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Embryonic stem cells as an in vitro model of the gastrulating mouse embryo

dinsdag, 8 mei, 2007 - 16:30
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
D
0.08
Erik Willems
doctoraatsverdediging

Embryonic stem (ES) cells are derived from a blastocyst embryo and can give rise to all cell types of the adult body. Pluripotency and self-renewal are the two features that makes ES cells attractive to fundamental as well as clinical research: they divide unlimitedly in a culture dish and retain their undifferentiated phenotype for extended periods of culture. Conversion of these cells to a specific phenotype, a process called differentiation, can be initiated by external stimuli added to the culture medium.

Since ES cells are derived from embryos, differentiation towards a certain fate could be induced by molecular mechanisms similar to the ones found in the embryo. Therefore, ES cell differentiation might not only be a tool to obtain mature cell types by applying the knowledge of the embryo, but also a way to understand the exact molecular mechanisms that lead to the formation of a specified cell in the embryo.

The main aim of this thesis was to develop in vitro assays based on ES cells that would allow us to study the molecular mechanisms that are required in the mouse for mesoderm formation, an event that is essential in early development. Defects in mesoderm generation lead to for example aberrant muscle or heart formation.

First, we had to set up a system that would allow us to monitor ES cell differentiation and that could detect alterations in the fate of differentiated cells. In the early embryo, most of the tissues formed can be marked quite specifically by the expression of a marker gene. Therefore, an accurate quantitative reverse transcriptase PCR approach was developed to detect the activity levels of these marker genes during ES cell differentiation.

By applying the ES cell differentiation system as an in vitro assay of the mouse embryo, we were able to confirm the roles of Nodal, Wnt, Bmp and FGF in the mouse embryo, suggesting the ES cell system indeed represented the mouse embryo in a culture dish.

By using different ES cell culture systems we could demonstrate that there was a balance between agonists (Wnt, Bmp, Nodal) and antagonists (Cer1, Lefty1, Sfrp1/5 and Dkk1) leading to the formation of mesoderm and neuroectoderm, respectively, in similarity to the anterior-posterior axis of the mouse embryo. Moreover, we identified several other roles for Nodal, Bmp4 and FGF signaling, which could not be revealed in the mouse embryo because of early lethality of their homozygous mutants. Mesoderm patterning appeared to be controlled by an equilibrium between Bmp4 and

Nodal, leading to the formation of posterior and anterior mesoderm respectively. Additionally, Nodal acted morphogenically to alter mesendoderm cells towards mesoderm or endoderm. Not only Bmp4 or Nodal had important functions during patterning of the mesoderm, as formation of mesendoderm by Nodal or posterior mesoderm by Bmp4 was impaired in absence of FGF signaling, revealing an important role for FGF in the epithelial to mesenchymal transition of newly formed mesoderm cells.

In conclusion, we were able to demonstrate that ES cells can indeed be used as an in vitro model of the mouse embryo, allowing the discovery of new functions that could not be identified in the in vivo situation. Alternatively, this work suggests that when the molecular signaling of the embryo is mimicked in ES cell differentiation, specific cell types can be obtained, directing this idea towards clinical approaches.