MITOTIC ANALYSIS AS A MEASURE OF GENOMIC INSTABILITY DURING THE PROPAGATION OF HUMAN PLURIPOTENT STEM CELLS

Genetic abnormalities in undifferentiated human embryonic stem (hES) cells have been reported in many studies and continue to be a significant challenge for the stable propagation of hES cells in sufficient quantities for therapeutic application. When cultured for long periods of time, hES cells are susceptible to genetic aberrations. The goal of this project is to observe and characterize the presence of abnormal mitotic figures in human pluripotent stem cells using the laboratory technique of immunocytochemistry. Future directions of this work will include quantifying abnormal mitotic figures under different culture conditions. It was predicted that supernumerary centrosomes, anaphase bridges, and multipolarity play a role in the genomic instability of human embryonic stem cells. Also, we predicted that single cell dissociation through enzymatic passaging increases genomic instability and links back to these abnormal mitotic figures. Basic cell culture techniques such as thawing, feeding, and passaging (manual and enzymatic) were used to propagate different cell lines. Comparing hES cell lines to embryonic carcinoma cells, we have observed similar abnormal mitotic figures, such as supernumerary centrosomes, anaphase bridges, and multi‐polarity, between the cell lines suggesting that both cell types are prone to genomic instability by the same causative mechanisms. In conclusion, we have found that hES cells and embryonic carcinoma cells are similar in the maintenance of their chromosomal integrity that could be a source of genetic instability of human embryonic stem cells in culture.