Somatic cell lines with targeted eGFP insertion into mouse Tph2 locus as a model for serotonergic transdifferentiation

The investigation of serotonergic (5-HT) neuron activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Differentiation of pluripotent stem cell lines or transdifferentiation of somatic cells carrying a 5-HT neuron-specific reporter provides a platform for such studies and can serve as a model for drug discovery. In this study we aimed to generate 5-HT neurons from somatic cells directly or via formation of induced pluripotent stem (iPS) cells using a serotonergic reporter mouse line. As a source of somatic cells we took primary mouse embryonic fibroblasts (MEFs) carrying the eGFP gene targeted to the endogenous Tph2 locus (Tph2:eGFP MEFs, Migliarini et al., 2013). Using these MEFs we first generated iPS cells by transfection with a single polycistronic lentivirus carrying Oct4, Sox2, cMyc, and Klf4 (OSKM) genes driven by a doxycycline-inducible promoter. The resulting iPS cells showed ES cell-like morphology and expressed the pluripotency-associated genes Nanog and Oct4, but not eGFP. To test the potential of Tph2:eGFP-iPS cells to produce 5-HT neurons we induced neuronal differentiation by formation of embryoid bodies following replating on PDL/Laminin substrate. First eGFP-positive (eGFP+) cells with neuronal morphology appeared 14 days after induction of differentiation. Immunohistochemical analysis revealed that all eGFP+ cells express the neuronal marker, Tuj1, and produce serotonin. We detected a high expression of 5-HT markers, such as Tph2 by qPCR in the eGFP+ cell population isolated by FACS, further confirming the 5-HT nature of eGFP-expressing cells. We next took advantage of Tph2:eGFP MEFs to monitor the transdifferentiation of somatic cells to 5-HT neurons by two different strategies: (1) direct conversion using a neuron-specific cocktail of reprogramming factors (Ascl1, Brn2, Myt1l, (ABM)) in combination with transcription factors important for serotonergic differentiation, (2) via induction of pluripotency genes in culture conditions permissive for the formation of 5-HT neurons. Induction of neuronal genes (ABM) led to the robust generation of neurons, but not of eGFP+ cells. In contrast, transfection of Tph2:eGFP MEFs with OSKM lentiviruses and cultivation in LIF-free conditions in neuronal differentiation medium led to the appearance of eGFP+ neurons after 25 days. In conclusion, we generated a useful model system to monitor 5-HT neurons in in vitro reprogramming experiments. To our knowledge, this is the first successful transdifferentiation of somatic cells to serotonergic phenotype. References: Migliarini et al., 2013, Mol Psychiatry 18(10):1106-18