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Embryonic
stem cells and their potential therapeutic applications
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What
is a “stem cell”?
Stem cells are the pluripotent cells that are present in an organism.
They have a self renewal capacity and can be maintained as undifferentiated
in vivo or in vitro. There are three known types of stem cells: Adult
stem cells, cord blood stem cells and embryonic stem cells.
Adult stem cells are present in numerous tissues and organs in a body.
Upon damage, they proliferate and help in regenerating and repairing the
damaged area. Although their transdifferentiation ability has been reported
in some animal studies, It is generally thought that their ability to
form different cell types are limited.
Cord blood stem cells are obtained from the umbilical cord just after
delivery. These cells, although they are obtained from newborns, are still
considered as being adult cells in origin. Therefore, their ability to
generate different cell types are likewise limited. On the other hand,
transplantation of cord blood stem cells can create an important therapeutic
option for several blood and immune system disorders. However, among the
the most important problems associated with cord blood stem cell transplantation
are: a limited amount of available stem cells in a source material and
the probability of immune rejection due to HLA imcompatilility. Cord blood
stem cells have a limited developmental potential in vitro and the number
of cells that are available for transplantation often limits the success.
As the number of cord blood banks increase, the number of desases that
can be treated with cord blood stem cell transplantation as well as success
rates are believed to increase in the near future.
How
are embryonic stem cells isolated?
Embryonic stem cells are obtained from the preimplantation embryos
during early developmental stages. Just before implanting in the
uterus, the embryo is called “blastocyst” and mainly
consists two types of cells: The cells on the outer part which will
form placenta upon implantation are named “trophectederm” cells,. On the other hand, the cells inside the cavity are called
as inner mass cells (ICM) and they form fetal tissues upon implantation.
In our laboratory, embryonic stem cells are isolated from inner
cell mass after mechanical or immunosurgical dissociation methods
(Picture 1). Isolated cells are then cultured in a special stem
cell culture conditions.
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Picture 1. An
embryo at the blastocyst stage. Cells inside the marked area are
the inner cell mass cells which are to be isolated and cultured. |
How
are embryonic stem cells cultured in a laboratory?
Although there have been numerous studies on the alternative culture
methods, embryonic stem cells require a special culture systems
consisted of mitotically inactivated feeder cell environment and
chemically defined media components such as leukemia indibitory
factor (LIF) and b-FGF. In the Istanbul Memorial Hospital Research
and Development Laboratory, mouse embryonic fibroblast cells are
obtained from 12-14th-day inbred Balb/c fetuses and used as feeder
cells.Picture 2 shows a stem cell colony cultured on mouse embryonic
fibroblast cells.
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Picture 2. A
stem cell colony. After mechanical and enzymatic dispersion of ICM
cells, they are, cultured on mitotically inactivated mouse embryonic
fibroblast layer for several days. |
What
are the morphological, genetical and immunological characteristics
of embryonic stem cells ?
Embryonic stem cells, when compared to other somatic cells, have
a distict higher nucleus-to-cytoplasm ratio and a prominent nucleoli
structure. These cells form three-dimensional colonies during their
culture on feeder cells. In picture 3, a typical human embryonic
stem cell morphology is seen. This picture belongs to one of the
human embryonic stem cell line (NS-3) isolated our laboratory.
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Picture 3. Morphology
of human embryonic stem cells (NS-3) |
Another characteristics
of embryonic stem cells is, similar to cancer cells, to divide and grow
indefinitely. However, unlike cancer cells, embryonic stem cells have
a normal karyotype and they maintain this normal chromosome structure
during long term culture. Picture 4 represents normal male (NS-3) and
female (NS-4) karyotype of human embryonic stem cells isolated in our
laboratory.
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4. Karyotypes representing human embryonic stem cell lines
NS3 (left) ve NS4(right) |
Picture 5. Alkaline
phosphatase staining of the lines NS-3 ve NS-4 |
Embryonic stem cells are further defined
by advanced molecular tests. In order to be immunologically characterized
as stem cells, These cells are screened for the expression of early
development and differentiation markers such as SSEA-1, 3, 4, TRA-1-60
and 81, OCT-4, alkaline phosphatase etc. Picture 5 shows the alkaline
phosphatase staining results of NS-3 and NS-4 lines. Stained sections
represent undifferentiated colonies. |
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Which
cell types can be obtained from embryonic stem cells upon differentiation?
In the absence of feeder cells and certain chemical agents, embryonic
stem cells can differentiate into all cell types that are present
in an organism. However, although the role of several chemicals
or conditions are partially understood in some differentiation pathways,
the exact differentiation mechanism are largely unknown hence are
not controllable at the moment.
In order to obtain different cell types we use a general embryoid
body formation protocol. In the absence of factors which inhibits
differentiation, embryonic stem cells can accumulate as three dimensional
aggregates in culture and form a sphere-like structure consisting
of three germ layers. (Pictures 6 and 7). Embryoid bodies are then
cultured in a special culture environments in order to obtain certain
cell types.
Nowadays, most of the differentiation studies are being performed
on cardiomyocytes, neurons, insulin secreting cells and blood cells.
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Picture 6. Stem
cell aggregates formed in the absence of feeder cells and chemical
agents.
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Picture 7. An
embryoid body. |
Therapeutic
potentials of embryonic stem cells
It has now been known that mouse stem cells have the potential to form
all the somatic and germ cells present in an adult mouse. As it has been
mentioned previously, their differentiation mechanisms as well as therapeutic
potentials upon transplantation are currently being investigated by many
scientists worldwide. In parallel, human embryonic stem cells also share
many common features with mouse embryonic stem cells and it is believed
that they can be used as a therapeutic source for human life-threatening
or degenerative disorders such as myocardial infarcts, Parkinson, Alzheimer
or diabetes in the near future. Since, in mice, it has been shown that
these cell can also form gamete cells in proper culture conditions, they
can also be used in infertility treatment.
Besides their tremendous therapeutic potential, there are several handicaps
which prevents them being included in the general practice.
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What
are the factors that limit the use of embryonic stem cells in the clinical
practice?
In tissue or organ transplantations, one of the most important factors
that affect the success rate is the tissue compatibility between donor
and the recipient. Therefore, when the embryonic stem cells are available
for human therapy, problems associated with tissue incompatibility are
believed to create similar limitations. In order to overcome this problem,
there exist several approaches: Establishment of multiple embryonic stem
cell banks, creation of a universal donor cell line with genetic engineering
methods and the isolation of a person-specific stem cell line by somatic
cell nucleus transfer methods (therapeutic cloning)
Although so far more than 80 different human embryonic stem cell lines
have been reported, only a few of these are characterized and registered
by NIH. Therefore, most of the research performed so far utilized only
a handful of these lines It is estimated that, when therapeutic conditions
are established in the future, in order to find the HLA-compatible stem
cell line with the patient, several hundreds of thousands different human
embryonic stem cell lines should be available. For this reason, collecting
established embryonic stem cell lines is a “bank” will facilitate
their utilization for clinical use in the future.
Another approach, as previously mentioned above, is to manipulate a human
embryonic stem cell line so that the cells of tissues generated from this
cell line can be transplanted without creating an immune response in the
recipient’s body. This can be done by genetically engineering the
genes (HLA genes) which are responsible for tissue compatibility and immune
response. This approach, if it is realized, can eliminate the need of
thousands of different human embryonic stem cell lines. Currently, the
technical problems as well as safetly and ethical issues associated with
genetic engineering of human cells for therapy limits this option.
Therapeutic cloning can be another approach to overcome the tissue rejection
problem in human stem cell therapy. (Picture 8). Especially in the last
5-10 years,there has been advances in somatic cell nucleus transfer techniques
Although the efficiency is still far from being clinically applicable
in humans, numerous laboratory as well as farm animals have been cloned
with this technique.
Picture
9. Therapeutic cloning by somatic cell
nucleus transfer: First stem requires the removal of genetic material
fom an oocyte (enucleation).Atfer that, a somatic cell nucleus from a
donor is inserted into the enucleated oocyte. After activation and embryo
cleavage steps, obtained blastocyst stage embryo is manipulated to isolate
stem cells and the isolated stem cells are cultured in otder to obtain
a variety of different cell types.
Reproductive cloning has been strictly forbidden
in many countries including US and Europe However, using the same somatic
cell nucleus transfer technique in order to obtain person-specific embryonic
stem cells is termed as therapeutic cloning and research involving this
technique are allowed in many countries. Furthermore, in some countries,
research on human therapeutic cloning is officially supported. As a result,
recently South Korean scientists has generated a human embryonic stem
cell line after somatic cell nucleus transfer The ethical as well as moral
issues regarding this approach are still under an intensive debate. From
the technical point of view, the applicability of this approach is still
very low since only one cell line has been obtained after manipulation
of more than 200 human oocytes.
Furthermore, since the technique is applied mostly on experimental animal
studies and farm animals, the protocols regarding human studies are lacking
and need to be well-established before any clinical trial. For this purpose,
our laboratory performs preliminary studies in order to increase the efficiency
of the protocols used in therapeutic cloning techniques. As being animal
models, mice and bovine oocytes are cureently being used for electrofusion
experiments.
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