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Digital archive of theses discussed at the University of Pisa


Thesis etd-02172010-224044

Thesis type
Tesi di dottorato di ricerca
Thesis title
Generation and characterization of a Tph2eGFP(frt) knockin embryonic stem cell line acting as an in vitro sensor for serotonergic neuron differentiation
Academic discipline
Course of study
tutor Prof. Pasqualetti, Massimo
  • eGFP
  • Embryonic stem cells
  • Knockin
  • Serotonin
  • Tph2
Graduation session start date
Release date
Serotonergic neurons are localized in the raphe nuclei of the brainstem where serotonin (5-HT) is synthesized through the activity of the rate-limiting enzyme Tryptophan hydroxylase 2, Tph2, selectively expressed in serotonergic neurons. In the Central Nervous System (CNS) serotonergic neurons provide a widespread axonal playing an important role in the control of several behavioral and physiological functions. Impairment to the normal 5-HT neurotransmission has been causally linked to neuropsychiatric disorders such as depression, anxiety, schizophrenia and autism.
Embryonic Stem (ES) cells represent an excellent model to study the differentiation of specific cell types, as well as for stem cell-based therapies designed to treat neurological disorders. In the last years few protocols using specific growth factors have been set up to obtain serotonergic neurons from differentiating mouse ES cells in vitro. However protocols using onerous reagents, such as growth factors, have restricted applicability in fields where neuron production on large scale are required, such as for example high-throughput screening. An affordable culture condition to obtain serotonergic neuron from differentiating ES cells, would then allow several application in this fields, from high throughput screening to the study several processes of serotonergic neuron development and functions, such as differentiation, migration and axonal outgrowth.
I tested several culture conditions using E14Tg2A murine ES cells and a simple treatment was identified to reproducibly and efficiently differentiate ES cells toward serotonergic neurons as assessed by the expression of serotonergic markers. On the whole, our differentiation strategy provides a simple and reliable method to generate an in vitro model to study the development and the biology of serotonergic neurons.