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Intrinsic and extrinsic factors controlling the differentiation of human midbrain progenitor cells

Christophersen, Nicolaj LU (2006) In Faculty of Medicine doctoral dissertation series 2006:124.
Abstract
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor deficits. Levodopa treatment provides marked symptomatic relief. However, within 5-10 years after the start of Levodopa treatment, most PD patients display a gradual loss of drug efficacy. Importantly, the neuropathology of PD is dominated by a marked loss of dopaminergic neurons located in the substantia nigra. This makes PD a suitable target for cell replacement therapy strategies. Clinical grafting trials have proved that cell replacement therapies may be effective in PD. So far, dopaminergic progenitors (and neurons) harvested from the embryonic ventral midbrain have been used to replace degenerated ventral midbrain dopaminergic neurons. The limited... (More)
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor deficits. Levodopa treatment provides marked symptomatic relief. However, within 5-10 years after the start of Levodopa treatment, most PD patients display a gradual loss of drug efficacy. Importantly, the neuropathology of PD is dominated by a marked loss of dopaminergic neurons located in the substantia nigra. This makes PD a suitable target for cell replacement therapy strategies. Clinical grafting trials have proved that cell replacement therapies may be effective in PD. So far, dopaminergic progenitors (and neurons) harvested from the embryonic ventral midbrain have been used to replace degenerated ventral midbrain dopaminergic neurons. The limited availability of tissue from embryos and the associated technical and ethical difficulties have lead to a search for alternative sources of transplantable cells. The search has identified stem and neural progenitor cells as possible sources. The idea is that these can be expanded in vitro for banking and then differentiated into dopaminergic neurons just prior to implantation into patients. This thesis focuses on the characterization of three potential human cell sources for the generation of transplantable midbrain dopaminergic progenitors and neurons. First, we studied neural stem cells obtained from the human embryonic and fetal forebrain. We observed induction of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in human fetal forebrain-derived neural stem cells differentiated under defined conditions in vitro. These tyrosine hydroxylase-expressing neurons were lost upon transplantation into a rat model of PD. To further characterize dopaminergic differentiation of human cells, we designed and tested a focused stem cell microarray(NeuroStem Chip) and performed gene expression profiling of human embryonic stem cells. Using this platform, we identified several genes expressed during proliferation of human embryonic stem cells and their early differentiation into dopaminergic neurons. We also employed the focused microarray to characterize immortalized cell lines derived from the human embryonic ventral midbrain. We observed significant up-regulation of mRNA levels of multiple dopaminergic neuron-related markers as well as novel secreted growth factors in one of the cell lines (MesC2.10 cells) undergoing differentiation. When grafting MesC2.10 cells into a rat model of PD, these cells ceased to express tyrosine hydroxylase. Work in this thesis also focused on the downstream targets of a known neuroprotective secreted growth factor (glial cell line-derived neurotrophic factor, GDNF) in midbrain dopaminergic neurons. We found that the protein Delta-like-1 homologue was highly up-regulated in the ventral midbrain following intrastriatal delivery of GDNF, and that it is also expressed in the midbrain during normal development. In conclusion, the studies included in this thesis provide new insights into the use of different human cell sources i) for cell replacement therapy in PD and ii) as platforms to identify genes expressed during normal development of midbrain dopaminergic neurons. (Less)
Abstract (Swedish)
Popular Abstract in Swedish

Parkinsons sjukdom (PS) karaktäriseras av långsam celldöd i hjärnan och gradvis uppträder minskad rörlighet och muskelstelhet som mest framträdande symptom. PS drabbar ungefär 0.1% av befolkningen, och inträder oftast i femtio- till sextioårsåldern. Varför sjukdomen uppkommer är till stor del okänt, men man vet att de dopamin producerande cellerna i mitthjärnan (särskilt i området substantia nigra dvs "svarta substansen") dör, med dopaminförlust i ett av hjärnans motoriska centra (striatum) som följd. Vid PS finns läkemedelsbehandlingar (t ex Levodopa), men de är mest effektiva de första åren efter att sjukdomen diagnostiserats. Vid PS har transplantat av hjärnceller från embryon visats ge... (More)
Popular Abstract in Swedish

Parkinsons sjukdom (PS) karaktäriseras av långsam celldöd i hjärnan och gradvis uppträder minskad rörlighet och muskelstelhet som mest framträdande symptom. PS drabbar ungefär 0.1% av befolkningen, och inträder oftast i femtio- till sextioårsåldern. Varför sjukdomen uppkommer är till stor del okänt, men man vet att de dopamin producerande cellerna i mitthjärnan (särskilt i området substantia nigra dvs "svarta substansen") dör, med dopaminförlust i ett av hjärnans motoriska centra (striatum) som följd. Vid PS finns läkemedelsbehandlingar (t ex Levodopa), men de är mest effektiva de första åren efter att sjukdomen diagnostiserats. Vid PS har transplantat av hjärnceller från embryon visats ge uttalade förbättringar. Emellertid är det svårt att få tillgång till tillräckligt med donatorsvävnad för det stora antal patienter som behöver cellterapi. Dessutom har användandet av denna typ av celler orsakat en etisk debatt. Detta har gjort att man nu söker efter en alternativ källa till donatorsceller. Möjliga källor är stamceller samt omogna nervceller. Tanken är att dessa celler kan förökas i laboratoriet och förvaras tills strax innan transplantationen då de differentieras till dopaminproducerande nervceller. Denna avhandling fokuserar på att beskriva tre möjliga källor till transplanterbara omogna dopaminproducerande celler samt nervceller. Först studerade vi stamceller från nervsystemet (neurala stamceller) med ursprung i framhjärnan från humana embryon och foster. Vi undersökte induktionen av enzymet, tyrosin hydroxylas, i neurala stamceller från humana embryon. Tyrosin hydroxylas är det enzym som begränsar hastigheten på dopaminsyntesen. De nervceller som uttryckte tyrosin hydroxylas gick förlorade när cellerna transplanterades till en råttmodell av PS. För att ytterligare karaktärisera utvecklingen (differentiering) av dopaminproducerande nervceller så skapade och testade vi en stamcells microarray (kallad NeuroStem Chip). Med denna teknik kunde vi utföra en beskrivning av hur generna uttrycks och fungerar. Genom att använda denna metod identifierade vi flera gener som är aktiva under förökningen av humana embryonala stamceller och den tidiga utvecklingen till dopaminproducerande nervceller. Vi använde också denna teknik för att karaktärisera immortaliserade cellinjer med ursprung i den främre mellanhjärnan hos humana embryon. Vi observerade en signifikant ökad aktivitet av mRNA-nivåer i många markörer relaterade till dopaminnervceller. Dessutom såg vi att det producerades nya proteiner som kan vara tillväxtfaktorer i en av cellinjerna (MesC2.10) när den genomgick differentiering. När dessa celler transplanterades till en råttmodell av PS så slutade cellerna att producera tyrosin hydroxylas. Denna avhandling fokuserar även på effekterna av ett välkänt nervskyddande ämne (glial cell line-derived neurotrophic factor, GDNF). GDNF kan hjälpa till att förhindra celldöd i PS. Vi fann att ett protein (Delta-like-1 homologue) ökad mängd i den främre mitthjärnan efter att GDNF tillförs. Detta protein uttrycks också under normal utveckling av hjärnan. Studierna inkluderade i denna avhandling ger nya kunskaper rörande användandet av olika typer av humana celler i) för transplantation vid PS och ii) som experimentella verktyg för att identifiera gener som uttrycks under den normala utvecklingen av dopaminproducerande nervceller från mitthjärnan. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Arenas, Ernest, Institutionen för Medicinsk Biokemi och Biofysik, Avdelingen för molekylär neurobiologi, Karolinska
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Medicin (människa och djur), Medicine (human and vertebrates), neurotrophic factors, Parkinson's disease, microarray, dopaminergic, Embryonic stem cells, neural stem cells
in
Faculty of Medicine doctoral dissertation series
volume
2006:124
pages
186 pages
publisher
Department of Experimental Medical Science, Lund Univeristy
defense location
Segerfalksalen, Wallenberg neurocentrum, Lund
defense date
2006-10-20 13:15:00
ISSN
1652-8220
ISBN
91-85553-46-6
language
English
LU publication?
yes
id
03520841-366e-49e1-8b49-1c233279a019 (old id 547306)
date added to LUP
2016-04-01 15:40:54
date last changed
2019-05-21 14:21:36
@phdthesis{03520841-366e-49e1-8b49-1c233279a019,
  abstract     = {Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor deficits. Levodopa treatment provides marked symptomatic relief. However, within 5-10 years after the start of Levodopa treatment, most PD patients display a gradual loss of drug efficacy. Importantly, the neuropathology of PD is dominated by a marked loss of dopaminergic neurons located in the substantia nigra. This makes PD a suitable target for cell replacement therapy strategies. Clinical grafting trials have proved that cell replacement therapies may be effective in PD. So far, dopaminergic progenitors (and neurons) harvested from the embryonic ventral midbrain have been used to replace degenerated ventral midbrain dopaminergic neurons. The limited availability of tissue from embryos and the associated technical and ethical difficulties have lead to a search for alternative sources of transplantable cells. The search has identified stem and neural progenitor cells as possible sources. The idea is that these can be expanded in vitro for banking and then differentiated into dopaminergic neurons just prior to implantation into patients. This thesis focuses on the characterization of three potential human cell sources for the generation of transplantable midbrain dopaminergic progenitors and neurons. First, we studied neural stem cells obtained from the human embryonic and fetal forebrain. We observed induction of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in human fetal forebrain-derived neural stem cells differentiated under defined conditions in vitro. These tyrosine hydroxylase-expressing neurons were lost upon transplantation into a rat model of PD. To further characterize dopaminergic differentiation of human cells, we designed and tested a focused stem cell microarray(NeuroStem Chip) and performed gene expression profiling of human embryonic stem cells. Using this platform, we identified several genes expressed during proliferation of human embryonic stem cells and their early differentiation into dopaminergic neurons. We also employed the focused microarray to characterize immortalized cell lines derived from the human embryonic ventral midbrain. We observed significant up-regulation of mRNA levels of multiple dopaminergic neuron-related markers as well as novel secreted growth factors in one of the cell lines (MesC2.10 cells) undergoing differentiation. When grafting MesC2.10 cells into a rat model of PD, these cells ceased to express tyrosine hydroxylase. Work in this thesis also focused on the downstream targets of a known neuroprotective secreted growth factor (glial cell line-derived neurotrophic factor, GDNF) in midbrain dopaminergic neurons. We found that the protein Delta-like-1 homologue was highly up-regulated in the ventral midbrain following intrastriatal delivery of GDNF, and that it is also expressed in the midbrain during normal development. In conclusion, the studies included in this thesis provide new insights into the use of different human cell sources i) for cell replacement therapy in PD and ii) as platforms to identify genes expressed during normal development of midbrain dopaminergic neurons.},
  author       = {Christophersen, Nicolaj},
  isbn         = {91-85553-46-6},
  issn         = {1652-8220},
  language     = {eng},
  publisher    = {Department of Experimental Medical Science, Lund Univeristy},
  school       = {Lund University},
  series       = {Faculty of Medicine doctoral dissertation series},
  title        = {Intrinsic and extrinsic factors controlling the differentiation of human midbrain progenitor cells},
  volume       = {2006:124},
  year         = {2006},
}