Characterization of human dopaminergic neurons in the developing mesencephalon and upon differentiation of stem cells- for replacement therapy in Parkinson´s disease
Nelander Wahlestedt, Jenny LU
(2013)
In Lund University Faculty of Medicine Doctoral Dissertation Series
2013:135.
- Abstract
- Cell-replacement therapy is a promising approach for treating patients with Parkinson ́s disease (PD). For this purpose, there is a need for developing a protocol that can generate high numbers of human transplantable mesencephalic dopaminer- gic (mesDA) neurons. For decades, studies have therefore been made in mouse and other model organisms in order to elucidate key-factors that can be used for proper characterization and patterning of cells to become mesDA neurons. However, limited numbers of studies have been performed in human to confirm the expression and role of these factors. In this thesis, I have analysed the developing human mesencephalon for expression of key-fate determining proteins, which are known to be important in mesDA... (More)
- Cell-replacement therapy is a promising approach for treating patients with Parkinson ́s disease (PD). For this purpose, there is a need for developing a protocol that can generate high numbers of human transplantable mesencephalic dopaminer- gic (mesDA) neurons. For decades, studies have therefore been made in mouse and other model organisms in order to elucidate key-factors that can be used for proper characterization and patterning of cells to become mesDA neurons. However, limited numbers of studies have been performed in human to confirm the expression and role of these factors. In this thesis, I have analysed the developing human mesencephalon for expression of key-fate determining proteins, which are known to be important in mesDA neuron development in the mouse. These key-factors were shown to exhibit a similar spatiotemporal expression pattern in the human brain, suggesting a conserved role for these proteins in mesDA neuron development across species. We were also able to confirm that human mesDA neurons are derived from radial glial cells in the floor plate (FP), positioned in the most ventral part of the mesencephalon. With the help of human specific mesDA markers we could optimize and develop a protocol that successfully patterns human embryonic stem cells (hESCs) into functional mesDA neuron progenitors. This protocol is one of the first to allow the generation of authen- tic mesDA neuron progenitors through a FP stage, mimicking early human mesDA neuron development. Furthermore, these cells survive transplantation and can restore motor deficits in a rat Parkinson ́s disease (PD) model. Thus, these cells show a prom- ising potential to be further developed for clinical use, treating patients with PD.
In addition to patterning cells to a mesencephalic (midbrain) fate, we were also able to regionalize hESCs to neural progenitors resembling those of the human em- bryonic forebrain and hindbrain. This protocol, in combination with the generation of a SOX1-GFP hESC reporter cell line and the expression of the cell-surface marker CORIN by human FP cells, allowed us to isolate pure populations of regionalized neu- roepithelial and FP cells for deep sequencing. From this study, we identified several microRNAs with potential roles in the specification and development of human neural progenitors populations, including mesDA neuron progenitors. This opens up the possibility to further explore the mechanisms behind the specification of cells within the human central nervous system and can potentially be used for further development and optimization of protocols specifying human neural progenitor subtypes. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
Hjärnan är ett av människans viktigaste organ och är det centra som styr våra känslor, minnen och sociala färdigheter. Hjärnan är också det organ som signalerar till musklerna i kroppen att utföra rörelser. När en person drabbas av Parkinsons sjukdom minskar signaleringen till musklerna, vilket medför att det blir svårt att röra sig och stelhet uppstår. Sjukdomen beror på att en viss typ av nervceller i mellanhjärnan har börjat degenerera. Dessa nervceller kallas dopaminerga celler, eftersom de kommuni- cerar genom frisättning av signalsubstansen dopamin. När nervcellerna släpper ut do- pamin skickas signaler till våra muskler och gör så att vi kan röra oss. Vid den tidpunkt då en patient får... (More) - Popular Abstract in Swedish
Hjärnan är ett av människans viktigaste organ och är det centra som styr våra känslor, minnen och sociala färdigheter. Hjärnan är också det organ som signalerar till musklerna i kroppen att utföra rörelser. När en person drabbas av Parkinsons sjukdom minskar signaleringen till musklerna, vilket medför att det blir svårt att röra sig och stelhet uppstår. Sjukdomen beror på att en viss typ av nervceller i mellanhjärnan har börjat degenerera. Dessa nervceller kallas dopaminerga celler, eftersom de kommuni- cerar genom frisättning av signalsubstansen dopamin. När nervcellerna släpper ut do- pamin skickas signaler till våra muskler och gör så att vi kan röra oss. Vid den tidpunkt då en patient får diagnosen Parkinsons sjukdom har en stor andel av de dopaminerga nervcellerna dött och signaleringen till musklerna i kroppen har minskat och rörelser blir därför långsamma och svåra att sätta igång. Idag finns det inget botemedel mot Parkinsons sjukdom och de läkemedel som finns tillgängliga för behandling lindrar endast symtomen tillfälligt, och är tyvärr associerade med svåra biverkningar samt att deras effektivitet minskar med tiden. Vad går det då att göra för att bota sjukdomen? En metod som många forskare idag anser vara lovande är celltransplantation. Då op- ereras friska dopaminerga nervceller in i hjärnan, för att ersätta de döda, och patien- terna får tillbaka förmågan att röra sig normalt. I hjärnan finns det flera olika celltyper som producerar dopamin, men bara de dopaminproducerande nervceller med exakt samma egenskaper som de som går förlorade vid Parkinsons sjukdom kan ge effektiv lindring. För att få fram ett stort antal celler av just denna nervcellstyp är det viktigt att komma underfund med hur dessa celler normalt bildas under embryoutvecklingen, för att sedan i laboratoriet kunna använda denna kunskap för att expandera upp rätt typ av celler. I denna avhandling har jag studerat utvecklingen av dessa nervceller och har bland annat kunnat bekräfta att en rad olika proteiner, som visat sig vara uttryckta av denna dopaminerga subtyp i mus, också uttrycks under human embryoutveckling. Detta har gett oss verktyget att kunna karaktärisera en “sann” transplantationsduglig dopaminerg nervcell och på så sätt hjälpt oss att optimera ett protokoll som gynnar bildandet av dessa celler från embryonala stamceller. Transplantation av dessa celler har visat sig kunna reparera motorfunktionen i djurmodeller med Parkinsons sjukdom och skulle därmed kunna utgöra en framtida källa till dopaminerga celler för trans- plantation till patienter. Detta protokoll har även visat sig kunna användas som en modell för tidig human embryonal nervcellsutveckling och har kunnat ge oss viktig information rörande utvecklingen av andra nervcellspopulationer i den humana hjär- nan, samt ge ytterligare kunskap om humana dopaminerga celler. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4147103
- author
- Nelander Wahlestedt, Jenny
LU
- supervisor
-
- Malin Parmar LU
- Anders Björklund LU
- opponent
-
- professor Cattaneo, Elena, Department of BioSciences and center for Stem Cell Research, University of Milan, Italy
- organization
- publishing date
- 2013
- type
- Thesis
- publication status
- published
- subject
- keywords
- Parkinson´s disease, stem cells, ventral mesencephalon, dopaminergic neurons, cell replacement therapy, human fetal tissue, radial glia, microRNA, BAC recombineering, reporter cell lines
- categories
- Higher Education
- in
- Lund University Faculty of Medicine Doctoral Dissertation Series
- volume
- 2013:135
- pages
- 140 pages
- publisher
- Developmental Neurobiology
- defense location
- Segerfalk lecture hall, BMC A10, Sölvegatan 17, Lund
- defense date
- 2013-12-12 09:15:00
- ISSN
- 1652-8220
- ISBN
- 978-91-87651-10-6
- language
- English
- LU publication?
- yes
- id
- 82784eec-af35-4e30-b4d8-3762ff59faa4 (old id 4147103)
- date added to LUP
- 2016-04-01 15:01:24
- date last changed
- 2019-05-22 04:21:02
@phdthesis{82784eec-af35-4e30-b4d8-3762ff59faa4,
abstract = {{Cell-replacement therapy is a promising approach for treating patients with Parkinson ́s disease (PD). For this purpose, there is a need for developing a protocol that can generate high numbers of human transplantable mesencephalic dopaminer- gic (mesDA) neurons. For decades, studies have therefore been made in mouse and other model organisms in order to elucidate key-factors that can be used for proper characterization and patterning of cells to become mesDA neurons. However, limited numbers of studies have been performed in human to confirm the expression and role of these factors. In this thesis, I have analysed the developing human mesencephalon for expression of key-fate determining proteins, which are known to be important in mesDA neuron development in the mouse. These key-factors were shown to exhibit a similar spatiotemporal expression pattern in the human brain, suggesting a conserved role for these proteins in mesDA neuron development across species. We were also able to confirm that human mesDA neurons are derived from radial glial cells in the floor plate (FP), positioned in the most ventral part of the mesencephalon. With the help of human specific mesDA markers we could optimize and develop a protocol that successfully patterns human embryonic stem cells (hESCs) into functional mesDA neuron progenitors. This protocol is one of the first to allow the generation of authen- tic mesDA neuron progenitors through a FP stage, mimicking early human mesDA neuron development. Furthermore, these cells survive transplantation and can restore motor deficits in a rat Parkinson ́s disease (PD) model. Thus, these cells show a prom- ising potential to be further developed for clinical use, treating patients with PD.<br/><br>
In addition to patterning cells to a mesencephalic (midbrain) fate, we were also able to regionalize hESCs to neural progenitors resembling those of the human em- bryonic forebrain and hindbrain. This protocol, in combination with the generation of a SOX1-GFP hESC reporter cell line and the expression of the cell-surface marker CORIN by human FP cells, allowed us to isolate pure populations of regionalized neu- roepithelial and FP cells for deep sequencing. From this study, we identified several microRNAs with potential roles in the specification and development of human neural progenitors populations, including mesDA neuron progenitors. This opens up the possibility to further explore the mechanisms behind the specification of cells within the human central nervous system and can potentially be used for further development and optimization of protocols specifying human neural progenitor subtypes.}},
author = {{Nelander Wahlestedt, Jenny}},
isbn = {{978-91-87651-10-6}},
issn = {{1652-8220}},
keywords = {{Parkinson´s disease; stem cells; ventral mesencephalon; dopaminergic neurons; cell replacement therapy; human fetal tissue; radial glia; microRNA; BAC recombineering; reporter cell lines}},
language = {{eng}},
publisher = {{Developmental Neurobiology}},
school = {{Lund University}},
series = {{Lund University Faculty of Medicine Doctoral Dissertation Series}},
title = {{Characterization of human dopaminergic neurons in the developing mesencephalon and upon differentiation of stem cells- for replacement therapy in Parkinson´s disease}},
volume = {{2013:135}},
year = {{2013}},
}