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Differentiation of Monkey Embryonic Stem Cells: RESULTS(3)

Additional culture in N2 medium resulted in the formation of suspended spherical structures (Fig. 5B) and adherent NPCs (Fig. 5C). The adherent cell population could be cultured and passaged on reaching approximately 70% confluency. The spherical structures responded to FGF-2 stimulation with an increase in size; quartered or halved sections grew back to their original size. The ICC characterization of the expanded cell populations from the entire EB outgrowth indicated that 66% ± 6% (1070/1622, two replicates) and 55% ± 4% (939/1710, two replicates, P > 0.05) of the cells were positive for the NPC markers, nestin and musashi1, respectively (Fig. 6, A and B). In an effort to enrich NPCs, mechanical isolation of tubular structures from EB outgrowths, as seen in Figure 5A, was undertaken with subsequent culture in N2 medium containing FGF-2. ventolin inhalers

Differentiation of Monkey Embryonic Stem Cells: RESULTS(1)

ES Cell Culture

The ES cells maintained on feeder layers of MEF formed colonies with individual cells containing large nuclei, prominent nucleoli, and distinctive cell boundaries (Fig. 2A). The cell line R366.4 has a 42,XY karyotype and was at passage numbers 31-36 when placed in culture, where it has been maintained for more than 8 mo in vitro. Maintenance of ES cells in an undifferentiated state was monitored by visual observations of their morphology and the expression of primate ES cell-specific antigens TRA-1-60, TRA-1-81, and SSEA-4 (Fig. 2, B-D). In contrast to mouse ES cells, monkey ES cells did not express SSEA-1 (Fig. 2E). Monkey ES cells also expressed alkaline phosphatase (Fig. 2F) and the POU domain gene product, Oct-4 (Fig. 2G). buy nexium online

Differentiation of Monkey Embryonic Stem Cells: MATERIALS AND METHODS(4)


The primary antibodies used were against SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (mouse monoclonal, kindly provided by Dr. Peter W. Andrews, University of Sheffield, Sheffield, U.K.), Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA), a-fetoprotein (mouse monoclonal, 1:500; Sigma), cardiac troponin I (mouse monoclonal, 1: 50; Santa Cruz Biotechnology), vimentin (mouse monoclonal, 1:5; Hybrid Bank C1B7, Developmental Studies Hybridoma Bank, Iowa City, IA), human nestin (rabbit polyclonal, 1:2000; Nakama et al., unpublished results), musashi1 (rat monoclonal, 1:1000), neuronal nuclear antigen (NeuN; mouse monoclonal, 1:50; Chemicon, Temecula, CA), microtu-bular associated protein-2C (MAP-2C; mouse monoclonal, 1:500; Chem-icon), glial fibrillary acidic protein (GFAP; mouse monoclonal, 1:100; Chemicon), choline acetyltransferase (ChAT; sheep polyclonal, 1:100; Chemicon), and TH (sheep polyclonal, 1:100; Chemicon). buy generic plavix

Differentiation of Monkey Embryonic Stem Cells: MATERIALS AND METHODS(3)

Spherical structures were also frozen using equilibrium, controlled-rate cooling after incubation in 1.5 M ethylene glycol and 0.1 M sucrose for 20-30 min. Cryovials were cooled to -70C at -20C/min, seeded and further cooled to -400C at -0.30C/min, and then cooled again to -1000C at -40C/min before storage in liquid nitrogen. Vials were thawed in a 370C water bath, and the spherical structures were washed through progressively lower concentrations of cryoprotectant media (1.5, 1.0, 0.5, and 0 M ethylene glycol) before viability analysis. amaryl diabetes medication

Differentiation of Monkey Embryonic Stem Cells: MATERIALS AND METHODS(2)

METHODS(2)Low-Temperature Storage of NPCs

The NPCs maintained in N2 medium were dissociated into small cell clumps by exposure to 0.05% trypsin and 0.04% EDTA in PBS and were neutralized in DMEM/F12 (1:1) containing 10% FBS. Cells were collected by centrifugation and transferred into a 1.2-ml cryovial (Nalge Nunc International) containing 1 ml of freezing medium (90% serum and 10% dimethyl sulfoxide [DMSO; Sigma]). Vials were slowly cooled (—FC/ min) to -800C and stored in liquid nitrogen. Thawing was in a 370C water bath, and the freezing medium was diluted gradually with 10 ml of culture medium. Cells were plated as described above or incubated in culture medium containing 0.1% trypan blue solution (Sigma) for evaluation of viability. buy avandia online

Differentiation of Monkey Embryonic Stem Cells: MATERIALS AND METHODS(1)

A multistep protocol (Fig. 1) for monkey ES cell culture and differentiation was adapted from mouse and human studies. Sequential culture procedures included expansion of ES cells followed by EB formation (step 1), production of NPCs from EB outgrowths or mechanically isolated cell populations (step 2), expansion of NPCs (step 3), and finally, differentiation of NPCs into neurons and glial cell phenotypes (step 4). buy celexa 20 mg

Differentiation of Monkey Embryonic Stem Cells: INTRODUCTION(3)

INTRODUCTION(3)Selection of NPCs (Step 2)

The EBs maintained for a total of 4 days (2 days in hanging drops and 2 days in suspension) were plated onto gelatin-coated culture dishes in ES medium. After 24 h of culture to allow cell attachment and surface spreading, ES medium was replaced with serum-free ITSFn medium containing DMEM/F12 (1:1) supplemented with insulin (10 |xg/ml), sodium selenite (6.7 pg/ml), transferrin (5.5 |xg/ml), and fibronectin (5 |xg/ml; Invitrogen). The resulting EB outgrowths were maintained in serum-free ITSFn medium for 7 days, with medium replenishment every 2 days. For immu-nocytochemistry (ICC), NPCs were plated onto polyornithine- and lami-nin-coated glass coverslips (polyornithine, 15 mg/ml; laminin, 1 mg/ml; Sigma). ddvap bed wetting

Differentiation of Monkey Embryonic Stem Cells: INTRODUCTION(2)

The directed differentiation of ES cells toward neural lineages has been studied in rodents, and several differentiation paradigms have emerged. For instance, ES cell aggregates cultured in medium containing retinoic acid undergo neuronal differentiation, and serum-free sequential culture with fibroblast growth factor (FGF)-2 has been used to isolate and enrich nestin-immunoreactive neural progenitors and neurons. Neuronal differentiation of monkey ES cells, including tyrosine hydroxylase (TH)-pos-itive phenotypes, has also been reported by coculturing monkey ES cells with bone marrow stromal cells. Recently, serum-free culture conditions have been adapted to the production of a highly enriched neural population of hES cells. buy effexor cheap

Differentiation of Monkey Embryonic Stem Cells: INTRODUCTION(1)


Embryonic stem (ES) cells were first derived from pluripotent cells present in the inner cell mass of mouse blastocysts. They are characterized by their ability to differentiate into multiple cell types representative of all three embryonic germ layers in vivo or in vitro and by the ability to form chimeras when introduced into embryos. The ES cell progeny contribute to all somatic cell lineages and to the germ lines in chimeric mice, and they can be propagated indefinitely without loss of euploid chromosomal complements. Thus, they have potential as a suitable cell source for replacement therapy in human cellular degenerative diseases. endep tablets

Role and Gonadotrophic Regulation of X-Linked: DISCUSSION(6)


Previous studies from our laboratory have shown that FSH increases follicular transforming growth factor a secretion and that this theca-derived growth factor induces follicular growth in vitro. In this context, recent studies using a coculture of bovine ovarian granulosa and theca cells have shown that the theca cells are essential in protecting granulosa cells from undergoing apoptosis, a process that is follicular stage-specific. These results suggest that theca cells may secrete one or more survival factors necessary for maintenance of granulosa cell viability, although the identity of this factor is yet to be defined. It is also possible that the regulation of follicular growth by FSH is mediated by the action of TNFa. buy Endep online

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