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).

BCR-ABL Activates STAT3 Via MEK Kinase 1 in Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4320-4320
Author(s):  
Yukinori Nakamura ◽  
Toshiaki Yujiri ◽  
Ryouhei Nawata ◽  
Kozo Tagami ◽  
Yukio Tanizawa
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Es Cells ◽  
Embryonic Stem ◽  
Myelogenous Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Mek Kinase

Abstract BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate STAT3 and to promote self-renewal in ES cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction (Oncogene22:7774, 2003). To investigate the role of MEKK1 in Bcr-Abl-induced transformation of ES cells, p210 Bcr-Abl was stably transfected into wild type (WT+p210) and MEKK1−/− (MEKK1−/−+p210) ES cells. Bcr-Abl enhanced both MEKK1 expression and activation in ES cells, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT+p210 cells showed constitutive STAT3 activation and formed compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1−/−+p210 cells, by contrast, showed less STAT3 activity than WT+p210 cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays an essential role in Bcr-Abl-induced STAT3 activation and in the capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.

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.

scholarly journals Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein.

1986 ◽  
Vol 103 (4) ◽  
pp. 1615-1623 ◽  
Author(s):  
B de Bernard ◽  
P Bianco ◽  
E Bonucci ◽  
M Costantini ◽  
G C Lunazzi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Protein A ◽  
Matrix Vesicles ◽  
Immune Reactivity ◽  
The Matrix ◽  
Immunohistochemical Evidence

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.

scholarly journals Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium.

1994 ◽  
Vol 126 (5) ◽  
pp. 1311-1318 ◽  
Author(s):  
R T Ballock ◽  
A H Reddi
Keyword(s):  
Alkaline Phosphatase ◽  
Serum Concentration ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Critical Role ◽  
Three Dimensional ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Type X Collagen

Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defined cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis.

197 INDUCED PLURIPOTENT STEM CELLS (iPS) DERIVED FROM EQUINE UMBILICAL CORD CELLS USING LENTIVIRUS VECTOR Stemcca

2014 ◽  
Vol 26 (1) ◽  
pp. 213
Author(s):  
M. Guastali ◽  
F. Bressan ◽  
R. Maziero ◽  
D. Paschoal ◽  
M. Sudano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Embryonic Stem Cells ◽  
Phosphatase Activity ◽  
Umbilical Cord ◽  
Alkaline Phosphatase Activity ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Induced Pluripotent

Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5 mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 105 cells, these cells were transduced with 50 μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8 ng mL–1 polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12 h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n = 10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.

Observation of Proliferation and Attachment of Three Different Cell Types on Nano-Fibrous Silk Film & Scaffold

2007 ◽  
Vol 342-343 ◽  
pp. 85-88 ◽  
Author(s):  
Hyun Sook Baek ◽  
Young Hwan Park ◽  
Ki Chang Seok ◽  
Jong Chul Park ◽  
Don Kyun Rah
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Viability ◽  
Phosphatase Activity ◽  
Silk Fibroin ◽  
Alkaline Phosphatase Activity ◽  
Mtt Assay ◽  
Cell Attachment ◽  
Three Dimensional ◽  
Cell Types ◽  
Different Cell Types

Attachment and viability of different cell types(fibrioblast, chondrocyte and osteoblast ) was observed on two forms of silk (mat & Three-dimensional scaffolds). The osteoblasts behaviors cultured on silk mat were significantly higher than that found on 3-D silk fibroin scaffold (3-D SF scaffold). In the MTT assay, the cell viability of fibroblasts, chondrocyte and osteoblasts seeded on 2-D nanofiber mat was (2-D mat) significantly higher than that found on 3-D SF scaffold. Similar result could be seen from SEM observation and cell attachment study. However, alkaline phosphatase activity was significantly increased on 3-D SF scaffold than on2-D nanofiber

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