MORPHOLOGICAL AND KARYOTYPE STUDIES OF MOUSE INNER CELL MASS COLONEIES DERIVED DURING EARLY PASSAGES

The colony from inner cell mass of embryo has potential to proliferation unlimitedly in vitro and remain undifferentiated. These colonies can maintain their normal karyotype in eupleuidy in a cell population. In order to prepare the embryonic stem (ES) cells, we cultured the blastocyst and evaluted the morphology of inner cell mass during the outgrowth, disaggregation, expantion and production of the cell colonies. The karyptype of these cells was studied in the early passage.

For this purpose, 20 mitomycin C-treated intact blastocyst, taken from 15-day rat embryos, were transferred to a layer of embryonic fibroblasts. The outgrowth of inner cell mass was mechanically disaggregated and the cells were passaged every 2-3 days in DMEM 1000 IU /ml containing LIF and 20 % FBS. The cells were sub-cultured for up to nine passages. The colony of the cells during the 7 th and 9th passages were processed for karyotyping by Giemsa staining.

The results showed that in the expantion stage the coloney appeared as a flat monolayer mass with strike boundaries and nondistinguished cytoplasm with a few nuclei. In coloney formation stage, the undifferentiated colonies of cell change their morphology to a round multilayer colony with clearly defined boundaries. Chromosomal status showed normal karyotype in 86% of cells at the 7 th passage and in 67% of cells at 9th passage. The anupleuidy of the chromosomes was mostly observed as monosomy and metacentric.

The overall findings from this study demonstrated that the ES undifferetiated colonies could be distinguish by morphological criteria, passage acceptability and division diplication rate. Furthermore, detection of ES cell colonieo during the expantion stage is the first signs of success in isolation of ES cell coloneies.