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Reference: Schneider MR, Adler H, Braun J, Kienzle B, Wolf
E, Kolb H-J. Canine Embryo-Derived Stem Cells – Towards Clinical Relevant Animal
Models for Evaluating Efficacy and Safety of Cell therapies. Stem Cells 2007;25:1850.
Summarized by: Ibrahim Odewale, Fall
2007
LAY SUMMARY
Embryonic stem cells (ESCs) can generate all types of cells, which could be
used to replace damaged cells or to regenerate damaged tissues. ESCs are of
special interest because of their potential clinical application in treating
diseases, and replacing pathological cells and tissues. Currently, ESCs have
been extensively studied in mouse; however, understanding the properties of
these cells in other animal models is necessary, in particular the animal close
to human is need for future development of technologies for humans. Canine (dog)
ESCs also have some advantages over mouse ESCs. Canines have a longer life-span
than mice; this will enable scientists to do long-term studies with ESCs that
are currently impossible in mouse models. Canine diseases are similar to human
diseases; this similarity makes them a better model for human diseases. The
significant size difference between canine and mouse will enable scientist to
study and anticipate potential problems when stem cell therapies are scaled up
for human delivery.
This study confirms the possibility of creating ESCs from canine embryo, and
the ability to induce differentiation into hematopoietic stem cell progenitors,
blood forming cells. The inner cell mass (ICM) was mechanically removed from
canine blastocysts at the 8 cell stage. The cells were cultured on
mitotically-inactivated mouse embryonic fibroblast. These cultured cells were
found to exhibit the shape and arrangement characteristic of ESCs, and expressed
ESC genes, such as NANOG, OCT4, SOX2 and SSEA-1. The expression pattern
parallels what is observed in mouse ESCs. When the cells were grown in
suspension media, they formed embryoid bodies, which is a step following ESC
development. The embryoid bodies gave rise to various cell types when they are
transferred to adhesive plates.
Differentiation into blood forming cells was achieved by growing the ES-like
cells on top of an irradiated stroma cells from OP9 mice. Differentiation was
confirmed by probing for CD34, which is a marker for hematopoietic
progenitor cells, and GATA2, a hematopoietic transcription factor. Upon
confirmation of the hematopoietic lineage the cells were further cultured to see
if they will give rise to hematopoietic colonies. Colony-forming units were
seen nine days after the culture, addition of hematopoietic growth factors
caused colony-forming units to form in 6 days rather than 9 days.
SCIENTIFIC SUMMARY
Embryonic stem (ES) cells are can differentiate into cells of the three germ
layers. ES cells are of special interest because of their potential clinical
application in treating diseases, and replacing pathological cells and tissues.
ES cells have the potential for tissue repair or regeneration.
The study of ES cells in canine is important because mouse models do not
adequately mimic all human diseases. Canines are long lived; therefore,
long-term studies which are impossible in mouse models can be done in canine.
Larger animals such as canines also acquire diseases that are similar to human
diseases; this similarity makes them a better model for human diseases. The
significant size difference between canine and murine will enable scientists to
study and anticipate potential problems when stem cell therapies are scaled up
for human. All the aforementioned reasons make ES cell study in canine an
important part of stem cell research because the techniques used will prove to
be more applicable to human subjects. This summary outlines the attempt to
establish canine ES cell lines, and the induction of the stem cells into
hematopoietic progenitor cells.
Inner cell mass (ICM) was mechanically removed from canine blastocysts at the
8 cell stage. The cells were then cultured on mitotically-inactivated mouse
embryonic fibroblast. They exhibited the characteristic ESC morphology, they
also expressed pluripotency markers. The cells were further subcultured in
suspension culture, which resulted in the formation of embryoid bodies that
further differentiated into various cell types, including neuron-like,
epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells
(Figure 1). The formation of these cell types confirms the pluripotency of the
cells. The ES-like cells were also cultured independent of the mouse feeder
layer, they where then characterized for both pluripotency markers. Lack of
differentiation was confirmed via alkaline phosphatase activity, and the
following pluripotency markers; NANOG, OCT4 and SOX2. The ES-like cells were
also studied with antibodies against stage-specific embryonic antigen SSEA-1 and
SSEA-4. The cells were positive for SSEA-1 but negative for SSEA-4, which is in
accordance with the expression pattern seen in mouse. The cells were induced
towards hemotopoietic progenitor cells by coculturing the cells with irradiated
OP9 bone marrow stroma cells. The cells were then subjected to
fluorescence-activated cell sorting (FACS) analysis using anti-canine CD34
antibody. Almost none of the cells stained positive on day 0 but more than
half of the cells expressed CD34 at days 6 and 9. RT-PCR was also done to show
the gene expression level of CD34 and GATA2, a transcription
factor for hematopoietic progenitor cells. The expression of both genes
mirrored the result of the FACS analysis with no expression on day 0 but
substantial expression on days 6 and 9.
The cocultured cells were harvested on day 0, day 6, and day 9, and then
cultured in 6-well plates to see if they will form colony-forming units (CFUs)
of the hemtopoietic lineage. As expected the cells from day 0 did not grow into
colonies, but cells from day 9 formed CFU-M, CFU-E, CFU-G, and CFU-GM. Cells
from day 6 formed colonies only when hematopoietic growth factors were added.
Although, undifferentiated phenotype was only maintained through passage 8
and the possibility of having an unlimited culture of the differentiated cells
is yet to be determined, this study further established the practicability of
canine stem cell techniques. These techniques can be used as a platform for
further stem cell studies in canine models, or they can be carried over to other
animal models. This study will also play a role in potential clinical
applications of organ and tissue regeneration in veterinary medicine.
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