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Developing a new tool for the study of human brain development

Radeviciute, Julija (2022) MOBN02 20221
Degree Projects in Molecular Biology
Popular Abstract
Developing a new tool for the study of human brain development

“If the human brain were so simple that we could understand it, we would be so simple that we couldn’t” physicist Emerson Pugh once said. Nevertheless, despite its complexity, researchers are trying to figure out one of the human body's most vital organs. Understanding the development of the brain is crucial to understanding how it works, and with this study, we aimed to help establish a valuable tool for neurodevelopmental studies.

During the human development pluripotent embryonic stem cells give rise to the more defined neural stem cells NSC which in turn later differentiate to the various cell types comprising the central nervous system. Induced pluripotent stem cells... (More)
Developing a new tool for the study of human brain development

“If the human brain were so simple that we could understand it, we would be so simple that we couldn’t” physicist Emerson Pugh once said. Nevertheless, despite its complexity, researchers are trying to figure out one of the human body's most vital organs. Understanding the development of the brain is crucial to understanding how it works, and with this study, we aimed to help establish a valuable tool for neurodevelopmental studies.

During the human development pluripotent embryonic stem cells give rise to the more defined neural stem cells NSC which in turn later differentiate to the various cell types comprising the central nervous system. Induced pluripotent stem cells (iPSC) is a recent advance which mimics the embryonic stem cells that in turn can be used to study the development of different tissues. Two well-known genes, PAX6 and NES which are expressed during the neural stem cell stage were edited to simultaneously express a fluorescence protein turning the cell nucleus red under the fluorescence microscope. This facilitates the selection of NPCs for further experiments during the research. In order to edit the gene, CRISPR-Cas9 system was used, which earned a Nobel prize recently for Emmanuelle Charpentier and Jennifer A. Doudna.

As a result, PAX6 reporter lines were not obtained since due to unknown reasons we could not validate the obtained cell cultures, while multiple iPSC clones of Nestin reporter were successfully derived and tested by differentiation to NPC lines. Two Nestin reporter lines were designed containing the two different linker sequences (IRES and P2A) to compare the results. Initial results show nearly identical levels of fluorescence in the two variants. Moreover, results showed an increase in fluorescence by 2.3 times from iPSC to NPC which was expected as the fluorescence levels correlate with the NES expression, which is low in iPSC and increases once the cells enter neural progenitor stage. The red fluorescence is the reason these cells can be separated from the unsuccessfully maturing cells, which can be easily excluded from the experiment increasing the accuracy of the research. The fluorescence signal in these lines is supposed to disappear after the cells turn to mature brain cells which will have to be tested in the future. In case of this successful event, the cell line could be used for neurodevelopmental experiments greatly contributing to the future understanding of the brain functioning mechanisms.


Master’s Degree Project in Molecular Biology, 45 credits, 2022.
Department of Biology, Lund University

Advisor: Dr. Pia Johansson
Cell and Gene Therapy core, Lund Stem Cell Center, Lund University (Less)
Please use this url to cite or link to this publication:
author
Radeviciute, Julija
supervisor
organization
course
MOBN02 20221
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9113656
date added to LUP
2023-04-20 15:13:41
date last changed
2023-04-20 15:13:41
@misc{9113656,
  author       = {{Radeviciute, Julija}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Developing a new tool for the study of human brain development}},
  year         = {{2022}},
}