APPROACHES FOR IMPROVEMENT OF HUMAN PLURIPOTENT STEM CELL-DERIVED STRIATAL NEURON DIFFERENTIATION PROTOCOLS AND QUANTITATIVE FLUORESCENCE MICROSCOPY METHODS.
Tesi di Dottorato
Data di Pubblicazione:
2018
Citazione:
APPROACHES FOR IMPROVEMENT OF HUMAN PLURIPOTENT STEM CELL-DERIVED STRIATAL NEURON DIFFERENTIATION PROTOCOLS AND QUANTITATIVE FLUORESCENCE MICROSCOPY METHODS / A. Laporta ; scientific tutor: C. Zuccato ; directed by: E. Cattaneo. DIPARTIMENTO DI BIOSCIENZE, 2018 Mar 23. 30. ciclo, Anno Accademico 2017. [10.13130/laporta-angela_phd2018-03-23].
Abstract:
Directed differentiation of human Embryonic Stem cells (hESC) and induced Pluripotent Stem
Cells (hiPS) is used to produce in vitro models to understand the mechanisms involved in neural
development and to study the cellular and molecular processes affected in neurodegenerative
diseases. Furthermore, these cells represent a potential source of in vitro generated mature
neurons that can be used in cell replacement therapies.
The laboratory where I performed my PhD thesis is interested in studying Huntington Disease
(HD), a rare inherited disorder caused by an expanded stretch of CAG trinucleotide repeats in
the huntingtin (HTT) gene, which results in neuronal dysfunction and death. In HD, the medium
spiny neurons (MSNs) of the striatum represent the population most severely affected. The
study of the different stages of striatal development in vitro from human pluripotent stem cells
(hPSC) could be instrumental for both the identification of the molecular processes that are
affected in HD and the generation of MSNs for cell replacement therapies. For this reason, the
main goal of my doctoral degree was to create in vitro models that recapitulate human striatal
development in vivo and ultimately generate authentic MSNs.
In the first part of my thesis, I confirmed previous data from the lab showing that H9 hPSC can
efficiently differentiate towards the striatal lineage (Delli Carri et al., 2013). Moreover, I
extended this finding by showing that this protocol can be successfully applied to other three
hPSC lines. Additionally, to better characterize the progenitor and neuronal subpopulations
generated at different stages of the in vitro differentiation, I developed an automated microscope
image quantification pipeline that enabled a high degree of accuracy in a diverse range of
molecular marker measurements. With this new method, I was able to monitor cell identity
transitions observed during in vitro differentiation and quantify the resulting neuronal
subpopulations.
Previous in vivo analysis of cell transitions in the human developing striatum allowed to identify
two transcription factors (TFs), Gsx2 and Ebf1, involved in neuronal identity progression.
Based on this, in the second part of my PhD work, I developed a strategy to improve MSNs
generation efficiency from hESC. Following in vitro differentiation, I monitored the effects of
the exogenous TFs expression by analysing the expression of various cell identity molecular markers by immunofluorescence. By using this strategy, I was able to improve the
differentiation of hPSCs into MSNs in vitro from 7% to 38%.
In the future, we are planning to take advantage of the tools and knowledge gathered in the
course of my PhD to develop a differentiation protocol in line with the GMP procedures
necessary for the cell replacement approach.
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
A. Laporta
Link alla scheda completa: