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I am interested studying fundamental questions of stem cell biology
using embryonic stem cell differentiation towards the endodermal
germ layer as a model. The main questions are:
- What are the constraints to developing a pure population
of endoderm progenitor cells?
- How can one control the differentiation of these progenitor
cells by manipulating the underlying transcriptional circuitry
of the cell?
The tools being applied include RT-PCR, FACS and RNA interference.
For tissues derived from the endodermal germ layer, such as the
liver, lung, and pancreas, current approaches for differentiation
from embryonic stem (ES) cells have yielded some promising results
(Abe 1997, Kubo 2004) but typically result in mixed populations
(Kubo 2004) or result in low yields of the cell of interest (Lin
2004, Ku 2004), probably due to a lack of control of differentiation.
My work seeks to understand how negative perturbation of a regulatory
transcription factor (TF), Foxa2, can be used to control the major
regulatory gene circuits that control endoderm differentiation,
and thus provide a refined level of control over the endoderm population.
The techniques used are end point and qRT-PCR, immunofluorescence,
FACS, and gene-silencing using RNA interference.
In vivo, Foxa2 has been shown to have
dramatic and irreversible effects on induction, maintenance, and
differentiation of the endoderm. Initially, the kinetics of major
endodermal regulatory TF is deciphered, in a serum-free monolayer-based
system and in the presence of the soluble factor activin, to better
understand dynamics of endodermal induction, maintenance and differentiation.
Next, a gene silencing strategy is used to silence Foxa2 in the
endoderm-enriched cultures at various times, with quantitative assessment
of gene silencing and effects on other major constituents of endodermal
gene circuits.
The effects of gene silencing on the endodermal gene circuits are
expected to vary with time and it is expected that major endodermal
TF will not be maintained. To understand these complex effects on
gene circuits in endoderm, a mathematical model taking into account
complex interactions between endodermal TF will be developed. Lastly,
a rational strategy will be developed for using developmentally
relevant soluble factors, such as activin or Wnt, to simultaneously
maintain endodermal TF's while preventing differentiation along
the liver lineage in Foxa2-silenced endodermal cells. Together,
these approaches are intended to address the fundamental problem
of lineage control within the endodermal cell compartment and to
take rational steps towards isolating and controlling an endodermal
progenitor cell.
My hobbies include tennis, big band jazz, anthropology, following
sports, community service.
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