Transient gene delivery for functional enrichment of differentiating embryonic stem cells

There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. One major challenge in the field of differentiating embryonic stem (ES) cells is the limitation to selectively purify and enrich these cells from a heterogeneous population. We developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells were purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis performed. We engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection of these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. In a second study, we explored improving the efficiency of a transient gene delivery system to differentiating ES cells by serum starving the cells for three days. We found that under serum starvation, expression of a constitutively-controlled plasmid increases from ~50% to ~83% of the population. When probed with the Cyp7A1 liver-specific reporter vector, the expression increases from ~1.4% to ~3.7% of the population. These trends were assessed using a Cy3-tagged oligonucleotide, which enabled rapid quantification of DNA uptake and was a valid predictor of ultimate cell transfection efficiency. These results suggest that modifications in media components prior to transfection of cells can have a profound effect on improving non-viral gene delivery. Efficiently genetically engineering cellular systems while circumventing the need to induce permanent gene changes will be critical for the generation of clinically-acceptable cellular material in the future.Ph.D.Includes bibliographical references (p. 82-90)by Eric J. Wallenstei