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Mahmoud Hashemi-Tabar, Fatemeh Javadnia, Mahmoud Orazizadeh, Maryam Baazm,
Volume 3, Issue 1 (7-2005)

Background: Recently, embryonic stem (ES) cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. Objectives: In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. Materials and Methods: The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoid bodies(EBs). The EBs were cultured for 8 - 20 days on collagen coated dish to induce the spontaneouse differentiation. Results: During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages. Conclusion: Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells.
Mahmoud Hashemi-Tabar, Fatemeh Javadnia, Mahmoud Orazizadeh, Ghasem Sakei, Maryam Baazm,
Volume 5, Issue 2 (7-2007)

Background: Since embryonic stem (ES) cells have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, ES cells could potentially provide an unlimited cell supply for human transplantation.
Objective: In order to study the differentiation of mouse embryonic stem (mES) cells, they were cultured in suspension by using ES media without Leukemia Inhibitory Factor (LIF) to induce spontaneous differentiation. Cellular morphology of differentiated derivatives was then evaluated.
Materials and Methods: Undifferentiated mES from our laboratory were cultured in three different settings by using ES media containing 0.1% / 1mM trypsin/EDTA and removing LIF; in the absence of murine embryonic fibroblast (MEF) feeder cells (group 1), in the presence of MEF feeder cells with a density of 0.5×105 cells/ml (group 2), and 0.5×106 cells/ml (group 3). Five days after the initiation of cell culture, and inducing mES cells to form embryoid bodies (EBs), they were removed from dish by centrifugation, and then they were cultured on collagen coated dishes for 20 days. The dishes were fixed and stained by Wright-Gimsa method at the end of the study period.
Results: In group 1, mES cells showed spontaneous differentiation to all derivatives of three germ cells, including: epithelia like, fibroblast like and neron-like cells. In group 2, almost all ES cells were found to be differentiated into granular progenitor cells including hematopoietic cell lineages. In group 3, various morphologies including nerve cell lineages and fibroblast-like cells were detected.
Conclusion: Differentiation of mES cells can be a dose response process, depending on the factors that may be released from MEF feeder layer to ES media in a coculture system. Our results indicated that in the presence of low numbers of MEF cells, mES cells can spontaneously differentiate into hematopoeitic cell lineages.

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