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Theses

To go to a specific year directly, please click on the year here: 2020, 2018, 2017, 2014, 2013


2021

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Shelby Shrigley: Exploring Patient-Specific Cell Replacement Therapy for Parkinson’s Disease OPEN ACCESS

Author: Shelby Shrigley
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2021-03-12
Place of defence: Segerfalksalen, Lund 
Opponent: Dr Morten Meyer
Date of publication: 2021
No of pages: 185
Type of document: PhD Thesis
Language: English

ISBN: 978-91-8021-026-3 / ISSN: 1652-8220
 

Abstract

Parkinson’s disease (PD) affects over six million people worldwide and is characterised by the progressive loss of dopaminergic (DA) neurons in the substantia nigra, accumulation of pathological alpha-synuclein (αSyn), and inflammation in the brain. This leads to motor impairments including rigidity, akinesia, bradykinesia, resting tremor, and postural instability. Cell replacement therapy aims to replace the midbrain DA neurons which have been lost in the disease to restore normal motor function. 
Previously, DA cells for transplantation have been derived from fetal mesencephalic tissue or human embryonic stem cells (hESCs). However, an alternative route to generate DA cells is via cellular reprogramming. This is a rapidly emerging field which allows somatic cells to be reprogrammed either into human induced pluripotent stem cells (hiPSCs) or directly into induced neurons (iNs) by forced expression of specific factors. This creates the possibility to use patient-specific cells which could reduce the risk of immune rejection and eliminate ethical concerns.
The overall aim of this thesis is to evaluate if patient-derived cells could be a suitable alternative in cell replacement therapy for PD. Firstly, an efficient protocol to directly reprogram human adult fibroblasts into iNs was developed. Following this, factors that could convert cells specifically towards a DA subtype were investigated and used to examine if cells from healthy donors and PD patients could be reprogrammed with similar efficiency. Next, a new humanized αSyn xenograft model of PD was established 
to assess the impact of host pathology on grafted cells. Finally, we explored if DA cells derived from a patient hiPSC line harbouring an αSyn triplication mutation could survive intracerebral transplantation and function on par with hESC-derived DA neurons. This patient line was also assessed for the presence of pathological features in different models of PD. These results will help to pave the way for future research assessing the potential of patient-derived cells for brain repair.

 

2020

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Marcella Birtele: Functional and Transcriptional Studies of Human Dopaminergic Neurons OPEN ACCESS

Author: Marcella Birtele
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2020-10-02
Place of defence: Segerfalksalen, Lund 
Opponent: Dr. Silvia Capello
Date of publication: 2020
No of pages: 190
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-965-7 / ISSN: 1652-8220

 

Abstract

Parkinson's Disease (PD) is the most common movement disorder and second most common neurodegenerative disease. The principal hallmark of the pathology is represented by a loss of mesen­ cephalic Dopaminergic neurons (mesDA) that reside in the Substantia Nigra pars compacta (SNpc). Another feature of the disease is represented by formation of abnormal protein  aggregates,  known as Lewy Bodies (LBs), mainly composed by the cx-synuclein protein. The etiology of mesDA death is still unknown, however LBs formation could represent one of the factor contributing to neuronal mesDA death and PD progression.
Cell Replacement Therapy for PD aims at restoring the function of the dopaminergic neurons through the transplantation of the lost cells in the brain. Recently, cell sources derived from stem cells such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) have been investigated and implicated in clinical trials for PD. Another route for generating neurons is represented by the direct reprogramming of terminally differentiated cells. With the overexpression of specific transcription factors (TFs) and/or micro RNA (miRNA) is possible to target somatic cells in vitro or resident brain cells in vivo for reprogramming into mesDA neurons.
The overall aim of my thesis has been to study functional and transcriptional profile of newly generated mesDA neurons in vitro and in vivo for cell-based therapies of PD. Indeed the transplanta­ tion outcome depends on the ability to generate mesDA neurons that are as similar as possible to the endogenous DA neurons. However, our knowledge of human DA neurons is limited by the inacces­ sibility of dev loping and adult brain tissues. In the first part of my thesis I focused on studying the properties of directly reprogrammed cells to determine their phenotypic and functional profile. In the second part of this thesis, I performed an extensive molecular, transcriptional and functional analysis of human fetal mesDA neurons to increase our understanding of DA neurons. Lastly, I focused on establishing a stem cell derived organoid system that allowed for the generation of authentic human DA neurons.


 

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Sara Nolbrant: Directing and Dissecting the Fate of Dopaminergic Neurons OPEN ACCESS

Author: Sara Nolbrant
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2020-01-31
Place of defence:  Belfragesalen, Lund 
Opponent: Prof. Maeve Caldwell
Date of publication: 2020
No of pages: 221
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-869-8 / ISSN: 1652-8220

 

Abstract

The brain is a complex organ with a limited inherent capacity to regenerate. Restoration of the damaged or diseased central nervous system therefore relies on therapeutic interventions. Cell replacement therapy offers an appealing strategy to treat many neurodegenerative diseases, amongst which Parkinson’s disease (PD) is considered a particularly promising candidate due to the focal degeneration of dopaminergic (DA) neurons in midbrain. Building on the success of early clinical trials using human fetal tissue as a cell source for brain repair in PD, protocols for developing DA neurons from human embryonic stem cells (hESCs) have been developed and are currently undergoing clinical translation. In the future, a potential alternative strategy for replacing lost DA neurons could be through direct conversion, where a somatic cell is directly reprogrammed to an induced neuron without surpassing the pluripotency stage.

The objectives of this thesis have been to direct and dissect the fate of DA neurons to generate cells that can be further developed for cell replacement therapy in PD. These efforts have resulted in a refined and clinically adapted hESC-based cell differentiation protocol and the identification of DA progenitor markers in vitro that can successfully predict the outcome of the grafting in vivo, thus securing the safety and efficacy of the grafted cell product. In addition, we have increased our understanding of the cellular diversity of the DA grafts by employing single cell sequencing for a unique and unbiased assessment of the composition of functionally mature fetal- and hESC-derived grafts. Finally, we developed a hESC-based system for direct conversion of human glial progenitors into induced DA neurons, and recognized these cells as promising substrates for making new neurons in the brain. Collectively, this work has advanced our development of a hESC-based DA differentiation protocol for clinical translation and has further explored direct neuronal reprogramming as a new avenue for brain repair.


2018

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Tiago Cardoso: Cell Replacement Therapy for Parkinson's Disease: Potential for Circuitry Repair OPEN ACCESS

Author: Tiago Cardoso
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2018-11-30
Place of defence:  Belfragesalen, Lund 
Opponent: Viviane Tabar, MD
Date of publication: 2018
No of pages: 168
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-711-0 / ISSN: 1652-8220

 

Abstract

The derivation of dopamine neurons from human embryonic stem cells (hESCs) now offers a promising alternative to fetal tissue for cell replacement therapy (CRT) in Parkinson´s disease (PD). Using the appropriate chemical cues in vitro, hESCs can be patterned towards bona-fide ventral midbrain (VM) DA neurons that survive, reinnervate, release DA and provide functional recovery when transplanted into rodent and non-human primate models of PD. However, the extent to which transplanted neurons integrate into the damaged host circuitry, which is necessary for regulated DA release, and hence to elicit a more complete circuitry repair, remains unknown. 
In this thesis, the potential for transplanted hESC-derived neurons to repair damaged circuitry in parkinsonian rats, as assessed by synaptic integration and targeted axonal outgrowth, was investigated. The role of graft- and host-dependent variables on synaptic integration and innervation were investigated in view of a better understanding of transplant biology, with potential to optimize the functionality of the graft.
We established a modified rabies-based tracing methodology that allows for the identification of monosynaptic inputs to a defined starter population in order to assess host-to-graft and graft-to-host synaptic integration in a cell transplantation model. In Paper I, we investigated the integration of hESC-derived neurons, and in Paper II during in vivo reprogramming, to investigate the integration of in situ converted neurons into pre-existing circuitry. Subsequently in Paper III, this methodology was utilized to reveal that transplanted hESC-derived VM neurons receive the correct set of presynaptic inputs from the host circuitry when placed in their homotopic location within the substantia nigra, while the profile of axonal outgrowth showed that transplanted neurons innervate across long-distances in a target-specific manner towards appropriate forebrain targets. Moreover, functional recovery as assessed by amphetamine-induced rotations matched the presence and timing of the arrival of graft-derived dopaminergic innervation in the dorsolateral striatum. In Paper IV, the role of graft neuronal phenotype and host environment on synaptic integration and graft-derived axonal outgrowth was investigated. These results show that graft-derived innervation is determined by cell intrinsic factors, as only the correct hESC derived VM neurons innervate the appropriate forebrain DA target regions. On the other hand, monosynaptic tracing showed that the pattern of integration is dependent on graft placement, as monosynaptic inputs to intrastriatal and intranigral grafts differed. Nonetheless, a certain level of anatomical and phenotypic overlap in presynaptic inputs to both ectopic and homotopic hESC-derived VM grafts was detected, suggesting that ectopically placed grafts may be modulated by functionally relevant structures involved in motor control, supporting the validity of this grafting paradigm in the clinic. Finally, transplantation of hESC-derived neurons in intact or 6-OHDA-lesioned animals revealed that the globus pallidus is differentially connected to transplanted neurons, identifying this structure as a possible important modulator of graft function in the DA-depleted parkinsonian brain.
Overall, the results of this thesis suggest that transplanted hESC-derived VM DA neurons have the capacity to achieve a more complete repair of the damaged host circuitry beyond simple DA neuron replacement. As such, they support the validity of ectopic grafting into the lesioned brain as a valid strategy for CRT in PD. Moreover, this work identifies host nuclei that may play an important role in graft modulation, hence prompting further functional experimentation.


2017

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Maria Pereira: Cell reprogramming as a way to produce new functional neurons

Author: Maria Pereira
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2017-12-15
Place of defence:  Segerfalksalen, Wallenberg Neuroscience Center, Lund 
Opponent: Prof. Benedikt Berninger
Date of publication: 2017
No of pages: 148
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-558-1 / ISSN: 1652-8220

Abstract

Cell reprogramming is a new and rapidly emerging field in which somatic cells can be converted into pluripotent stem cells or other somatic cell types, by overexpression of specific genes. Through viral overexpression of neural genes, it is nowadays possible to directly reprogram different somatic cell sources into neurons in vitro, in a process called direct neuronal reprogramming. By using subtype­ specific neuronal genes, it is also possible to produce different subtypes of neurons. This brings new opportunities for the treatment of brain disorders, as it could be used to generate new and functional cells to replace damaged neurons.

In this thesis, the potential of cell reprogramming for  the generation  of  new  neurons is explored, both in vitro and in vivo. The first part of the  thesis  (Paper I) shows  how  an improved  protocol  for in vitro reprogramming  of  human  skin  cells into  neurons  can be used  to  produce  high yields of  neurons in  culture,  that survive  transplantation in the  rodent  adult  brain. The second part of  the  thesis  (Paper II, III, IV) is aimed at exploring  different  strategies  to  reprogram  non-neuronal  cells  into  neurons  in vivo. An extensive analysis of  the  physiological,  molecular  and  genetic  properties  of  these  neurons was done and we show that these cells are functional, mature over-time and express specific neuronal markers in vivo. Taken together, the work hereby presented contributes to a better understanding of the potential of this technique as well as the properties of reprogrammed  neurons in vitro and in vivo.


2014

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Ulrich Pfisterer: Direct Conversion of Human Fibroblasts to Induced Neurons

Author: Ulrich Pfisterer
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2014-09-19
Place of defence:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Dr. Marius Wernig
Date of publication: 2014
No of pages: 143
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-024-1 / ISSN: 1652-8220

Abstract

During direct cellular reprogramming, forced expression of key transcription factors (TFs) di­rectly converts one terminally differentiated cell type into that of another fate, exemplified in this theses by the conversion of fibroblasts into functional induced neurons (iNs).
Direct conversion of mouse fibroblasts to functional neurons was established in 2010, and the starting point of my doctoral thesis was the aim to transfer iN-technology to human cells and to ex­plore the potential of this technique in regards to generate subtype-specific neurons as well as to use these cells for transplantation studies. In Paper I, we described the possibility to convert human fetal and postnatal fibroblasts into functional human induced neurons (hiNs). Generating a mixed popula­tion of glutamatergic and GABAergic hiNs, we were exploring the ability to direct hiNs towards a distinct neuronal subtype and therefore selected TFs, which are expressed in midbrain dopaminergic (mDA) neurons and their progenitors. When over-expressing these fate-specifying TFs during direct neuronal conversion, we could show for the first time, that hiNs acquire a dopaminergic (DA) sub­ type.
This led us to explore, whether functional hiNs could be generated from adult sources. In Paper II, we succeeded in generating functional hiNs from fibroblasts of different adult donors; in addition we found that the donor age does not affect the conversion potential  of  the fibroblasts.
The successful generation of functional hiNs in vitro caught our interest to whether new neurons could be obtained also via direct conversion in vivo. In Paper III, we demonstrated that transplanted fibroblasts convert to hiNs in the adult rat brain and that residing astrocytes in the mouse brain can be converted into iNs.
For hiNs to become a clinically relevant cell source for cell therapy, additional analysis regarding their survival and maturation after transplantation but also the improvement of conversion  efficien­cies is important. In Paper IV we addressed this in part by devising a  more  efficient  conversion protocol as well as by examining the impact of different maturation times of hiNs in culture prior to grafting. Besides using hiNs for transplantation studies, in Paper V we demonstrated  that hiNs could be used to develop a phenotypic screening assay for the identification of small molecules and associ­ated signaling pathways, important for direct neuronal conversion .
Overall, this thesis work has opened up possibilities to generate hiNs for brain repair, to obtain patient-specific hiNs for disease-modeling in vitro and to utilize hiNs in high content screening assays.

 

 

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Olof Torper: Generation of induced neurons via direct conversion in vivo and in vitro
Author: Olof Torper
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2014-02-28
Place of defence:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Prof. Ola Hermansson
Date of publication: 2014
No of pages: 125
Type of document: PhD Thesis
Language: English
ISBN: 978-91-87651-44-1 / ISSN: 1652-8220

Summary

Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signal­ ing. Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy.

In this thesis I focus on the direct reprogramming where one terminally dif­ ferentiated cell is directly converted into another without passing a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the forced expression of Asc/1, Brn2 and Mytl L (ABM). By including the additional factors, Foxa2 and Lmxl a subtype spe­ cific neurons could be obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons. Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo. These cells expressed pan-neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and convert these into neurons in vivo. Using the same strategy we cloned the three factors, Asc/1, Lmxl a and Nurrl (ALN) together with GFP, into Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells. By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan­ neuronal markers NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their func­ tional properties in terms of electrophysiology and synaptic formation.

Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are associated with logisti­ cal and ethical concerns. They are readily a'!'ailable cells that can be obtained from the skin of a patient and direct conversion offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted. Further, in vivo reprogramming offers an alternative to traditional cell 

herapy by creating new neurons in the brain removing the need of an exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke is limited and treatments for neu­ rological disorders like Parkinson's disease (PD) progressively decline in effective­ ness and are associated with severe side effects.

In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced expression of tran­ scription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2 glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly used in clinical trials. Future studies should focus on factors involved in the specific­ ity of the required cell and how well the cell that is formed correspond genetically, functionally and viably to its endogenous counterpart.


2013

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Jenny Nelander Wahlestedt: Characterization of human dopaminergic neurons in the developing mesencephalon and upon differentiation of stem cells - for cell replacement therapy in Parkinson's disease

Author: Jenny Nelander Wahlestedt
Supervisor: Malin Parmar
Department: Experimental Medical Science
Date of defence: 2013-12-12
Place of defence:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Prof. Elena Cattaneo
Date of publication: 2013
No of pages: 140
Type of document: PhD Thesis
Language: English
ISBN: 978-91-87651-10-6 / ISSN: 1652-8220

Summary

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 SOXJ-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 pos­ sibility 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.