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Established Cell Lines ◽  
Established Cell

Despite meticulous attempts for more than two decades, establishment of authentic porcine embryonic stem cell (ESC) from pig has never been successful. Although putative porcine ESC-like cells have been reported, such cell lines easily lose the ability of self-renewal, becoming extinct or differentiating after only a limited number of passages in culture. Porcine ESC-like cells exhibiting the property of self-renewal rather than pluripotency are considered a valuable resource in applications such as drug screening and toxicology testing in humans and livestock, and in veterinary medicine. In the present study, we evaluated the effect of glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 and Erk signalling inhibitor PD184352 for use in establishing ESC-like cell lines derived from the inner cell mass (ICM) of porcine blastocysts produced in vitro. These ICM-derived cell lines were initially cultured and passaged in conventional human ES medium. They displayed so-called ESC-like morphology; for example, the isolated colonies began to grow as a monolayer with coarse cell–cell boundaries, in which the cells exhibited polygonal boundaries, high nuclear/cytoplasmic ratios, abundant lipid-like inclusions, alkaline phosphatase activity, and expression of markers of undifferentiated cells such as OCT4 and NANOG. After transfer to culture in ES medium containing the inhibitors, the morphology of the colony was dramatically changed, displaying a closely packed and smooth-edged colony with tight cell–cell boundaries. Remarkably, growth of the established cell lines is leukemia inhibitory factor (LIF)-dependent. The inclusion of inhibitors supports self-renewal, thus enabling continuous culture for over 100 passages while maintaining an undifferentiated state. High-passage-number cells continued to express undifferentiated marker genes and showed alkaline phosphatase activity and telomerase activity with an X chromosome status of XaXi. We further investigated the potential for differentiation of the established cell lines. The cells could easily form embryoid body-like spheres in suspension culture. When either the spheres or ESC-like cells were inoculated under the kidney or testis capsules of nude mice, classical teratoma formation was not observed after 2 to 3 months. However, histological analyses revealed apparent invasive proliferation derived from porcine cells. Although further analyses are required to characterise the property of the porcine ESC-like cells, we have recently succeeded in establishment of green fluorescent protein (GFP)-expressing stable cells lines, which will be useful for further investigation.

Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

2011 ◽  
Vol 251 (2) ◽  
pp. 110-118 ◽  
Author(s):  
Dorien A.M. van Dartel ◽  
Jeroen L.A. Pennings ◽  
Liset J.J. de la Fonteyne ◽  
Karen J.J. Brauers ◽  
Sandra Claessen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation

The influence of titanium surfaces treated by alkalis on macrophage and osteoblast-like cell adhesion and gene expression in vitro

RSC Advances ◽  
2015 ◽  
Vol 5 (99) ◽  
pp. 81378-81387 ◽  
Author(s):  
Ting Ma ◽  
Xi-Yuan Ge ◽  
Sheng-Nan Jia ◽  
Xi Jiang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Cell Adhesion ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Related Gene ◽  
Titanium Surfaces

The effect of alkali-treated titanium surfaces on inflammation-related gene expression of macrophages and alkaline phosphatase activity of osteoblast-like cells.

scholarly journals Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Andrea C. Romero ◽  
Eugenio Vilanova ◽  
Miguel A. Sogorb
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
Stem Cell ◽  
Cell Viability ◽  
Embryonic Stem Cell ◽  
Total Duration ◽  
Embryonic Stem ◽  
End Points ◽  
Cell Test

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicityin vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes:Pnpla6,Afp,Hdac7,Vegfa, andNes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression ofNes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting forin vitrorisk assessment embryotoxicity.

scholarly journals 169 CULTURE OF MURINE EMBRYONIC STEM CELLS ON NWPF DISCS

2005 ◽  
Vol 17 (2) ◽  
pp. 235 ◽  
Author(s):  
G. Cetinkaya ◽  
S. Arat ◽  
H. Odaman Mercan ◽  
M.A. Onur ◽  
A. Tumer
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Signalling Pathways ◽  
Feeder Layers ◽  
The Matrix ◽  
Matrix Bound

Murine embryonic stem cells derived from the inner cell mass of mouse blastocysts can be maintained in culture for extended periods by using feeder layers and leukemia inhibitory factor (LIF). Maintenance of undifferentiated status occurs via LIF-mediated signalling pathways. In this study we cultured embryonic stem (ES) cells in Knockout-DMEM with serum replacement on a three-dimensional matrix, non-woven polyester fabric (NWPF), which is formed from non-arrayed polyethylene teraphthalate fibers. The surface of the fibers was modified by immobilizing LIF. While stimulating the matrix-bound form of LIF in vitro, we also tried to induce LIF-mediated signalling pathways continually. Our goal was to constitute a synthetic microenvironment that would support the undifferentiated growth of murine ES cells. Experimental groups were examined according to colony morphology, alkaline phosphatase activity, SSEA-1 antibody immunoreactivity, and SEM analyses. It was shown that three dimensional macroporous fibrous matrix, NWPF could support growth of undifferentiated ES cells. However, the ratio of undifferentiated colonies was higher on feeder layers than an polymeric surfaces (93% on mouse embryonic fibroblasts; 63,7% on hydrolized polymeric surface, P < 0,05). Results showed that LIF-immobilized surfaces supported undifferentiated growth of ES cells better than hydrolyzed surfaces. Colonies cultured on LIF-immobilized surfaces, had higher alkaline phosphatase activity and undifferentiated phenotype ratio than those on hydrolyzed surfaces. When the soluble or the matrix-bound form of LIF was used, the number of undifferentiated colonies increased in the polymeric groups (77.8% soluble LIF; 81.6% matrix bound LIF P < 0,05). On NWPF discs, ES cells formed big cell aggregates which had high alkaline phosphatase activity but low SSEA-1 immunoreactivity . When they were passaged to feeder layers, SSEA-1 activity increased. We managed to obtain undifferentiated colonies on NWPF discs by using LIF but the skeletal structure of polymeric matrix would be more convenient for differentiation studies. This study was performed in TUBITAK-RIGEB and supported by a part of grant from Hacettepe University (0102601001).

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