Stem cells

STEM CELLS

 The history of research on adult stem cells

began about 40 years ago. In the 1960s,
researchers discovered that the bone
marrow contains at least two kinds of stem
cells. One population, called
hematopoietic stem cells, forms all the
types of blood cells in the body. A second
population, called bone marrow stromal
cells, was discovered a few years later.
 Stromal cells are a mixed cell population
that generates bone, cartilage, fat, and
fibrous connective tissue.
 Where are adult stem cells found and
what do they normally do?

 An adult stem cell is an

undifferentiated cell found among
differentiated cells in a tissue or organ,
can renew itself, and can differentiate
to yield the major specialized cell types
of the tissue or organ.
 Where adult stem cells are
found?
Adult stem cells have been identified in many organs
and tissues. However, there are a very small
number of stem cells in each tissue.

 Stem cells are thought to reside in a specific area of

each tissue where they may remain quiescent (non-
dividing) for many years until they are activated by
disease or tissue injury.

 The adult tissues reported to contain stem cells

include brain, bone marrow, peripheral blood, blood
vessels, skeletal muscle, skin and liver.
Role of adult stem cells
 The primary role of adult stem cells in a

living organism are to maintain and repair the

tissue in which they are found.
 Some scientists now use the term somatic

stem cell instead of adult stem cell. Unlike

embryonic stem cells, which are defined by
their origin (the inner cell mass of the
blastocyst), the origin of adult stem cells in
mature tissues is unknown.
 What are the tests used for identifying adult stem
cells?
 One or more of the following methods are normally
used for the identification of adult stem cells:

• Labeling the cells in a living tissue with molecular markers

and then determining the specialized cell types they generate.

• Removing the cells from a living animal, labeling them in cell

culture, and transplanting them back into another animal to
determine whether the cells repopulate their tissue of origin

• Isolating the cells, growing them in cell culture, and

manipulating them, often by adding growth factors or
introducing new genes, to determine what differentiated cells
types they can become.
 A single adult stem cell should be able to
generate a line of genetically identical cells
—known as a clone—which then gives rise
to all the appropriate differentiated cell
types of the tissue.
 Adult stem cells may exhibit two types of
differentiation pathways;
• Normal differentiation; to form specialized
cell types of the tissue in which they reside.
• Transdifferentiation or plasticity; cells may
also exhibit the ability to form specialized cell
types of tissues other than the place where
they reside.
 Normal Differentiation
The following are examples of differentiation
pathways of adult stem cells

• Hematopoietic stem cells give rise to all types of

blood cells: red blood cells, B lymphocytes, T
lymphocytes, natural killer cells, neutrophils,
basophils, eosinophils, monocytes, macrophages,
and platelets.
• Bone marrow stromal cells (mesenchymal stem
cells) give rise to a variety of cell types: bone
cells (osteocytes), cartilage cells (chondrocytes),
fat cells (adipocytes), and other kinds of
connective tissue cells such as those in tendons.
• Neural stem cells in the brain give rise to its three
major cell types: nerve cells (neurons) and two
categories of non-neuronal cells- astrocytes and
oligodendrocytes.

• Epithelial stem cells in the lining of the digestive

tract occur in deep crypts and give rise to several
cell types: absorptive cells, goblet cells, Paneth
cells, and entero-endocrine cells.

• Skin stem cells occur in the basal layer of the

epidermis and at the base of hair follicles. The
epidermal stem cells give rise to keratinocytes,
which migrate to the surface of the skin and form
a protective layer. The follicular stem cells can
give rise to both the hair follicle and to the
epidermis.
 Trans-differentiation or plasticity of adult
stem cells
 Certain adult stem cell types are pluripotent. This ability to
differentiate into multiple cell types is called plasticity or
transdifferentiation.

The following list offers examples of adult stem cell

plasticity that have been reported during the past few
years
 Hematopoietic stem cells may differentiate into:
• three major types of brain cells (neurons, oligodendrocytes, and
astrocytes)
• skeletal muscle cells
• cardiac muscle cells
• liver cells.
 Bone marrow stromal cells may differentiate into: cardiac muscle
cells and skeletal muscle cells.

 Brain stem cells may differentiate into: blood cells and skeletal
muscle cells.
 What are embryonic stem cells?
Aare derived from embryos originated from eggs
that have been fertilized in vitro in an in vitro
fertilization clinic and donated to research
institutions with donors consent.
The embryos from which human embryonic
stem cells are derived are typically four or
five days old and are a hollow microscopic ball
of cells called the blastocyst which includes
three stages;
• Trophoblast, which is the layer of cells that
surrounds the blastocyst
• Blastocoel, which is the hollow cavity inside the
blastocyst
• Inner cell mass, which is a group of
approximately 30 cells at one end of the blastocoel.
 How are embryonic stem cells grown in the
laboratory?

 Growing cells in the laboratory is known as cell

culture.

 Human embryonic stem cells are isolated by

transferring the inner cell mass into a plastic
laboratory culture dish that contains a nutrient broth
known as culture medium.

 The inner surface of the culture dish is typically

coated with mouse embryonic skin cells that have
been treated so they will not divide. This coating
layer of cells is called a feeder layer.
The reason for having the mouse cells in the bottom of
the culture dish is to give the inner cell mass cells a
sticky surface to which they can attach. In addition,
these feeder cells release nutrients into the culture
medium.

Over the course of several days, the ESC proliferate

and begin to crowd the culture dish.
Cells are removed gently and plated into several fresh
culture dishes. The process of replating the cells is
repeated many times and for manymonths, and is
called subculturing. Each cycle of subculturing the
cells is referred to as a passage.
 Embryonic stem cells that have proliferated
in cell culture for six or more months without
differentiating, are pluripotent, and appear
genetically normal and are referred to as an
embryonic stem cell line.
 Once cell lines are established, or even
before that stage, batches of them can be
frozen and shipped to other laboratories for
further culture and experimentation.
What laboratory tests are used to identify embryonic
stem cells?

At various points during the process of

generating embryonic stem cell lines,
scientists test the cells to see whether they
exhibit the fundamental properties that make
them embryonic stem cells.
These tests include;
• growing and subculturing the stem cells for many
months. This ensures that the cells are capable of
long-term self-renewal.
• Scientists inspect the cultures through a microscope
to see that the cells look healthy and remain
undifferentiated.

• Using specific techniques to determine the presence

of surface markers that are found only on
undifferentiated cells.

• Another important test is for the presence of a protein

called Oct-4, which is typically made by
undifferentiated cells. Oct-4 is a transcription factor,
helps turn genes on and off at the right time, which
is an important part of the processes of cell
differentiation and embryonic development.

• Examining the chromosomes under a microscope.

This is a method to assess whether the
chromosomes are damaged or if the number of
chromosomes has changed.
 However, microscopy examination of
chromosomes, does not detect genetic
mutations in the cells.
 Determining whether the cells can be
subcultured after freezing, thawing, and
replating.

Testing whether the human embryonic stem

cells are pluripotent by;
• allowing the cells to differentiate spontaneously in
cell culture
• manipulating the cells so they will differentiate to
form specific cell types
• injecting the cells into an immunosuppressed
mouse to test for the formation of a benign tumor
called a teratoma.
Teratomas typically contain a mixture of many
differentiated or partly differentiated cell types
This is an indication that the embryonic stem
cells are capable of differentiating into multiple
cell types.
 How are embryonic stem cells are stimulated to
differentiate?

As long as the embryonic stem cells in culture are

grown under certain conditions, they can remain
undifferentiated (unspecialized). But if cells are
allowed to clump together to form embryoid
bodies, they begin to differentiate spontaneously.

 Although spontaneous differentiation is a good

indication that a culture of embryonic stem cells is
healthy, it is not an efficient way to produce
cultures of specific cell types.
To generate cultures of specific types of differentiated
cells—heart muscle cells, blood cells, or nerve cells,
for example—scientists try to control differentiation of
embryonic stem cells by;

• Change the chemical composition of the culture medium

• Alter the surface of the culture dish
• Modify the cells by inserting specific genes

 If scientists can reliably direct the differentiation of

embryonic stem cells into specific cell types, they may
be able to use the resulting, differentiated cells to
treat certain diseases at some point in the future.
 Diseases that might be treated by transplanting cells
generated from human embryonic stem cells include
Parkinson's disease, diabetes, traumatic spinal cord
injury, Purkinje cell degeneration, Duchenne's
muscular dystrophy, heart disease, and vision and
hearing loss.
 What are the similarities and differences
between embryonic and adult stem cells?

Adult and embryonic stem cells differ in the number

and type of differentiated cells types they can become.

Embryonic stem cells can become all cell types of the

body because they are pluripotent.
Adult stem cells are generally limited to differentiating
intodifferent cell types of their tissue of origin. However,
some evidence suggests that adult stem cell plasticity
may exist, increasing the number of cell types a given
adult stem cell can become.
 Large numbers of embryonic stem cells can be
relatively easily grown in culture, while adult stem
cells are rare in mature tissues and methods for
expanding their numbers in cell culture have not
yet been worked out. This is an important
distinction, as large numbers of cells are needed for
stem cell replacement therapies.
 A potential advantage of using stem cells from an
adult is that the patient's own cells could be
expanded in culture and then reintroduced into the
patient. The use of the patient's own adult stem
cells would mean that the cells would not be
rejected by the immune system.
 Embryonic stem cells from a donor introduced into a
patient could cause transplant rejection.
 What are the potential uses of human stem
cells?

What are the obstacles that must be overcome

before these potential uses will be realized?

 There are many ways in which human stem

cells can be used in basic research and in
clinical research. However, there are many
technical hurdles between the promise of
stem cells and the realization of these uses,
which will only be overcome by continued
intensive stem cell research.
 Potential uses

 Studies of human embryonic stem cells may yield

information about the complex events that occur
during human development. A primary goal of this
work is to identify how undifferentiated stem cells
become differentiated. Scientists know that turning
genes on and off is central to this process. Some of
the most serious medical conditions, such as cancer
and birth defects, are due to abnormal cell division
and differentiation.

 A better understanding of the genetic and molecular

controls of these processes may yield information
about how such diseases arise and suggest new
strategies for therapy.
Human stem cells could also be used to test new drugs.
For example, new medications could be tested for safety
on differentiated cells generated from human
pluripotent cell lines.
Other kinds of cell lines are already used in this way.
Cancer cell lines, for example, are used to screen
potential anti-tumor drugs. But, the availability of
pluripotent stem cells would allow drug testing in a
wider range of cell types.
 Perhaps the most important potential application of
human stem cells is the generation of cells and
tissues that could be used for cell-based
therapies.

 Today, donated organs and tissues are often used to

replace ailing (ill) or destroyed tissue, but the need
for transplantable tissues and organs far outweighs
the available supply.

 Stem cells, directed to differentiate into specific cell

types, offer the possibility of a renewable source of
replacement cells and tissues to treat diseases
including Parkinson's and Alzheimer's diseases,
spinal cord injury, stroke, burns, heart disease,
diabetes, osteoarthritis, and rheumatoid arthritis.
For example, it may become possible to generate
healthy heart muscle cells in the laboratory and then
transplant those cells into patients with chronic heart
disease.
Preliminary research in mice and other animals indicates
that bone marrow stem cells, transplanted into a
damaged heart, can generate heart muscle cells and
successfully repopulate the heart tissue. Other recent
studies in cell culture systems indicate that it may be
possible to direct the differentiation of embryonic stem
cells or adult bone marrow cells into heart muscle cells (
Figure 4).
 In people who suffer from type I diabetes, the cells
of the pancreas that normally produce insulin are
destroyed by the patient's own immune system. New
studies indicate that it may be possible to direct the
differentiation of human embryonic stem cells in cell
culture to form insulin-producing cells that eventually
could be used in transplantation therapy for
diabetics.
Heart muscle repair with adult stem cells.
Also, to avoid the problem of immune rejection,
scientists are experimenting with different
research strategies to generate tissues that
will not be rejected.
Thank you