Objective: The process of ESCs derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to optimize a protocol for isolating and culturing of mouse embryonic fibroblast (MEF) as feeder layer and mESC culture on MEF.Materials and Methods: Mature NMRI male mice were coupled for one night with two groups of females (either naïve or those pre-injected with different doses of Folligon (PSMG). At 12.5 days after gestation (E12.5), MEF cells were obtained from mice embryos using two different methods Chemical (using trypsin/EDTA) or mechanical (using 18G syringe). Different combination of supplements/media was used to optimize MEF culture. The viability of cells was measured by trypan blue and MTT-assay and the doubling time was calculated. To obtain blastocyst, time-mated females were sacrificed at E3.5, and then blastocysts were flushed-out of the uterous and collected using an inverted microscope. The blastocysts were implanted on mitomycin inactivated MEF feeders. After 4-6 days the inner cell mass of hatched blastocysts were isolated from trophoblast cells by pipetting the ICM. Results: We obtained blastocysts from both hormone injected and not injected mice with the same success rates. Obtained MEF cells grow better in cultivation medium with 15% FBS and 0.1% 2ME and they retain higher viability after freeze/thawing. Implanted blastocysts, obtained by both procedures, hatched and expanded for derivation of mESCs similarly. The blastocysts were positive for alkaline phosphatase.Conclusion: The protocol described above seems to be the most efficient and practical procedure for blastocysts isolation and MEF culture as its feeder layer for establishing a mESC line in our laboratory.

Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs and the combination of hLIF and bFGF was unable to improve the culture condition.

In this paper we would like to present a numerical study of the effect of magnetic fields on natural convection (magneto-convection) flow of electrically conducting fluid. The 2D square cavity which was studied is subjected to a sinusoidal temperature conditions. The left and the right walls were respectively heated and cooled with a sinusoidal temperature while the top wall was kept thermally insulated. The equations are solved numerically by employing finite element method (MEF) using the software COMSOL Multiphysics. We presented the results in wide range of Hartmann number and Rayleigh number in terms of isotherm contours, velocities fields streamlines,, and in an average and local Nusselt number which varies sinusoidally. Our results are shown to be in good conformity with the available benchmark solutions.

Objective: Our aims were to derivate horse ES cell lines by repeated passage of ICM cells and assess hLIF and bFGF effects on maintenance of their pluripotenciesMaterials and Methods: A basic medium was prepared for the culture of ICMs and horse embryonic stem cells using KODMEM supplemented with 10% FBS, 0.1mM Non-essential Amino Acids, 2mM L-glutamine, 1% Insulin-Transferrin-Selenium, 100mg/ml of streptomycin, 100 units/ml of penicillin and 0.1mM 2b- mercaptoethanol on γ-irradiated MEFs. ICM cells were recovered mechanically from day-7 blastocysts and cultured in basic medium on feeder cells. The culture medium was changed every day after first passage and the passage was performed by mechanical dissociation with a needle every 6-8 days until passage 15. The putative horse ESCs were cultured in the basic medium supplemented with human leukemia inhibitory factor (hLIF 40ng/ml) only, basic fibroblastic growth factor (bFGF 4ng/ml) only, or bFGF plus hLIF with MEF feeder cells. Colony morphology was evaluated, continuously. Finally, for determination of cell pluripotency, the hESCs were analyzed for markers of pluripotency. Immunostaning of the putative horse ESCs was done for Oct4, SSEA-1, GCTM-2, TRA1-60 and TRA1-81.Results: Derived ICMs, from day-7 blastocysts, were cultured with basic medium on MEF feeder cell monolayer. They grew as flat colonies including cells with different morphology (P0). Different morphological cells were transferred for next passages separately (P1). Cells in basic medium only, and basic medium added LIF could keep colony form similar to each other until passage 15. Although the cells in basic medium containing bFGF could produce some colonies in early passages, they could not maintain their morphology in later passages. Those colonies which were plated in basic medium added hLIF and bFGF did not grow after a few passages. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct4, SSEA-1, GCTM-2, TRA1- 60 and TRA1-81. These results indicated that those cell colonies were not completely purified embryonic stem cells, but some of them were still pluripotent.Conclusion: Our study show the primary horse ESCs are able to self-renew when they are cultured in the basic medium on γ-irradiated MEFs. These results also indicated that cell colonies were not completely purified embryonic stem cells, but some of them were still pluripotent. ESC-like cell morphology of horse putative ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggests that hLIF is neither prerequisite nor negative for maintenance of horse ESCs; bFGF seem to be negative for maintenance of horse ECSs. The combination of LIF and bFGF is unable to improve the culture condition. Thus, the new factors need to be investigated further to maintain horse ESCs in purified population.