Læknaneminn - 01.04.2008, Page 105

marker). We measured expression
of aforementioned markers in H9
cells (positive control), hepatocytes
(positive control) and three different
cultures of EBs (EB5, 6 and 7).
Our results showed no expression
of sox-17, strong expression of
AFP, brachyury expression in the
undifferentiated H9s, EB6 and
EB7 and oct-4 expression in the
undifferentiated H9, as well as EB5-
7.
Establishment of ES cell lines
expressing green fluorescence
protein (GFP) under the control of
albumin promoter (pAlb-GFP ES
cell line) was verified by subsequent
G418 selection and clonal expansion
of stable transfectants. Nucleofection
with linearized DNA proved stable,
although not extremely efficient.
Efficiency of generation of single cell
clones of G418 resistant cells proved
to be less than 1%. This is accordance
with hESCs inherent affinity for
colonal growth.
Understanding of hESCs is still
in the preliminary stages and
ongoing research is key to gathering
knowledge of factors controlling
growth and differentiation of hESCs.
Culturing methods, differentiation
tracking and single cell cloning
experimental methods are under
constant development as the means
to the end of fully harnessing the
potential of hESCs.
The Role of Smadl
in Adult Murine
Hematopoiesis
Gudrun Eiriksdottir, Sofie Singbrant
and Stefan Karlsson
Department of Molecular Medicine
and Gene Therapy, Lund Stem Cell
Center, Lund University Hospital,
Lund, Sweden
Objective:
Hematopoiesis refers to the highly
dynamic process of blood cell
formation. All types of blood
cells originate from a common
hematopoietic stem cell (HSC),
which by definition can both self-
renew and thereby give rise to new
stem cells, and also differentiate into
mature cells of all blood lineages.
The exact molecular mechanisms
controlling the HSC fate decisions
are, however, poorly understood.
One important signaling pathway is
mediated by the transforming growth
factor-|3 (TGF-P) superfamily, where
several members including bone
morphogenetic proteins (BMPs), are
involved in the regulation of HSCs.
Because of possible redundancy
between the different signaling
pathways, a more systematic analysis
of the intracellular signal-mediating
Smad proteins in the regulation of
HSC fate in vivo is needed. Effects
of BMP signaling, which is mediated
by Smadl and 5, in the regulation of
human HSCs have been reported. It is
therefore of great interest to study the
effect of their deficiency utilizing a
murine knockout model. In contrast
to the essential roles of Smadl and
Smad5 during embryogenesis,
recent findings suggest that Smad5 is
dispensible for the adult mouse. This
study is focused on the role of Smadl
in adult murine hematopoiesis.
Materials and Methods:
To circumvent the embryonic lethal
phenotype we used a Cre-mediated
conditional knockout model. Mice
with floxed Smadl alleles were mated
with transgenic mice expressing the
Cre-recombinase gene under control
of the interferon inducible promoter
Mxl, which results in efficient
recombination of floxed genes in
the hematopoietic system upon
interferon induction. The impact
of the genetic deficiency on the
regeneration of the hematopoietic
system, and the proliferation and
differentiation capacity of the HSCs
was investigated both in vivo and in
vitro. After interferon-induced Cre-
mediated recombination in Smadlfl/
fl mice which caused deletion of
the Smadl-allele, bone marrow
was collected and transplanted into
lethally irradiated recipients.
Results:
Peripheral blood analysis 6 weeks
after transplantation revealed that
Smadl deficient HSCs show normal
competitive repopulation ability and
lineage distribution in vivo short-
term after BM transplantation. Long-
term effect of Smadl deletion is
currently being evaluated.
Conclusion:
The question remains whether Smad 1
truly is dispensible in adult murine
hematopoiesis, or successfully
compensated for by Smad5. It is
true that these proteins share great
homology and probably cooperate
in many ways, but whether they are
capable of completely compensating
the loss of each other will be addressed
in a future experiment on Smadl/
Smad5 double-knockout mice.