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Direct conversion from mouse fibroblasts to neurons in vitro and grafted in an epileptic model

Chi, Hao (2015) MOBT01 20142
Degree Projects in Molecular Biology
Abstract
Epilepsy is one of the most common neurological disorders. The most common way to treat epilepsy is by taking antiepileptic drugs, however, still a considerable portion of patients are still suffering from intractable epilepsy so new treatment methods are needed. Cell grafting to treat neurological diseases has been tried for decades. Yet, good cells source has been hard to find. Direct conversion of neurons from a different germ line cell type (Example: Fibroblast, mesodermal) is a new field in cell conversion research, and these so called induced neurons (iN) could be a good cell source for transplantation. It may has many advantages over other cell sources, as the neurons are not going through a stage of pluripotency but are directly... (More)
Epilepsy is one of the most common neurological disorders. The most common way to treat epilepsy is by taking antiepileptic drugs, however, still a considerable portion of patients are still suffering from intractable epilepsy so new treatment methods are needed. Cell grafting to treat neurological diseases has been tried for decades. Yet, good cells source has been hard to find. Direct conversion of neurons from a different germ line cell type (Example: Fibroblast, mesodermal) is a new field in cell conversion research, and these so called induced neurons (iN) could be a good cell source for transplantation. It may has many advantages over other cell sources, as the neurons are not going through a stage of pluripotency but are directly converted to an adult phenotype, thus reducing the likelihood of tumor formation. The conversion time might be reduced and possibly a higher chance that the cells are directed to be the desirable type. In addition, the fibroblast is an abundant cell source with strong self-proliferation abilities, which means a large amount of neurons could be produced if the conversion ratio is high. In this study, we explore the intrinsic electrophysiological properties of the iN in vitro and graft the cells to an in vitro model of hyperexcitable epileptic tissue to investigate synaptic integration in a pathological environment. Results: Conversion ratio is estimated to be 3.2%~7.5%. The converted cells are vulnerable and could not easily adapt into the hippocampal tissue forming real connections. Further trials are needed to find ways to enhance their survival ratio in a tissue environment and increase forming connections with endogenous cells. (Less)
Popular Abstract
Epilepsy is one of the most common neurological disorders, characterized by epileptic seizures (excessive or hypersynchronous brain activites). More than 1% of the world population suffer from the disease. Among the various forms of epilepsy, Temporal lobe epilepsy (TLE) is the most common type, accounting for around 30% of all types of epilepsy. Currently, the available treatments for TLE are far from optimal. One of the most common medicines to treat epilepsy is the antiepileptic drugs. However one third of the patients still experience seizures after taking the drugs. Basically, the only left option for these patients is surgical intervention, which could results in severe side effects. Therefore, novel treatments for TLE, as well as... (More)
Epilepsy is one of the most common neurological disorders, characterized by epileptic seizures (excessive or hypersynchronous brain activites). More than 1% of the world population suffer from the disease. Among the various forms of epilepsy, Temporal lobe epilepsy (TLE) is the most common type, accounting for around 30% of all types of epilepsy. Currently, the available treatments for TLE are far from optimal. One of the most common medicines to treat epilepsy is the antiepileptic drugs. However one third of the patients still experience seizures after taking the drugs. Basically, the only left option for these patients is surgical intervention, which could results in severe side effects. Therefore, novel treatments for TLE, as well as other types of epilepsy, are necessary.

Using induced neurons (iN) to treat epilepsy may be an alternative option. Loss of neurons, especially the inhibitory ones, is a pathological hallmark of TLE. Besides that, the synaptic connections within hippocampus are also reorganized in TLE, resulting in the structure to be hyperexcitable and vulnerable to seizure invasion. Therefore, we want the induced neurons to be inhibitory (presumably GABAergic) and able to integrate into hippocampal tissue in order to lower the excitation of the structure. The origin of the induced neurons can be induced pluripotent stem cells (IPS) or embryonic stem cells (ESC). Recently, it has been proved that it is possible to directly convert fibroblasts to neurons, which is potentially safer and more reproducible comparing with the conventional induction methods. In this study, we established the conversion platform of mouse embryonic pulmonary epithelia cells to neurons and studied the electrophysiological properties of the neurons in vitro. Furthermore, we grafted the cells in situ in hippocampal organotypic slices, which is a classical epileptic model.

The results showed that different passage numbers of fibroblasts have different conversion efficiencies. Fibroblasts of lower passage numbers were more easily converted than those of higher passage numbers. The conversion efficiency could be further improved by either increasing the multiplicity of infection (MOI) in virus transduction or replating the cells after the conversion is initiated. Electrophysiology recordings revealed that induced neurons generated from mouse fibroblasts are heterogeneous. Different categories of iN were characterized by their electrophysiology profiles. The survival ratio of iN on hippocampal organotypic cultures was low and they were also poorly connected to other neurons.

Advisor: My Andersson, faculty of medicine
MasterĀ“s Degree Project 60 credits in molecular biology 2014~2015
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Chi, Hao
supervisor
organization
course
MOBT01 20142
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
5466304
date added to LUP
2015-06-05 12:23:00
date last changed
2015-06-05 12:23:00
@misc{5466304,
  abstract     = {Epilepsy is one of the most common neurological disorders. The most common way to treat epilepsy is by taking antiepileptic drugs, however, still a considerable portion of patients are still suffering from intractable epilepsy so new treatment methods are needed. Cell grafting to treat neurological diseases has been tried for decades. Yet, good cells source has been hard to find. Direct conversion of neurons from a different germ line cell type (Example: Fibroblast, mesodermal) is a new field in cell conversion research, and these so called induced neurons (iN) could be a good cell source for transplantation. It may has many advantages over other cell sources, as the neurons are not going through a stage of pluripotency but are directly converted to an adult phenotype, thus reducing the likelihood of tumor formation. The conversion time might be reduced and possibly a higher chance that the cells are directed to be the desirable type. In addition, the fibroblast is an abundant cell source with strong self-proliferation abilities, which means a large amount of neurons could be produced if the conversion ratio is high. In this study, we explore the intrinsic electrophysiological properties of the iN in vitro and graft the cells to an in vitro model of hyperexcitable epileptic tissue to investigate synaptic integration in a pathological environment. Results: Conversion ratio is estimated to be 3.2%~7.5%. The converted cells are vulnerable and could not easily adapt into the hippocampal tissue forming real connections. Further trials are needed to find ways to enhance their survival ratio in a tissue environment and increase forming connections with endogenous cells.},
  author       = {Chi, Hao},
  language     = {eng},
  note         = {Student Paper},
  title        = {Direct conversion from mouse fibroblasts to neurons in vitro and grafted in an epileptic model},
  year         = {2015},
}