Generation of human astrocytes for disease modeling. A study based on stem cells, direct conversion and genome engineering to dissect the role of astrocytes in leukodystrophies
Quist, Ella LU
(2023)
In Lund University, Faculty of Medicine Doctoral Dissertation Series
- Abstract
- Astrocytes are one of the major cell types in the central nervous system and are indispensable for brain development and function. The human brain and human astrocytes have unique structures and functions that are not present in other animal species. Studies of fundamental astrocyte biology in humans and their role in neurological disease have been hindered by shortage of native human astrocytes for research purposes and inadequate animal and cell models. With advancements in stem cell technology, the possibility to generate astrocytes in vitro from human pluripotent stem cells (hPSCs), ultimately derived from patient cells, emerged. However, traditional differentiation protocols of hPSCs to functional astrocytes, based on external cues to... (More)
- Astrocytes are one of the major cell types in the central nervous system and are indispensable for brain development and function. The human brain and human astrocytes have unique structures and functions that are not present in other animal species. Studies of fundamental astrocyte biology in humans and their role in neurological disease have been hindered by shortage of native human astrocytes for research purposes and inadequate animal and cell models. With advancements in stem cell technology, the possibility to generate astrocytes in vitro from human pluripotent stem cells (hPSCs), ultimately derived from patient cells, emerged. However, traditional differentiation protocols of hPSCs to functional astrocytes, based on external cues to mimic development, are complex and time-consuming. In contrast, ectopic overexpression of cell lineage-specific transcription factors can fast forward this process.
Here we have developed a rapid and efficient method to generate functional and mature astrocytes from hPSCs through overexpression of the gliogenic transcription factors Sox9 and Nfib. We have performed extensive phenotypic and functional characterization to confirm an astrocytic identity of the obtained cells. This method reduces the time to generate mature astrocytes from months to weeks. By combining our method with CRISPR/Cas9 genome editing we demonstrate that our method is feasible for disease modeling of the leukodystrophies Alexander disease (AxD) and Megalencephalic leukoencephalopathy with subcortical cysts (MLC).
Furthermore, we have developed an efficient method to directly convert human fibroblasts to astrocytes. We show that our method can be used with fibroblasts obtained from the entire human lifespan. We also, for the first time, show a co-culture system of astrocytes and neurons obtained through direct conversion of the same starting fibroblast populations. Finally, we provide proof-of-principle that our direct conversion method can be used for disease modeling by directly converting AxD patient fibroblasts to astrocytes.
The methods developed in this thesis allow for rapid generation of patient specific astrocytes which have the potential to uncover the role of astrocytes in neurological disorders and reveal novel targets for therapeutic interventions.
(Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/e132c96a-23b5-4b8b-ab82-10081b069705
- author
- Quist, Ella
LU
- supervisor
-
- Henrik Ahlenius LU
- Isaac Canals LU
- Zaal Kokaia LU
- opponent
-
- Associate Professor Erlandsson, Anna, Uppsala University
- organization
- publishing date
- 2023
- type
- Thesis
- publication status
- published
- subject
- keywords
- Astrocytes, Stem Cells, transcription factor programming, direct conversion, genome engineering, CRISPR/Cas9, leukodystrophy
- in
- Lund University, Faculty of Medicine Doctoral Dissertation Series
- issue
- 2023:2
- pages
- 92 pages
- publisher
- Lund University, Faculty of Medicine
- defense location
- BMC I1345, Sölvegatan 17 i Lund
- defense date
- 2023-01-19 13:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-8021-341-7
- project
- Dissecting the role of Astrocytes in Leukodystrophies using Stem Cells, Reprogramming and Genome Engineering
- language
- English
- LU publication?
- yes
- id
- e132c96a-23b5-4b8b-ab82-10081b069705
- date added to LUP
- 2022-12-15 14:02:12
- date last changed
- 2023-01-02 12:12:33
@phdthesis{e132c96a-23b5-4b8b-ab82-10081b069705,
abstract = {{Astrocytes are one of the major cell types in the central nervous system and are indispensable for brain development and function. The human brain and human astrocytes have unique structures and functions that are not present in other animal species. Studies of fundamental astrocyte biology in humans and their role in neurological disease have been hindered by shortage of native human astrocytes for research purposes and inadequate animal and cell models. With advancements in stem cell technology, the possibility to generate astrocytes in vitro from human pluripotent stem cells (hPSCs), ultimately derived from patient cells, emerged. However, traditional differentiation protocols of hPSCs to functional astrocytes, based on external cues to mimic development, are complex and time-consuming. In contrast, ectopic overexpression of cell lineage-specific transcription factors can fast forward this process.<br/><br/>Here we have developed a rapid and efficient method to generate functional and mature astrocytes from hPSCs through overexpression of the gliogenic transcription factors Sox9 and Nfib. We have performed extensive phenotypic and functional characterization to confirm an astrocytic identity of the obtained cells. This method reduces the time to generate mature astrocytes from months to weeks. By combining our method with CRISPR/Cas9 genome editing we demonstrate that our method is feasible for disease modeling of the leukodystrophies Alexander disease (AxD) and Megalencephalic leukoencephalopathy with subcortical cysts (MLC).<br/><br/>Furthermore, we have developed an efficient method to directly convert human fibroblasts to astrocytes. We show that our method can be used with fibroblasts obtained from the entire human lifespan. We also, for the first time, show a co-culture system of astrocytes and neurons obtained through direct conversion of the same starting fibroblast populations. Finally, we provide proof-of-principle that our direct conversion method can be used for disease modeling by directly converting AxD patient fibroblasts to astrocytes. <br/><br/>The methods developed in this thesis allow for rapid generation of patient specific astrocytes which have the potential to uncover the role of astrocytes in neurological disorders and reveal novel targets for therapeutic interventions.<br/>}},
author = {{Quist, Ella}},
isbn = {{978-91-8021-341-7}},
issn = {{1652-8220}},
keywords = {{Astrocytes; Stem Cells; transcription factor programming; direct conversion; genome engineering; CRISPR/Cas9; leukodystrophy}},
language = {{eng}},
number = {{2023:2}},
publisher = {{Lund University, Faculty of Medicine}},
school = {{Lund University}},
series = {{Lund University, Faculty of Medicine Doctoral Dissertation Series}},
title = {{Generation of human astrocytes for disease modeling. A study based on stem cells, direct conversion and genome engineering to dissect the role of astrocytes in leukodystrophies}},
url = {{https://lup.lub.lu.se/search/files/131462364/Ella_Quist_WEBB.pdf}},
year = {{2023}},
}