<p>Human embryonic stem cells (hESC) have potential applications as tools for drug screening to identify small molecule regulators of self-renewal or differentiation. Elucidating the mechanisms governing lineage commitment in hESC will allow for efficient derivation of specified cell types for clinical use. Recognizing the early steps in loss of pluripotency is key to achieving both goals of drug screening and derivation of therapeutically relevant cell types. Here we report the use of a real time cell cycle fluorescent reporter for the first time in hESC that indicates onset of differentiation in a lineage unbiased manner. Pluripotent hESC possess a short cell cycle length, due primarily to a truncated G1 phase. G1 lengthens concomitant with differentiation. Stable hESC lines expressing the live cell cycle reporter exhibit fluorescence only during G1. Due to the short length of pluripotent G1 phase, G1 fluorescence is only weakly and transiently detected, however it is quickly increased to easily detectable levels upon onset of differentiation. We hypothesize that lengthened G1 phase can be used as an indicator of differentiation status of individual human embryonic stem cells.</p> <p>Cells with lengthened G1 are typically negative for pluripotency markers OCT4, Tra-1-60 and SSEA-3 following differentiation. Differentiated cells with lengthened G1 also demonstrate increased levels of lineage-specific differentiation markers at both the protein and mRNA level. Automated image analysis of hESC indicates this mutually exclusive relationship between lengthened G1 and pluripotency exists both on the cellular level and in colonies as a whole. Here we have shown that lengthened G1 indicates both loss of pluripotency and gain of lineage markers.</p> / Master of Science (MSc)