Isolation and differentiation of mouse embryonic stem cells

Recently, embryonic stem (ES) cells have become very important resources in basicmedical researches. These cells can differetiate into derivatives of all primary germ layers.

In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and themorphological aspects, population doubling time, alkalin phosphatse and differentiation properties ofthe cells were investigated.

The balstocysts from NMRI mice were cultured for 3 days up to time thatinner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinizedevery 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed andstained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at thesame time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoidbodies(EBs). The EBs were cultured for 8 - 20 days on collagen coated dish to induce thespontaneouse differentiation.

During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony ofES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasmincluding a few nuclei. In colony formation stage, the morphology changed from flat monolayer toround multilayer with strike define boundaries. Undifferentiated cells were seen as intensely smallcells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of coloniestend to differetiate by separation from each other and became larger and diffused on substrate byattaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of EScolonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneouslydifferentiate into various types of cells such as nerve and hematopoitic lineages.

Despite strike morphology of ES colonies, it is difficult to distinguish thedifferentiated from undifferentiated cell colonies in the colony formation stage. New ES cells arecapable to give rise into EBs and are susceptible of spontaneously differentiation in various type ofcells.