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.

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].

APPROACHES FOR IMPROVEMENT OF HUMAN PLURIPOTENT STEM CELL-DERIVED STRIATAL NEURON DIFFERENTIATION PROTOCOLS AND QUANTITATIVE FLUORESCENCE MICROSCOPY METHODS.

A. Laporta
2018-03-23

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.
ZUCCATO, CHIARA
Settore BIO/11 - Biologia Molecolare
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].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/571234
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