From c-Photina mouse embryonic stem cells to high-throughput screening of differentiated neural cells via an intermediate step enriched in neural precursor cells.

The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina mES stably expressing a Ca(2+)-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates. The c-Photina mES cell line is particularly well suited for the study of the pharmacological modulation of target genes that induce Ca(2+) mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC(1280) library monitoring the agonistic or antagonistic activities of compounds. They also demonstrate the possibility to generate and freeze bulk preparations of cells at an intermediate stage of differentiation and enriched in neural precursor cells, which retain the ability to form fully functional neural networks once thawed. The proposed cell model is of high value for HTS purposes because it offers a more physiological environment to the targets of interest and the possibility of using frozen batches of neural precursor cells.

A photoprotein in mouse embryonic stem cells measures Ca2+ mobilization in cells and in animals.

Exogenous expression of pharmacological targets in transformed cell lines has been the traditional platform for high throughput screening of small molecules. However, exogenous expression in these cells is limited by aberrant dosage, or its toxicity, the potential lack of interaction partners, and alterations to physiology due to transformation itself. Instead, primary cells or cells differentiated from precursors are more physiological, but less amenable to exogenous expression of reporter systems. To overcome this challenge, we stably expressed c-Photina, a Ca(2+)-sensitive photoprotein, driven by a ubiquitous promoter in a mouse embryonic stem (mES) cell line. The same embryonic stem cell line was also used to generate a transgenic mouse that expresses c-Photina in most tissues. We show here that these cells and mice provide an efficient source of primary cells, cells differentiated from mES cells, including cardiomyocytes, neurons, astrocytes, macrophages, endothelial cells, pancreatic islet cells, stably and robustly expressing c-Photina, and may be exploited for miniaturized high throughput screening. Moreover, we provide evidence that the transgenic mice may be suitable for ex-vivo bioimaging studies in both cells and tissues.