Generation of astrocytes from embryonic stem cells

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Human exposure to chemicals by environmental pollution, food or drug constituents has been linked to developmental neurotoxicity (DNT) in epidemiological studies. In vitro models open up new possibilities to study toxicity on a molecular level, which is expected to improve human risk assessment. A starting point for the development of these models may be pluripotent stem cells as they can replicate development of an embryo in vitro.

In this thesis, I discuss how DNT can be modelled in vitro using stem cell derived, differentiating neural cultures. Transcriptional profiling is suggested as a sensitive endpoint to detect toxic effects of substances. For this purpose, we developed comprehensive lists of marker genes for cells of different developmental stages within developing neural cultures. These were used to describe the effect of chemicals on embryonic stem cells (ESC) that differentiate to neurons.

Until now, in vitro neurotoxicology has mainly focussed on neurons, the primary effector cells of the brain. However, other cells, such as astrocytes also play a role in generation of toxicity in the brain, either by causing an overshooting inflammatory response upon activation by pathogens or toxicants, or by metabolic activation of xenobiotics. At the start of this thesis, no protocols that described the generation of pure and functional astrocytes from ESC were known. Therefore, I developed two methods for the differentiation of mouse embryonic stem cell derived astrocytes (MEDA).

The first method aimed at producing subtypes of astrocytes to study possible differences in astrocyte subpopulations. It relies on a 2-step protocol and yielded mixed subpopulations of astrocytes. While most cells (81 ± 16%) express the astrocyte marker S100β, only a subpopulation of these MEDA (31 ± 18 %) was positive for the standard astrocyte marker GFAP.

With the second protocol, homogeneous astrocyte populations were obtained in very short time. ESC were first differentiated into pure populations of neural precursor cells. These precursor cells were then differentiated within 3-5 days to GFAP-positive MEDA. The fast transition into astrocytes makes them ideally suited for studies of developmental toxicity, as drugs interfering with astrocyte development can be picked up quickly.

Both types of stem cell derived astrocytes were characterised in depth as to their inflammatory competence, metabolic activity, and their ability to provide trophic support to developing neuronal cultures. They were also compared to primary astrocytes isolated from mouse brain. To our knowledge, this work comprised the first functional characterisation of astrocyte subpopulations, and we found that GFAP-negative astrocytes contribute to inflammatory responses, and are able to support neurons in the same way as their GFAP-positive counterparts. In co-cultures with neurons, we found that MEDA were able to prolong neuronal survival. Furthermore, when plated on astrocytes, neurons grew at low cell-densities allowing single cell analysis of individual neurons. We propose that MEDA are an adequate alternative to primary isolated astrocytes.

The new cell models generated during the course of this thesis are expected to be useful for research on brain disease and the development of novel test systems to detect (developmental) neurotoxicity.

Zusammenfassung in einer weiteren Sprache

In epidemiologischen Studien konnte ein Zusammenhang zwischen Umweltgiften und Chemikalien in Medikamenten und Nahrungsmittelzusätzen und Entwicklungsstörungen des menschlichen Gehirns (developmental neurotoxicity, DNT) hergestellt werden. In vitro-Modelle eröffnen neue Möglichkeiten die giftige Wirkung von Substanzen auf molekularer Ebene zu untersuchen, wodurch eine verbesserte Risikoabschätzung für den Menschen erwartet wird. Grundlage für diese Modelle könnten pluripotente Stammzellen sein, mit denen sich die Embryonalentwicklung des Gehirns in vitro nachstellen lässt.
In dieser Arbeit stelle ich vor wie DNT durch aus Stammzellen gewonnene Gehirnzellen untersucht werden kann. Transcriptions-Profiling wird als sensible Methode für die Erfassung toxischer Effekte von Substanzen vorgestellt. Dafür wurden umfassende Listen zelltypspezifischer Marker-Gene für verschieden Stadien der Gehirnentwicklung ausgearbeitet. Mit diesen Marker-Genen wurden die Effekte von Chemikalien auf ESC, die zu Neuronen differenzieren, untersucht.

Die in vitro-Neurotoxikologieforschung hat sich bisher hauptsächlich auf Nervenzellen als die Haupteffektorzellen des Gehirns konzentriert. Astrozyten sind jedoch auch beteiligt an der Entstehung von toxischen Prozessen im Gehirn, z. B. durch eine überschießende Immunantwort nach einer Substanz-induzierten Aktivierung, oder auch durch eine Aktivierung von xenobiotischen Substanzen durch Stoffwechselprozesse. Am Anfang dieser Arbeit war keine Möglichkeit bekannt, reine Kulturen von funktionalen Astrozyten aus embryonalen Stammzellen (ESC) zu generieren. Ich habe im Laufe dieser Arbeit zwei Protokolle zur Gewinnung funktionaler Astrozyten entwickelt. Mit der ersten Methode konnten gemischte Subpopulationen von Astrozyten hergestellt werden, welche sich in der Expression des Astrozyten-Markers GFAP unterschieden. Diese Subtypen wurden auf potentielle funktionale Unterschiede untersucht.

Im zweiten Protokoll wurde eine reine Kultur aus neuralen Stammzellen gewonnen, aus welcher sich in kürzester Zeit (3-5 Tage) reine Kulturen von GFAP-positiven Astrozyten differenzieren ließen. Diese äußerst schnelle Umwandlung zu reinen Populationen machen diese Astrozyten zu einer idealen Plattform, um beispielsweise den Einfluss von Substanzen auf die Entwicklung von Astrozyten zu untersuchen.

Beide Astrozytenkulturen wurden umfassend auf ihre inflammatorische Kompetenz, ihre metabolische Aktivität und die Fähigkeit sich entwickelnde Neuronenkulturen zu unterstützen charakterisiert und mit aus Mäusehirnen isolierten Astrozyten verglichen. Nach unserem Wissen, erlaubten es unsere Kulturen, zum ersten Mal eine funktionelle Charakterisierung von Astrozyten-Subpopulationen durchzuführen.

In Cokulturen mit Neuronen konnten wir feststellen, dass abgesonderte Faktoren von ESC-derivierten Astrozyten (MEDA) die Überlebensdauer von Neuronen positiv beeinflussten. Weiterhin ließen sich in Cokulturen Neuronenkulturen in sehr niedriger Zelldichte herstellen, was eine Einzelzellanalyse von Neuronen ermöglichte. Durch meine Arbeit konnte ich herausfinden, dass MEDA eine gute Alternative zu primär-isolierten Astrozyten darstellen könnten.

Die während dieser Arbeit entstandenen zellbasierten Modelle werden zum Verständnis von der Entstehung von Gehirnkrankheiten beitragen und die Entwicklung von Testsystemen für die Erfassung von (Entwickungs-)Neurotoxizität ermöglichen.

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570 Biowissenschaften, Biologie
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ISO 690KÜGLER, Philipp, 2011. Generation of astrocytes from embryonic stem cells [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Kugler2011Gener-17434,
  year={2011},
  title={Generation of astrocytes from embryonic stem cells},
  author={Kügler, Philipp},
  address={Konstanz},
  school={Universität Konstanz}
}
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December 14, 2011
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