Modeling biological systems in vitro has contributed to clarify complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated towards specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2(GFP), in which the vital reporter GFP replaces the Tryptophan hydroxylase 2 (Tph2) gene. Tph2(GFP) mES cells selectively express GFP upon in vitro differentiation towards the serotonergic fate, they synthetize serotonin, possess excitable membranes and show the typical morphological, morphometrical and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP we highlighted by time-lapse video-microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that pre-differentiated Tph2(GFP) cells are able to terminally differentiate, integrate and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2(GFP) mES cell line as a useful tool allowing accurate developmental and dynamic studies, and represents a reliable platform for the study of serotonergic neurons in health and disease.
A Tph2GFP Reporter Stem Cell Line To Model in Vitro and in Vivo Serotonergic Neuron Development and Function
MARINO, ATTILIO;MIGLIARINI, SARA;BRILLI, ELISA;PELOSI, BARBARA;MADDALONI, GIACOMO;PRATELLI, MARTA;PELLEGRINO, MARIO;PASQUALETTI, MASSIMO
2017-01-01
Abstract
Modeling biological systems in vitro has contributed to clarify complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated towards specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2(GFP), in which the vital reporter GFP replaces the Tryptophan hydroxylase 2 (Tph2) gene. Tph2(GFP) mES cells selectively express GFP upon in vitro differentiation towards the serotonergic fate, they synthetize serotonin, possess excitable membranes and show the typical morphological, morphometrical and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP we highlighted by time-lapse video-microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that pre-differentiated Tph2(GFP) cells are able to terminally differentiate, integrate and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2(GFP) mES cell line as a useful tool allowing accurate developmental and dynamic studies, and represents a reliable platform for the study of serotonergic neurons in health and disease.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
