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Konstantinos Anastassiadis
Mechanisms that control the totipotency and differentiation of mouse ES cells
Previous and current research
We are interested in exploiting the mechanisms that control self- renewal and
differentiation of mouse embryonic stem cells. These cells originate from the
Inner Cell Mass (ICM) of the blastocyst and give rise to all tissues of the embryo
including the germline. ES cells are a powerful experimental system to understand
the mechanisms that underlie developmental processes.
In previous work we focused on factors that control the maintenance of the totipotent
character. The transcription factor Oct-3/4 is expressed in the ICM, in the epiblast
and only in germ cells later in development. Embryos with both alleles of the
Oct-3/4 deleted die around implantation and blastocyst outgrowths do not sustain
the ICM but differentiate into trophectoderm. On the other hand, overexpression
of Oct-3/4 drives differentiation into primitive endoderm. Thus, the expression
level of Oct-3/4 plays a critical role for the maintenance of ES cell totipotency.
Self-renewal and maintenance of totipotency is one side of a coin, differentiation
is the other. Embryonic stem cells can differentiate into all 3 germ layers in
culture. During differentiation the totipotent ES cells give rise to progenitor
cells that in their turn give rise to terminally differentiated cells. The process
of differentiation is guided by the various factors that are added to the culture
media and is not yet well understood.
It is important to understand this process in order to guide and control it.
For this reason we have developed (and continue to develop) various genetic experimental
tools. We have modified the tetracycline-regulated system for a highly predictable
application. For example ES cells carrying tetracycline controlled SV40 large
T-Antigen have been made, differentiated into neurons and immortalized with tetracycline
regulated T-Antigen expression. The differentiation status of the cells was investigated
by immunostaining for neuronal specific markers, at various time points, both
in induced cells and in cells where T-Antigen expression was switched off. Induced
cells were positive for markers that characterize neural precursors and negative
for markers that characterize post-mitotic neurons. Cells that the expression
of T-Antigen was switched off, started expressing markers that characterize post-mitotic
neurons. In other words, we have developed a system that enables conditional
immortalization of cell populations that arise during ES cell differentiation.
This technical advance opens a door to new mechanistic studies on gene expression
hierarchies in differentiation.
Future prospects and goals
- Differentiation of ES cells using lineage selection. The goal is to achieve a homogenous population of progenitors or differentiated cells.
- Analyze the gene expression profile and the chromatin status of the conditionally immortalized progenitors.
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Selected publications
Anastassiadis, K., J. Kim, N. Daigle, R. Sprengel, H.R. Schöler, A.F. Stewart (2002): A
predictable ligand regulated expression strategy for stably integrated
transgenes in mammalian cells in culture. Gene, 298: 159-172.
Voss, A.K., T. Thomas, P. Petrou, K. Anastassiadis, H. Schöler, P. Gruss (2000): Taube
nuss is a novel gene essential for the survival of pluripotent cells of
early mouse embryo. Development, 127(24): 5449-5461.
Pesce, M., K. Anastassiadis, H.R. Schöler (1999): Oct-4: Lessons of totipotency from embryonic stem cells. Cells Tissues Organs, 165: 144-152.
Nichols, J., B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Schöler, A. Smith (1998): Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95: 379-391.