Investigations of Murine Embryonic Stem Cell Maintenance by Analyses of Culture Variables and Gene Expression

Abstract

The clinical realization of many gene and cell therapies requires robust, scalable methods for expanding stem cells ex vivo without compromising their developmental potential. This requirement is usually complicated by the low frequency of stem cells in most tissues (< 0.1 %) and the lack of assays for quantifying stem cells. Murine embryonic stem cells (ESC) provide a useful model for stem cell bioprocess research since ESC can be propagated indefinitely under defined conditions and assayed functionally. Nevertheless, conventional ESC cultures require daily medium exchange and an understanding of their environmental tolerance ranges is still lacking. We have now begun to explore these using the embryoid body (EB) assay to quantify R1 ESC integrity as a function of culture variables. We are also exploring the feasibility of using mRNA-based assays to assess the effect of altered culture conditions on ESC maintenance. Analysis of microarray data has revealed a subset of genes whose change in expression was consistently correlated with loss of ESC pluripotency following the induction of differentiation by LIF removal, DMSO or retinoic acid treatment. Regression models were fitted to the data and their parameter estimates used to quantify the differential expression of genes, providing new candidates for future investigation of the mechanisms by which ESC may lose pluripotency.