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Neurogenesis from Neural Stem Cells, Ependymal Cells and Fibroblasts

Devaraju, Karthikeyan LU (2014) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2014:32.
Abstract (Swedish)
Popular Abstract in Swedish

Stroke är en ledande orsak till dödlighet och handikapp i hela världen. Stroke leder till förlust av nervceller och även andra celler i hjärnan på grund av bristande blodtillförsel. För tillfället finns väldigt få möjligheter att behandla funktionshinder relaterade till stroke. Det har visats att stroke leder till ökad neurogenes, nybildning av nervceller i hjärnan. Denna ökade neurogenes är dock förmodligen inte tillräcklig för att återställa förlorad funktion. Därför finns ett behov av att utveckla nya terapier för att ersätta förlorade nervceller genom att förbättra neurogenes i hjärnan och/eller transplantatio av nervceller. Stroke som drabbar kortex leder till svårare handikapp jämfört med... (More)
Popular Abstract in Swedish

Stroke är en ledande orsak till dödlighet och handikapp i hela världen. Stroke leder till förlust av nervceller och även andra celler i hjärnan på grund av bristande blodtillförsel. För tillfället finns väldigt få möjligheter att behandla funktionshinder relaterade till stroke. Det har visats att stroke leder till ökad neurogenes, nybildning av nervceller i hjärnan. Denna ökade neurogenes är dock förmodligen inte tillräcklig för att återställa förlorad funktion. Därför finns ett behov av att utveckla nya terapier för att ersätta förlorade nervceller genom att förbättra neurogenes i hjärnan och/eller transplantatio av nervceller. Stroke som drabbar kortex leder till svårare handikapp jämfört med de som påverkar striatum , och hurvida kortikal neurogenes sker efter stroke är kontroversiellt . Därför kan celltransplantationkan vara nyckeln till reparation av kortex efter stroke.



Tidigare studier har identifierat många positiva men mycket få negativa regulatorer av nybildning av nervceller efter stroke. Det har också föreslagits att ependymala celler kan bidra till stroke-inducerad neurogenes.

Nervceller till transplantation kan genereras från olika källor såsom fostrets hjärna, embryonala stamceller och inducerade pluripotenta stamceller. Dessa är dock förknippade med etiskt svåra frågor och har risker associerade med avstötning och uppkomst av cancer. Direkt omvandling av patientens egna hudceller till nervceller, och använda dessa till transplantation, skulle kunna övervinna dessa problem och potentiellt återställa funktionen i stroke-skadad hjärna.



I denna avhandling har vi använt transgena modeller, virala vektorer, elektroporation-medierad genleverans och överuttryck av transkriptionsfaktorer för att påvisa neurogenes från neurala stamceller, ependymalceller och fibroblaster.



Vi visar att LNK, en känd hämmare av hematopoietiska stamcellers självförnyelse, också uttrycks i hjärnan. Överuttryck eller avlägsnande av Lnk leder till minskad eller ökad neurogenes respektive in vitro. Efter stroke ses en ökad proliferation av neurala stamceller hos djur utan LNK uttryck. Detta observerades dock inte i djur med status eplipeticus , en allvarlig form av epilepsi som också är associerad med ökad neurogenes. Vi visade att uppreglering av STAT1/3 efter stroke leder till ökat Lnk uttryck. Varpå LNK hämmar neurala stamcellers svar på ökad IGF1 stimulering efter stroke, genom att minska AKT fosforylering. Vi har därigenom identifierat LNK signalering som en ny mekanism för att påverka neurogenes efter stroke .



Vi undersökte sedan hurvida ependymalceller i ventrikelväggen på vuxna råttor bidrar till neurogenes efter stroke. Vi identifierade FoxJ1 som en markör av ependymala celler i råttor. Vi använde sedan FoxJ1 promotorn och piggyBac systemet för att genetiskt märka dessa celler med fluorescerande reporterproteiner, GFP eller RFP, genom elektroporering. Genom att följa de märkta cellerna i intakt och strokeskadad hjärna, identifierade vi att FoxJ1 uttryckande celler bidrar till neurogenes i luktbulberna men väldigt lite i striatum. Således har FoxJ1 uttryckande celler förmodligen bara en mindre roll i reparation av striatum efter stroke.



Vi testade sedan om humana fetala lungfibroblaster kan direkt omvandlas till kortikala nervceller. Vi överuttryckte olika kombinationer av transkriptionsfaktorer som är kända för att vara inblandade i utvecklingen av kortikala nervceller. Vi upptäckte att överuttryck av olika uppsättningar av dessa faktorer i fibroblaster konverterade dem till kortikal-liknande nervceller. Dessa nervceller uttryckte markörer för kortikala nervceller och visades vara funktionella med hjälp av elektrofysiologi.



Sammanfattningsvis, visar dessa resultat,att hämning av LNK, en negativ regulator av neurogenes från hjärnans egna neurala stamceller, och intrakortikal transplantation av kortikala nervceller direkt omvandlade från fibroblaster kan utvecklas till nya terapeutiska strategier för stroke i framtiden. (Less)
Abstract
Stroke is a major cause of death and disability around the world. Stroke leads to loss of neurons and also other cells in the brain due to lack of blood supply. Currently no therapies are available to treat stroke-related disability. It has been shown that stroke leads to increased neurogenesis, birth of new neurons, within the brain. This increased neurogenesis is not sufficient to restore lost function. There is a need to develop therapies for neuronal replacement by improving neurogenesis within the brain and / or transplanting neurons. Cortical strokes lead to more disability after stroke as compared to those affecting the striatum, and whether cortical neurogenesis occurs after stroke is controversial. Cell transplantation may be the... (More)
Stroke is a major cause of death and disability around the world. Stroke leads to loss of neurons and also other cells in the brain due to lack of blood supply. Currently no therapies are available to treat stroke-related disability. It has been shown that stroke leads to increased neurogenesis, birth of new neurons, within the brain. This increased neurogenesis is not sufficient to restore lost function. There is a need to develop therapies for neuronal replacement by improving neurogenesis within the brain and / or transplanting neurons. Cortical strokes lead to more disability after stroke as compared to those affecting the striatum, and whether cortical neurogenesis occurs after stroke is controversial. Cell transplantation may be the key to cortical repair after stroke.



Reports have identified positive but very few negative regulators of neurogenesis after stroke, and suggested that ependymal cells can also contribute to stroke-induced neurogenesis. Transplantation of neurons generated from different sources such as fetal brain, embryonic stem cells and induced pluripotent stem cells are associated with ethical issues and carry the risk of immune rejection and tumorigenicity. Direct conversion of patient’s own skin cells to neurons could overcome these problems and potentially restore function after transplantation in stroke-damaged brain.



In this thesis we have used transgenic models, viral vectors, electroporation-mediated gene delivery and overexpression of transcription factors to demonstrate neurogenesis from neural stem cells, ependymal cells in the lateral ventricular wall and fibroblasts.



We show that Lnk, a known inhibitor of hematopoietic stem cell self-renewal, is also expressed in the brain. Overexpression or removal of Lnk expression leads to decreased or increased neurogenesis in vitro respectively. When brain is damaged by stroke there is increased proliferation of neural stem cells in animals without Lnk expression. This was not observed in status epilepticus, a severe form of epilepsy. We determined that upregulation of Stat1/3 after stroke leads to increased Lnk expression. Subsequently Lnk inhibits cellular response to increased IGF1 stimulation after stroke, by decreasing Akt phosphorylation. We have identified Lnk signaling as a novel mechanism of influencing neurogenic response to stroke.



We next determined if ependymal cells in lateral ventricular wall of adult rat brain contribute to neurogenesis after stroke. We identified FoxJ1 as a marker of ependymal cells in rats similar to mice, and used FoxJ1 promoter in piggyBac system to genetically label these cells with fluorescent reporter proteins GFP or RFP by electroporation. Tracing the lineage of the labeled cells in intact and stroke-damaged brain, we identified that FoxJ1 expressing cells contribute to olfactory bulb neurogenesis while the striatal neurogenic response was not significant. Thus, FoxJ1 expressing cells probably have only a minor role in repair after stroke.



We then tested whether human fetal lung fibroblasts could be directly converted to cortical neurons. We overexpressed sets of transcription factors that are known to be involved in cortical neuron development. We found that overexpression of different sets of these factors in fibroblasts converted them to cortical-like neurons. These neurons expressed markers of cortical neurons and were functional by electrophysiology.



In summary, these results raise the possibility that inhibition of Lnk, a negative regulator of neurogenesis from the brain’s own neural stem cells, and intracortical transplantation of cortical neurons directly converted from fibroblasts could be developed into novel therapeutic strategies for stroke in the future. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Lendahl, Urban, Department of Cell and Molecular Biology, Karolinksa Institute, Stockholm, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
adult neurogenesis, stroke, neural stem cells, Lnk, ependymal cells, FoxJ1, cortical reprogramming
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2014:32
pages
116 pages
publisher
Neurology, Lund
defense location
Segerfalksalen, Wallenberg Neurocentrum, Lund.
defense date
2014-03-21 09:00
ISSN
1652-8220
ISBN
978-91-87651-56-4
language
English
LU publication?
yes
id
6634fbd2-2cf6-4784-8f24-6fc8d7a7eddc (old id 4330891)
date added to LUP
2014-02-25 14:32:15
date last changed
2016-09-19 08:44:49
@phdthesis{6634fbd2-2cf6-4784-8f24-6fc8d7a7eddc,
  abstract     = {Stroke is a major cause of death and disability around the world. Stroke leads to loss of neurons and also other cells in the brain due to lack of blood supply. Currently no therapies are available to treat stroke-related disability. It has been shown that stroke leads to increased neurogenesis, birth of new neurons, within the brain. This increased neurogenesis is not sufficient to restore lost function. There is a need to develop therapies for neuronal replacement by improving neurogenesis within the brain and / or transplanting neurons. Cortical strokes lead to more disability after stroke as compared to those affecting the striatum, and whether cortical neurogenesis occurs after stroke is controversial. Cell transplantation may be the key to cortical repair after stroke.<br/><br>
<br/><br>
Reports have identified positive but very few negative regulators of neurogenesis after stroke, and suggested that ependymal cells can also contribute to stroke-induced neurogenesis. Transplantation of neurons generated from different sources such as fetal brain, embryonic stem cells and induced pluripotent stem cells are associated with ethical issues and carry the risk of immune rejection and tumorigenicity. Direct conversion of patient’s own skin cells to neurons could overcome these problems and potentially restore function after transplantation in stroke-damaged brain.<br/><br>
<br/><br>
In this thesis we have used transgenic models, viral vectors, electroporation-mediated gene delivery and overexpression of transcription factors to demonstrate neurogenesis from neural stem cells, ependymal cells in the lateral ventricular wall and fibroblasts.<br/><br>
<br/><br>
We show that Lnk, a known inhibitor of hematopoietic stem cell self-renewal, is also expressed in the brain. Overexpression or removal of Lnk expression leads to decreased or increased neurogenesis in vitro respectively. When brain is damaged by stroke there is increased proliferation of neural stem cells in animals without Lnk expression. This was not observed in status epilepticus, a severe form of epilepsy. We determined that upregulation of Stat1/3 after stroke leads to increased Lnk expression. Subsequently Lnk inhibits cellular response to increased IGF1 stimulation after stroke, by decreasing Akt phosphorylation. We have identified Lnk signaling as a novel mechanism of influencing neurogenic response to stroke.<br/><br>
<br/><br>
We next determined if ependymal cells in lateral ventricular wall of adult rat brain contribute to neurogenesis after stroke. We identified FoxJ1 as a marker of ependymal cells in rats similar to mice, and used FoxJ1 promoter in piggyBac system to genetically label these cells with fluorescent reporter proteins GFP or RFP by electroporation. Tracing the lineage of the labeled cells in intact and stroke-damaged brain, we identified that FoxJ1 expressing cells contribute to olfactory bulb neurogenesis while the striatal neurogenic response was not significant. Thus, FoxJ1 expressing cells probably have only a minor role in repair after stroke.<br/><br>
<br/><br>
We then tested whether human fetal lung fibroblasts could be directly converted to cortical neurons. We overexpressed sets of transcription factors that are known to be involved in cortical neuron development. We found that overexpression of different sets of these factors in fibroblasts converted them to cortical-like neurons. These neurons expressed markers of cortical neurons and were functional by electrophysiology.<br/><br>
<br/><br>
In summary, these results raise the possibility that inhibition of Lnk, a negative regulator of neurogenesis from the brain’s own neural stem cells, and intracortical transplantation of cortical neurons directly converted from fibroblasts could be developed into novel therapeutic strategies for stroke in the future.},
  author       = {Devaraju, Karthikeyan},
  isbn         = {978-91-87651-56-4},
  issn         = {1652-8220},
  keyword      = {adult neurogenesis,stroke,neural stem cells,Lnk,ependymal cells,FoxJ1,cortical reprogramming},
  language     = {eng},
  pages        = {116},
  publisher    = {Neurology, Lund},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Neurogenesis from Neural Stem Cells, Ependymal Cells and Fibroblasts},
  volume       = {2014:32},
  year         = {2014},
}