144 A targeted serotonergic neuron reporter stem cell line to model in vitro serotonergic neuron development and function
G. PACINI1, A. MARINO1, B. PELOSI1, E. BRILLI1, S. MIGLIARINI1, A.
FERRARI2 & M. PASQUALETTI1
Institute(s): 1University of Pisa, Cell and Developmental Unit, Department of Biology, Pisa, Italy, 2ETH Zurich, Zurich, Switzerland
ABSTRACT
Modeling biological systems in vitro has contributed to clarify complex mechanisms regulating development and function of specific cell types in simplified and controlled experimental conditions. To date studies on serotonergic neuronal development and activity in vitro and its relationship to disease has been limited due to the lack of suitable in vitro model systems allowing to trace and to follow differentiating serotonergic neurons in heterogeneous cell populations and to study their development over the time. It has recently emerged that embryonic stem cell (ESC) based models are not only able to mimic but also provide novel insight in developmental neurobiology providing suitable tools to dissect the establishment of more complex neuronal features such as regional patterning and hodological properties. In this study we described the generation and characterization of a serotonergic neuron reporter stem cell line, namely the Tph2eGFP knockin murine ESC line in which the vital reporter eGFP replaces the Tryptophan hydroxylase 2 (Tph2) gene. We successfully obtained specific eGFP labeling of serotonergic neurons from in vitro differentiated Tph2eGFP ESCs and the expression of the reporter was exclusively confined to serotonergic cell lineage. Serotonergic neurons obtained from Tph2eGFP cells shared both morphological and molecular features typical of mature serotonergic raphe neurons. Moreover the morphometrical variability of serotonergic neurons well modeled the heterogeneity of the serotonergic neurons present in vivo.
Finally, the presence of sodium and calcium voltage-dependent channels in serotonergic neurons differentiated in vitro was functionally demonstrated.
The expression of the vital reporter allowed to use timelapse video- microscopy to highlight several dynamic developmental events of serotonergic neuron such as differentiation, cell migration and axonal outgrowth. Concerning neuronal migration, we observed that serotonergic neurons moved in vitro proceeding by nucleokinesis and we underlined a correlation between eGFP expression levels and their neuronal migration activity. Eventually, Tph2eGFP ESC-derived cells were further characterized by in vivo grafting and we demonstrated that pre-differentiated Tph2eGFP cells integrated and survived the host brain innervating different areas and showing features resembling that of endogenous serotonergic neurons.
On the whole, the present study introduced the Tph2eGFP ESC line as a novel tool acting as accurate and faithful in vitro sensor for serotonergic neuron differentiation particularly suitable for live cell screening assays, and put such a model system as simple and reliable platform for the study of serotonergic neurons both in health and disease condition.
146
Figure 1: Molecular analysis of in vitro differentiated Tph2eGFP ESCs showed molecular features mirroring to that of developing serotonergic neurons present in the raphe region. Immunofluorescence analysis on eGFP expressing cells differentiated in vitro from Tph2eGFP using appropriate protocols, revealed that they expressed markers of post-mitotic neurons (NeuN-positive, a-c), they synthesize serotonin as 5-HT immunoreactivity can be highlighted in all eGFP positive neurons (d-f), and they showed expression of markers for serotonergic neuron phenotype such as the serotonin transporter (SERT, g-i) and the vescicular monoamine transporter 2 (VMAT2, j-l). eGFP expression was restricted to serotonergic neuronal lineage as double immunofluorescence for eGFP and Tyrosine hydroxilase (Th) showed that any eGFP+ cell co-localized with Th positive cell (m-o). eGFP positive neurons were devoid of markers for glial lineages such as Olig2 (p-r) and GFAP (s-u).
Scale bars: 10 µm.
Figure 2. in vitro differentiated eGFP+ Tph2eGFP cells reflect morphological and morphometrical features of serotonergic raphe neurons. Comparison between neurons obtained in vitro from differentiating Tph2eGFP ES cell culture (a-d) showing different soma size and eGFP+ serotonergic neurons present in vivo in the raphe nuclei of Tph2::eGFP(FRT) heterozygous mice (e-f). Morphometrical quantification of the heterogeneity in serotonergic soma neuronal size of in vitro (j, grey line) and in vivo (j, red line) differentiated eGFP-positive cells, by means of probability density plot, highlighting that the repertoire of soma size range of serotonergic neurons differentiated in vitro closely matches those observed in vivo. Scale bars: 10 µm.
148
followed over the time. The growth cone of one single elongating neurite can be followed as indicated by arrows.
Figure 4. Presence of functional voltage-dependent channels in eGFP+ neurons differentiating from Tph2eGFP cells. Representative calcium influx response to depolarization stimulus in individual eGFP positive neurons differentiated in vitro from Tph2eGFP cells using appropriate protocols. In (a) the time course of fluorescence intensity in 3 different living serotonergic neurons loaded with Fluo4-AM calcium indicator dye showed calcium influx after KCl induced depolarization suggesting the presence of functional calcium voltage-dependent channels. (b-e) Images representing the field analyzed in false colours:
eGFP endogenous fluorescence (b), basal intracellular calcium level as assayed by Fluo4-AM dye loading (c), calcium influx due to 120 mM KCl induced depolarization (d), further calcium raising induced by ionomycin addition (e).
Figure 5. in vivo analysis of pre-differentiated Tph2eGFP cells following transplantation into mice brain. (a-f) Confocal images showing the presence of eGFP positive cells 25 weeks after transplantation of pre-differentiated Tph2eGFP cells in the dorsal raphe of wilde- type mice. Sagittal sections of dorsal raphe were stained with specific antibodies against eGFP (green) and 5-HT (red) and double immunofluorescence showed the presence of co- labeled cells. (g-i) Grafted cells emitted a lot of fibers showing immunoreactivity for both eGFP (g) and 5-HT (h).
