Thesis section: Restorative neurology

Restorative neurology
Essay
In Neuropsychiatry
Submitted for partial fulfillment of Master Degree

By
Samy moussa Seliem
M.B.B.CH

Supervisors of

Prof. Mohammed Yasser Metwally
Professor of Neuropsychiatry

Faculty of Medicine-Ain Shams University

www.yassermetwally.com

Prof. Naglaa Mohamed Elkhayat
Professor of Neuropsychiatry


Dr. Haitham Hamdy salem
Lecturer of Neuropsychiatry


Faculty of Medicine

Ain Shams University

2011

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Contents

Subject

1. Introduction and aim of the work
2. Stem cell
3. Stem cell therapy in Parkinson disease
4. Stem cell therapy in in stroke
5. Stem cell therapy in demyelinating disease
6. Stem cell therapy in in amyotrophic lateral sclerosis

7. Stem cell therapy in muscular dystrophy

8. Stem cell therapy Huntington chorea

9. Stem cell therapy for Alzheimer's Disease

10.Stem cell therapy in degenerative diseases in children

11.Stem cell therapy in retinal degeneration

12.Stem cell therapy in spinal cord injury

13.Stem cell therapy in peripheral nerve injury

Summary

References

i

Introduction
Stem cells are unspecialized cells in the human body that are
capable of becoming specialized cells, each with new
specialized cell functions. The best example of a stem cell is the
bone marrow stem cell that is unspecialized and able to
specialize into blood cells, such as white blood cells and red
blood cells, and these new cell types have special functions,
such as being able to produce antibodies, act as scavengers to
combat infection and transport gases. Thus one cell type stems
from the other and hence the term “stem cell.” Basically, a stem
cell remains uncommitted until it receives a signal to develop
into a specialized cell. Stem cells have the remarkable properties
of developing into a variety of cell types in the human body.
They serve as a repair system by being able to divide without
limit to replenish other cells. When stem cell divides, each new
cell has the potential to either remain as a stem cell or become
another cell type with new special functions, such as blood cells,
brain cells, etc. (Bongso and Lee, 2005).

Stem cells also known as progenitor cells which are cells
that have not undergone differentiation to acquire specific
structure or role. They have the potential to self-renew, divide
and differentiate into specialized cell types. They are also,
sometimes, termed ‘pluripotent’ or ‘undifferentiated’ cells

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because they can differentiate and develop into various cell lines
(Metwally, 2009).

Scientists and researchers are interested in stem cells for
several reasons. Although stem cells do not serve any one
function, many have the capacity to serve any function after
they are instructed to specialize. Every cell in the body, for
example, is derived from first few stem cells formed in the early
stages of embryological development. Therefore, stem cells
extracted from embryos can be induced to become any desired
cell type. This property makes stem cells powerful enough to
regenerate damaged tissue under the right conditions (Crosta,
2010).

Perhaps, the most important reason that stem cell
development is so appealing to neurologists can be found in the
statement “The adult human brain, in contrast to other organs
such as skin and liver, lacked the capacity for self repair and
regeneration” (Lin et al., 2007).

The types of stem cells include: Bone marrow-derived
mesenchymal stem cells (BMSCs), embryonic stem cells
(ESCs), Adult (somatic) stem cells, and neural stem cells
(NSCs). BMSCs also termed bone marrow stromal cells are
another example of a somatic stem cell being studied for its
therapeutic potential in the central nervous system (CNS) and in
other tissue (Abdallah and Kassem, 2008).

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BMSCS generate neurotransmitter-responsive cells with
electro-physiological properties similar to neurons (Diana and
Gabriel, 2008).

ESCs are pluripotent cells isolated from the inner cell mass
of day 5-8 blastocyte with indefinite self-renewal capabilities as
well as of the ability to differentiate into all cell types derived
from the three embryonic germ layers. The primary therapeutic
goal of ESCs research is cell replacement therapy (Aoki et al.,
2007).

Adult (somatic) stem cells: it has a capacity to differentiate
into tissue-specific types and represent a potential source of
autologus cells for transplantation therapy that eliminate
immunological complications associated with allogenic donor
cells as well as bypass ethical concern associated with ESCs,
All types are generally characterized by their potency, or
potential to differentiate into different cell types (such as skin,
muscle, bone, etc) (Lin et al., 2007).

Scientists discovered ways to obtain or derive stem cells
from early mouse embryos more than 20 years ago. Many years
of detailed study of biology of mouse stem cells led to the
discovery, in 1998, of how to isolate stem cells from human
embryos and grow the cells in the laboratory. These are called
human embryonic stem cells. The embryos used in these studies
were created for infertility purposes through in vitro fertilization

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procedures and when they were no longer needed for that
purpose, they were donated for research with the informed
consent of the donor (Ordrico et al., 2001).

The concept that the adult mammalian CNS contains NSCs
was first discovered from evidence of neuronal turnover in the
olfactory bulb and hippocampus in the adult organism cells with
more restricted neural differentiation capabilities committed to
specific subpopulation lineage, have been generated from
human ESCs or directly isolated from neurogenic regions of
fetal and adult CNS, such as the subventricular zone, which
provides neuroblasts to replenish inhibitory interneurons in the
olfactory bulb (Lin et al., 2007).

Stem cell differentiation must be turned on, given direction,
and turned off as needed in order to properly supply the basic
building blocks of tissues in different organ systems. This
requirement for precise regulation applies to an even greater
degree to the differentiation of neuronal progenitor cells,
because effective neural function depends on establishing
precise linkage and interactions between different individual
neurons and classes of neurons (Metwally, 2009).

Most tissue repair events in mammals are dedifferentiation
independent events brought about by the activation of pre-
existing stem cells or progenitor cells. By definition, a
progenitor cell lies in between a stem cell and a terminally
differentiated cell (Crosta, 2010).

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With the therapeutic application of NSCs for
neurorestoration in mind, a clearer picture is emerging. Both in
normal neurodevelopment and stem cell biology, the precursor
cells display preprogrammed behavior modified by cues from
the local environment. The fundamental assumption is that
differentiation and predictable behavior of NSCs can be
achieved if the appropriate cocktail of soluble/diffusible or
contact-mediated signals is present. In addition, several
corollary considerations are quickly evident. For example, can
we use NSCs from different sources in an equivalent fashion?
The answer to this important question requires that we
understand the developmental potential of all the types of NSCs
(Marquez et al., 2005).

Medical researchers believe that stem cell therapy has the
potential to dramatically change the treatment of human disease.
A number of adult stem cell therapies already exist, particularly
bone marrow transplants that are used to treat leukemia. In the
future, medical researchers anticipate being able to use
technologies derived from stem cell research to treat a wider
variety of diseases including cancer, Parkinson's disease, spinal
cord injuries, Amyotrophic lateral sclerosis, multiple sclerosis,
and muscle damage, amongst a number of other impairments
and conditions (Goldman and Windrem, 2006).

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Aim of the work:

The aim of this work is to study and summarize recent
progress in stem cell therapies aimed at neurodegenerative
disorder and illustrate how some of aforementioned methods
and strategies are being utilized to formulate clinically viable
treatments.

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Stem cells

Definition: A stem cell is a cell that has the ability to divide
(self replicate) for indefinite periods, often throughout the life of
the organism. Under the right conditions, or given the right
signals, stem cells can give rise (differentiate) to the many
different cell types that make up the organism. That is, stem
cells have the potential to develop into mature cells that have
characteristic shapes and specialized functions, such as heart
cells, skin cells, or nerve cells (Charron et al., 2009).
The word “stem” actually originated from old botanical
monographs from the same terminology as the stems of plants,
where stem cells were demonstrated in the apical root and shoot
meristems that were responsible for the regenerative
competence of plants. Hence also the use of word “stem” in
“meristem” (Kiessling and Anderson, 2003).

Historical overview of stem cell therapy:
The stem cell is the origin of life. As stated first by the great
pathologist (Rudolph Virchow), “All cells come from cells”.
The fertilized egg is formed from fusion of the haploid progeny
of germinal stem cells. The fertilized egg is totipotent; from it
forms all the tissues of the developing embryo. During
development of the embryo, germinal stem cells are formed,
which persist in adult to allow the cycle of life to continue. In

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the adult, tissue is renewed by proliferation of specialized stem
cells, which divide to form one cell that remains a stem cell and
another cell that begins the process of differentiation to
specialized function of a mature cell type, normal tissue renewal
is accomplished by the differentiating progeny of stem cells, the
so-called transit amplifying cells. For example, blood cells are
mature cells derived from hematopoietic stem cells in the bone
marrow; the lining cells of the gastrointestinal tract are formed
from transit amplifying cells, progeny of stem cell in the base of
intestinal glands (Crosta, 2010).
Nineteenth century pathologists first hypothesized the
presence of stem cells in the adult as “embryonal rests” to
explain the cellular origin of cancer and the studies indicate that
the most cancers arise from stem cells or their immediate
progeny, the transit-amplifying cells. Cancer results from an
imbalance between the rate at which cells are produced and the
rate at which they terminally differentiate or die. Understanding
how to control the proliferation and differentiation of stem cells
and their progeny is not only the key to controlling and treating
cancer, but also to cell replacement and gene therapy for many
metabolic, degenerative, and immunological diseases (Virchow,
1985).

Stem cell properties:

Stem cells have a capacity for self-renewal giving rise to
more stem cells, and the ability to differentiate into tissues of

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various lineages under appropriate conditions. They may be
totipotent, pluripotent or multipotent, depending on type. Only
the embryo is totipotent. Embryonic stem cells (ESCs) are
pluripotent, as they are capable of differentiating into many
tissue types, whereas differentiation of adult stem cells is
generally restricted to the tissue in which they reside, as with
hepatocytes in the liver, and haemopoietic stem cells in blood
(figure 1) (Bongso and Lee, 2005).

Figure (1): Stem cell self-renewal and differentiation (Bongso and
Lee, 2005).

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A) Stem cell self renewal:
The defining feature of a true stem cell is the capacity for
self-renewal. Self renewal occurs when a cell that has been
activated to divide does so asymmetrically. The result produces
one cell that is exactly like the mother cell and one cell that
takes on biological functions that are different from those of the
mother cell. Without self-renewal, each activation event would
result in the progressive loss of the originating stem cell
population (Andeson et al., 2001).

B) The stem cell life cycle:

Stem cell activation is generally followed by a clonal
expansion of the daughter cell that is produced. This is
associated with a series of biological processes that include
proliferation, migration, differentiation, and at some point cell
death. Regulation of these downstream events determines the
net effect that, each stem cell activation has on new tissue
formation (Song et al., 2007).

C) Stem cell plasticity:
The term plasticity means that a stem cell from one adult
tissue can generate the differentiated cell types of another tissue.
At this time, there is no formally accepted name for this
phenomenon in the scientific literature. It is variously referred
to as “plastisity” “unorthodox differentition” or
“transdifferentiation” (figure 2) (Joanna et al., 2009).

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To show that the adult stem cells can generate other cell
types requires them to be tracked in their new environment,
whether it is in vitro or in vivo. In general, this has been
accomplished by obtaining the stem cells from a mouse that has
been genetically engineered to express a molecular tag in all its
cells. It is then necessary to show that the labeled adult stem
cells have adopted key structural and biochemical
characteristics of the new tissue they are claimed to have
generated (Gussoni et al., 2002).
Also it is necessary to demonstrate that the cells can
integrate into their new tissue environment, survive in the tissue,
and function like the mature cells may assume the characteristic
of cells that have developed from the same primary germ layer
or a different germ layer, for example, much plasticity
experiments involve stem cells derived from bone marrow,
which is a mesodermal derivative. The bone marrow stem cells
may then differentiate into another mesodermally derived tissue
such as skeletal muscle, cardiac muscle or liver (Kocher et al.,
2001).
Stem cell lineage differentiation and commitment is
conventionally viewed as progressively
downstream, unidirectional and irreversible. The
notion of unidirectional tissue-lineage commitment of stem cells
is being challenged by evidence of plasticity, or lineage
conversion, in adult stem cells. Mechanisms allowing for
such plasticity include transdifferentiation
which describes the conversion of a cell of one
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tissue lineage into a cell of an entirely distinct lineage, with
concomitant loss of the tissue-specific markers and function of
the original cell type, and acquisition of markers and function of
the trans-differentiated cell type (Bianco et al., 2005).
Alternatively, adult stem cell may differentiate into a tissue
that, during normal embryonic development, would arise from a
different germ layer. For example, bone marrow derived cells
may differentiate into neural tissue, which is derived from
embryonic ectoderm and neural stem cell lines cultured from
adult brain tissue may differentiate to form hematopoietic cells,

Figure (2): Evidence of plasticity of stem cell (Joanna et al., 2009).

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or even give rise to many different cell types in embryo. In
both cases cited above, the cells would be deemed to show
plasticity, but in the case of bone-marrow stem cells generating
brain cells, the finding is less predictable (Song et al., 2007).
Alternative mechanisms for explaining apparent stem cell
plasticity involve cell-cell fusion between a stem cell and a
tissue specific cell, the existence of multiple stem cell
populations in one pool of cells, and the ability of the stem cells
to differentiate to a more primitive, less specialized cell lineage,
and then re-differentiate down another lineage (Bongso and
Lee, 2005).
The differentiation potential of stem cells:
Many of the terms used to define stem cells depend on the
behavior of the cells in the intact organism (in vivo), under
specific laboratory conditions (in vitro), or after transplantation
in vivo, often to a tissue that is different from the one from
which the stem cells were derived (Joanna et al., 2009).
So they are three classes of stem cells exist: totipotent,
pluripotent multipotent and unipotent.
1) Totipotent:
Totipotency is the ability of a cell to divide and produce all
of the undifferentiated cells within an organism, from the Latin
word totus, meaning entire; For example, the fertilized egg is
said to be totipotent, because it has the potential to generate all
the cells and tissues that make up an embryo and that support its
development in uterus. After fertilization, the cell begins to

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divide and produce other totipotent cells; these totipotent cells
begin to specialize within a few days after fertilization. The
totipotent cells specialize into pluripotent cells, which they
develop into the tissues of the developing body. Pluripotent
cells can further divide and specialize into multipotent cells,
which produce cells of a particular function (Svendsen and
Ebert, 2008).
Adult mammals, including humans, consist of more than 200
kinds of cells. These include nerve cells (neurons), muscle cells
(myocytes), skin (epithelial) cells, blood cells (erythrocytes,
monocytes, lymphocytes, etc.), bone cells (osteocytes) and
cartilage cells (chondrocytes). Other cells, which are essential
for embryonic development but are not incorporated into the
body of the embryo, include the extraembryonic tissues,
placenta, and umbilical cord. All of these cells are generated
from a single, totipotent cell, the zygote or fertilized egg
(Joanna et al., 2009).

2) Pluripotent:
Pluripotent stem cells can give rise to any type of cell in the
body except those needed to develop a fetus or adult because
they lack the potential to support the extraembryonic tissue
(e.g., the placenta). Most scientist use the term pluripotent to
describe stem cells that can give rise to cells derived from all
three embryonic germ layers (endoderm, mesoderm, and

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ectoderm). These three germ layers are the embryonic source of
all cells of the body (figure 3) (Svendsen and Ebert, 2008).

Figure (3): Pluripotent stem cells (Svendsen and Ebert, 2008).

The term “pluri” is derived from the Latin word plures,
means several or many. Thus, pluripotent cells have the
potential to give rise to any type of cell, a property observed in
the natural course of embryonic development and under certain
laboratory conditions. Pluripotent stem cells are isolated from
embryos that are only several days old; cells from these stem

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cell lines can be cultured in the lab and grown without limit
(Sonja et al., 2006).
3) Multipotent:
Multipotent cells, in contrast, can only give rise to a small
number of cell types and they can produce only cells of a
closely related family cell. As haematopiotic stem cells that
differentiate to red blood cells, white blood cells and platelets.
A hematopoietic cell, or a blood stem cell, can develop into
several types of blood cells but cannot develop into liver cells or
other types of cells; the differentiation of the cell is limited in
scope. A multipotent blood cell can produce red and white
blood cells (figure 4) (Svendsen and Ebert, 2008).

Figure (4): Multipotent stem cell (Svendsen and Ebert, 2008).

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4) Unipotent:
Unipotent stem cells, a term that is usually applied to a cell
in adult organisms, means that the cells in question are capable
of differentiating along only one lineage. The term “uni” is
derived from the Latin word unus, which means one. Also, it
may be that the adult stem cells in many differentiated,
undamaged tissues are typically unipotent and give rise to just
one cell type under normal conditions. This process would
allow for a steady state of self renewal for the tissue. However,
if the tissue becomes damaged and the replacement of multiple
cell types is required, pluripotent stem cells may become
activated to repair the damage (Avasthe et al., 2008).

F igu re (5): Differentiation of human stem cells (Bongso and Lee,
2005).

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Classification of stem cells according to their sources:
Stem cells can be classified into four broad types based on
their origin, stem cells from embryos; stem cells from the fetus;
stem cells from umbilical cord; and stem cells from the adult.
Each of these can be grouped into subtypes (Andeson et al.,
2001).

1) Embryonic stem cells:
In mammals; the fertilized oocyte, zygote, 2-cells, 4-cells, 8-
cells and morula resulting from cleavage of the early embryo
are examples of totipotent cells (ability to form a complete
organism) (figure 6) (Avasthe et al., 2008).

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Figure (6): Development and differentiation of human tissues (Avasthe
et al., 2008).

The inner cell mass (ICM) of the 5 to 6 days old human
blastocyte is the source of pluripotent embryonic stem cells
(HESCs) and consisting of 50–150 cells (figure 7) (Bongso and
Lee, 2005).

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Figure (7): Human blastocyst
showing inner cell mass and
trophectoderm (Bongso and Lee,
2005).

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Figure (8): How human embryonic stem cells are derived? (Bongso
and Lee, 2005).

Characteristics of human embryonic stem cells:
They can maintain undifferentiated phenotype and these
cells are able to renew themselves continuously through many
passages leading to the claim that they are immortal, also these

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cells are pluripotent, meaning that they are able to create all
three germ layers of the developing embryo and thus they can
develop into each of the more than 200 cell types of the adult
body (figure 9) (Junying et al., 2006).

Figure (9): Characteristics of embryonic stem cells (Junying et al.,
2006).

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Nearly all research to date has taken place using mouse
embryonic stem cells (MES) or Human embryonic stem cells
(HESCs). Both have the essential stem cell characteristics, yet
they require very different environments in order to maintain an
undifferentiated state. Mouse ES cells are grown on a layer of
gelatin and require the presence of Leukemia Inhibitory Factor
(LIF) (Bongso and Lee, 2005).
HESCs are grown on a feeder layer of mouse embryonic
fibroblasts (MEFs) and require the presence of basic Fibroblast
Growth Factor (bFGF or FGF-2). Without optimal culture
conditions or genetic manipulation, embryonic stem cells will
rapidly differentiate (Avasthe et al., 2008).

Identification of the human embryonic stem cells:
Laboratories that grow human embryonic stem cell lines use
several kinds of tests to identify the human embryonic stem
cells.
These tests include:
1- Growing and sub-culturing 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 (Lawrence et al., 2006).
2- 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

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protein called oct-4, which undifferentiated cells
typically make. Oct-4 is a transcription factor, meaning
that it helps turn genes on and off at the right time, which
is an important part of the processes of cell
differentiation and embryonic development.
3- 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.
It does not detect genetic mutations in the cells.
4- Determining whether the cells can be subculture after
freezing, thawing, replanting (junying et al., 2006).
Differentiation of human embryonic stem cells:
In order to start differentiation, the HESCs must be removed
from the feeder layer and the cell replated and will form
embryoid bodies (Ebs), spherical aggregates in which the
HESCs undergo mixed spontaneous differentiation toward
lineages of all three dermal layers. Another protocol of
differentiation directly without formation of embryoid bodies
stage have resulted in more controlled differentiation and better
yield of the required cells (figure 10) (Joanna et al., 2009).

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Figure (10): Fluorescent markers can be used to identify stem cells hidden
among ordinary adult cells. Here, human embryonic stem cells are
recognized by the marker proteins they express (green) (Joanna et al.,
2009).

Ethical considerations:
The promise of stem cell therapy has ignited public dispute
on the ethics of using aborted embryos for medical purposes.
Individual attitudes are usually influenced by religious and
liberal views but also by concerns that the practice of embryonic
tissue transplantation will increase the pressure to perform
abortions and create a black market in which pregnancy and
aborted tissues will be sold to the highest bidder. The regulated
banking of stem cell lines may solve some of the ethical issues.
As in other cases in which medical and scientific advances
found society without the means to deal with their ethical, legal,
and social consequences, it is important to discuss these issues
in public, with the active participation of the medical and
scientific community (Christopher, 2008).

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2) Fetal stem cells:
The identification of human fetal stem cells has raised the
possibility of using autologus cells for in utero treatments. The
human fetal stem cells population extracted from fetal blood
contains adherent cells that divide in culture for 20 to 40
passages and can differentiate into mesenchymal lineages
including bone and cartilage, but also have the ability to form
oligodendrocytes and hematopoiotic cells. These cells, which
can be found circulating only during the first trimester, are
similar to hematopoiotic populations in fetal liver and bone
marrow (Avasthe et al., 2008).

3) Umbilical cord stem cells:
These are cells harvested from the cord blood. Cord blood is
rich in the stem cells and after appropriate human leukocyte
antigen [HLA] matching may be used to treat a variety of
conditions. Characteristics of these cells are identical to adult
stem cells except that they are not derived from adults and that
their concentration is far more in umbilical blood as compared
to adults. The use of umbilical cord stem cells in orthopedics is
still in a nascent stage and most studies currently focus on the
use of the stem cell (Crosta, 2010).

4) Adult stem cell:
It is an undifferentiated cell that is found in a differentiated
tissue, it can renew itself and become specialized to yield all the

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specialized cell types of the tissue from which it originated.
Adult stem cells, like all stem cells, share at least two
characteristics. First, they can make identical copies of
themselves for long period of time; this ability to proliferate is
referred to as long term self renewal. Second, they can give rise
to mature cell types that have characteristic morphologies
(shapes) and specialized functions (Charron et al., 2009).
Typically, stem cells generate an intermediate cell type or
types before they achieve their fully differentiated state. The
intermediate cell is called a precursor cells in fetal or adult
tissues are partially differentiated cells that divide and give rise
to differentiated cells. Such cells are usually regarded as
“committed” to differentiating along a particular cellular
development pathway, although this characteristic may not be as
definitive as once thought (Bianco et al., 2005).

Adult stem cells are rare. Their primary functions are to
maintain the steady state functioning of a cell, called
(homeostasis) and with limitation to replace cells that die due to
injury or disease. For example, only an estimated 1 in 10,000 to
15,000 cells in the bone marrow is a hematopoietic (blood-
forming) stem cell (HSC). Furthermore, adult stem cells are
dispersed in tissues throughout the nature of animal and behave
very differently, depending on their local environment. For
example, HSCs are constantly being generated in the bone
marrow where they differentiate into mature types of blood

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cells. Indeed, the primary role of HSCs is to replace blood cells
(Abdallah and Kassem 2008).
Unlike embryonic stem cells, which are defined by their
origin (the inner cell mass of the blastocyte), adult stem cells
share no such definitive means of characterization. In fact, no
one knows the origin of adult stem cells in any mature tissue.
Some have proposed that stem cells are somehow set aside
during fetal development and restrained from differentiating.
Definition of adult stem cells vary in the scientific literature
range from a simple description of the cells to a rigorous set of
experimental criteria that must be met before characterizing a
particular cell as an adult stem cell. Most of the information
about adult stem cells comes from studies of mice. The list of
adult tissues reported to contain stem cells is growing and
includes bone marrow, peripheral blood, brain, spinal cord,
dental pulp, blood vessels, skeletal muscle, epithelia of skin and
digestive system, cornea, retina, liver, and pancreas
(Christopher, 2008).
Ideally, adult stem cells should also be clonogenic. In other
words, a single adult stem cell should be able to generate a line
of genetically identical cells, which then gives rise to all the
appropriate, differentiated cell types of the tissue in which it
resides. Again, this property is difficult to demonstrate in vivo;
in practice, scientists show either that a stem cell is clonogenic
in vitro, or that a purified population of candidate stem cells can
repopulate the tissue (Avasthe et al., 2008).

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Sources of adult stem cells:
(I) Bone Marrow-Derived Stem/Progenitor Cells:
Adult bone marrow-derived stem cells are presently the cell
types most widely used in stem cell therapy. A heterogeneous
subset there of, termed autologous bone marrow-derived
mononuclear cells (ABMMNCs), comprises the following types
of stem cells, (Mesenchymal stem cells, Hematopoietic stem
cells and Endothelial progenitor cells), that have potential
therapeutic uses (figure 11) (Svendsen and Ebert, 2008).

Figure (11): Some of the known sources of adult stem cells (Svendsen and
Ebert, 2008).

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(a) Mesenchymal stem cells (MSCs):
MSCs are a proper stem cell which can be greatly and
efficiently expanded in culture and can differentiate to several
specific mesenchymal cell lineages. Mesenchymal (Stromal)
stem cells (MSCs) are found in various niches of adult tissue.
MSCs are rare in bone marrow (<0.01% of nucleated cells, by
some estimates) and 10 times less abundant than hematopoietic
progenitor cells but MSCs can be readily grown in culture.
However, more recently, other sources of MSCs have been
described including placenta, adipose tissue, cord blood and
liver (junying et al., 2006).
The human Mesenchymal stem cells (HMSCs) from bone
marrow can be cloned and expanded in vitro more than 1
million-fold and retain the ability to differentiate to several
mesenchymal lineages. Researchers have not yet found
conditions that allow continuous, indefinite HMSC growth, yet
it is possible to produce billions of MSCs in vitro for cellular
therapy from a modest bone marrow aspirate drawn through the
skin. MSCs need to be expanded ex vivo because they
apparently are very contact inhibited, and there is little evidence
of in vivo expansion as MSCs labeled with membrane dyes, that
would be diluted and undetected from dividing cells after about
3 divisions, are found months later even in repairing tissue
(Sottile et al., 2002).

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Advantages of Mesenchymal Stem Cells:
Ease of isolation, high expansion potential, genetic stability,
reproducible characteristics in widely dispersed laboratories,
compatibility with tissue engineering principles and potential to
enhance repair in many vital tissues. There they may be the
current preferred stem cells model for cellular therapeutic
development (Diana and Gabriel, 2008).
Biology of mesenchymal stem cells (MSCs):
The anatomical locations of phenotype of MSCs have no yet
been well defined in vivo. Some have used expression of Stro-1
and VCAM-1 to analyses putative MSC in vivo in human. A
general consensus among researchers in the field is that MSC
can be successfully defined based on staining with surface
markers such as CD44, CD90, CD73, CD105 and CD166.
However, none of these antigens are unique to MSC. Using
markers such as Stro-1 (human) and Sca-1 (mouse), several
reports indicate that MSC reside adjacent to endothelium in the
bone marrow and possibly other tissues (Zannettino et al.,
2007).
(b) Hematopoietic stem cells (HSCs):
HSCs are presented in umbilical cord blood with a frequency
of just under one in 1 million mononuclear cells (one in 3
million MNCs) or mobilized peripheral blood (one in 6 million
MNCs). They are capable of unlimited cell proliferation in bone
marrow and must undergo at least 20 to 23 divisions on their

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way to produce mature blood cells, even assuming no cell death
along the way (Emerson et al., 2008).
Biology of heamtopiotic stem cells:
Much effort has been focused on discovering cell surface
markers that can identify those cells that have true functional
stem cell properties. Perhaps clinically most familiar is CD34, a
glycoprotein present on the cell surface of stem and progenitor
cells which is used to enrich stem cells mobilization and
collection for HSCs, but even within the CD34+ population,
only a small percentage are HSCs (Emerson et al., 2008).
For decade scientists and hematologists have struggled with
the difficulty that HSCs cannot be purified based on
phenotypical characteristics and perhaps more importantly,
cannot be expanded and cloned ex vivo. Recent evidence has
emerged suggesting that HSCs can be expanded ex vivo. But
there is still no evidence to support the idea of clonality. For
these reasons HSCs are not ideally suited for in vitro
experiments designed to test plasticity. In this regard HSCs
differ dramatically from MSCs in bone marrow and neural stem
cells (NSCs) in the central nervous system, both of which can
be clonally derived and tested for multiple differentiation
pathways (figure 12) (Joanna et., 2009).

26

Figure (12): Hematopoietic and stromal stem cell differentiation (Joanna et al., 2009).
(c) Endothelial progenitor cells (EPCs):
A subset of bone marrow-derived hematopoietic progenitor
cells: endothelial progenitor cells (EPCs). These cells can give
rise to endothelial recovery and new capillary formation after
ischemia (Einstein and Ben-Hur, 2008).
(II) Neural stem cells:
The concept that adult mammalian CNS contains NSCs was
first inferred from evidence of neuronal turnover in olfactory

27

bulb and hippocampus in the adult organism. The multipotency
of NSCs was demonstrated in vitro in 1990 by their ability to
differentiate into neurons, astrocytes, and oligodendrocytes as
well as various forms of neural precursors. In addition, in vivo
delivery of these cells to animal models of neurodegenerative
diseases was associated with varying degrees of functional
recovery. Currently, there is no set of markers or protein
expression profiles that precisely define and fully characterize
undifferentiated NSCs. Neural stem cells (NSCs) and neural
precursor cells (NPCs) can be isolated from the developing or
adult CNS and can be safely expanded in chemically defined
culture media for an extended (Song et al., 2007).
(a) Adult neural precursor cells (NPCs):
New neurons are derived in adulthood from a population of
adult NPCs, which are primarily found in the subependymal
layer of the ventricular zone and the dentate gyrus of the
hippocampus, although they are also probably found in other
sites. However, the behavior of the neural precursor cells
(NPCs) found in all these sites is different, and may relate as
much to the environment in which they find themselves as to
their intrinsic properties, eg; nigral NPCs appear to only
differentiate into astrocytes in situ or when grafted to the adult
nigra, but when they are cultured in vitro or transplanted into the
hippocampus they can form neurons (Gronthos et al., 2003).

28

Properties of neural stem cells:
(1) Immunosupressive effect of NSCs:
Although NSCs may exert their therapeutic effects by
directly replacing missing cells, transplantation rarely results in
significant numbers of transplant-derived terminally
differentiated neurons. The beneficial effect of NSCs in disease
models may be attributable to alternative biologic properties.
The first indication of an anti-inflammatory effect of NPCs
came from transplantation experiments in rats with experimental
autoimmune encephalomyelitis (EAE). It was shown
transplantation of NPCs reduced brain inflammation and clinical
disease severity, it was suggested that the benefit of NPC
transplantation was mediated by an anti-inflammatory effect
(Raisman and Li, 2007).
The exact mechanisms by which transplanted NPCs
attenuate brain inflammation are unclear. Some suggests an
immunomodulatory effect by which NPCs promote apoptosis of
type 1 T-helper cells, shifting the inflammatory process in the
brain toward a more favorable climate of dominant type 2 T-
helper cells. Alternatively, a nonspecific bystander
immunosuppressive effect of NPCs on T-cell activation and
proliferation has been suggested. The suppressive effect of
NPCs on T cells was accompanied by a significant suppression
of pro-inflammatory cytokines. This nonspecific anti-
inflammatory mechanism may be of major importance in the
application of transplantation therapy in immune-mediated

29

diseases because it can protect the host CNS and graft from
additional immune attacks (Einstein and Ben-Hur, 2008).
(2) Neuroprotictive effects of transplanted NSCs:
Neuroprotective effect was observed in other non
autoimmune experimental disease models. Neural stem cells
rescued dopaminergic neurons of the mesostriatal system in a
Parkinson disease (PD) model in rodents. These findings led to
the concept that NSCs are endowed with inherent mechanisms
for rescuing dysfunctional neurons. This effect was found to be
important in other neurologic diseases. Neural stem cells seeded
on a synthetic biodegradable scaffold and grafted into the hemi-
sectioned adult rat spinal cord induced significant improvement
in animal movement by reduction of necrosis in the surrounding
parenchyma and by prevention of inflammation, glial scar
formation, and extensive secondary cell loss (Einstein and Ben-
Hur, 2008).
(3) Neurotrophic effects of transplanted NSCs:
After sectioning of the adult spinal cord, NSC
transplantation induced a permissive environment for axonal
regeneration. Similarly, in a model of retinal degeneration, NPC
transplantation promoted neural growth in the optic nerve. In
both cases, this effect was mediated by induction of matrix
metalloproteinases that degrade the impeding extracellular
matrix and cell surface molecules, enabling axons to extend
through the glial scar. Transplantation of olfactory-ensheathing
cells into the sectioned spinal cord also promoted axonal

30

regeneration in long fiber tracts, with a return of lost function.
This was explained by the creation of proper realignment,
enabling axonal growth through a permissive tract. In addition,
the cells increased axonal sprouting, remyelination, and
vascularization of the injured spinal cord (Raisman and Li,
2007).
Isolation of human NSCs:
To date, they are primary isolated and propagated in vitro as
cells that form free-floating neurospheres when cultured in
serum-free medium on non adherent surfaces in the presence of
mitogenic factors such as basic FGF or FGF-2 and epidermal
growth factors, although there have also been reports of
monolayer cultures (McBride et al., 2004).
(III) Pancreatic stem/progenitor cells:
There is strong evidence that new pancreatic islets can
derive from progenitor cells present within the ducts and islets,
in a process called “neogenesis”. Furthermore, when these
pseudo-islets were transplanted into non-obese diabetic (NOD)
mice, diabetes reversal was observed. Candidate pancreatic
stem/progenitor cells have also been described within acini, but
contamination with endocrine and ductal cells in cultures could
not be excluded in these experiments (Limmbert et al., 2008).
The isolation of a distinct stem/progenitor cell within the
endocrine pancreas depends on the identification of a specific
progenitor marker. The exciting observation that nestin positive
islets cells display endocrine differentiating capacity led to the

31

hypothesis that this intracytoplasmic filament protein might
correspond to a pancreatic stem/progenitor cell marker. More
recently, in two important studies a population of cells in the
developing and adult mouse pancreas was identified, which
under differentiation conditions, released insulin in a glucose-
dependant manner. After differentiation, these cells expressed
specific developmental pancreatic endocrine genes (e.g. Ngn3,
Pax-4, Pax-6 and PDX-1) and contamination with mature beta
cells was ruled out (Limbert et al., 2008).
While mature beta cell replication appears to be major
physiological beta-cell regenerative process, identification of
pancreatic cells with progenitor features might open an
important and promising strategy for cell replacement and
regeneration therapy. Anyhow, to be clinically relevant, in vitro
proliferation of progenitor cells from human pancreas must
produce large amounts of cells, in order to allow cells isolated
from one single donor to be sufficient to treat a given diabetic
patient. It would be even better to have one single donor for
several diabetics. For these reasons, acinar isolated
stem/progenitor cells might be of interest, considering that
exocrine tissue constitutes 90% of pancreatic tissue and is
discarded during islet isolation (Kushner et al., 2005).
(IV) Other sites:
First identified in human bone marrow, a population of
mesenchymal progenitor/stem cells (MSC) with well
characterized immunophenotype and distinct from

32

hematopoietic stem cells, was shown to possess a high
proliferation rate and great plasticity. Under specific culture
conditions these cells differentiate into mesenchymal tissues,
such as bone, cartilage, muscle, tendon, adipose and stroma, as
well as neuronectodermal tissues (Limbert et al., 2008).
Adult tissues and organs known to have stem cells
Source Description
Brian Stem cells of the brain can differentiate into the three kinds of
nervous tissue-astrocytes, oligodendrocytes, and neurons-and in
some cases, blood-cell precursor.

Bone marrow These occur as hematopoietic stem cells, which give rise to all blood
cells, and as stroma cells, which differentiate into cartilage and bone.

Endothelium These stem cells are called hemangioblasts and are known to
differentiate into blood vessels and cardiomyocytes. They may
originate in bone marrow, but this is uncertain.

Skeletal muscle These stem cells may be isolated from muscle or bone marrow. They
mediate muscle growth and may proliferate in response to injury or
exercise.

Skin Stem cells of the skin are associated with the epithelial cells, hair
follicle cells, and the basal layer of the epidermis. These stem cells
are involved in repair and replacement of all types of skin cells.

Digestive Located in intestinal crypts, or invaginations. These stem cells are
system responsible for renewing the epithelial lining of the gut.

Many types are believed to exist, but examples have yet to be
Pancreas isolated. Some neural cells are known to generate pancreatic β cells.

The identity of liver stem cells is still unclear. Stem cells from bone
Liver marrow may repair some liver damage, but most repairs seems to be
carried out by the hepatocytes (liver cells) themselves.
Table (1): Sources of adult stem cells (Limbert et al., 2008).

33

Identification of the adult stem cells:
The scientists often use one or more of the following three
methods to identify and test adult stem cells:
1- Labeling the cells in a living tissue with molecular markers
and then determining the specialized cell types they
generate. Then
2- Removing the cells from living animals, labeling them in
cell culture, and transplanting them back into another animal
to determine whether the cells repopulate their tissue of
origin. Then
3- 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 (Raisman and Li, 2007).
The similarities and differences between embryonic and adult
stem cells:
The adult and embryonic stem cells differ in the number and
types of differentiated cells types they can become. Embryonic
stem cells can become all cell types of the body because are
pluripotent. Adult stem cells are generally limited to
differentiating into different 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 (figure 13). 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

34

their numbers in cell culture have not yet been worked out. This
is an important distinction, as a large number of cells are needed
for stem cell replacement therapies (Limbert et al., 2008).
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 patient’s own
adult stem cells would means that the cell would not be rejected
by the immune system. This represents a significant advantage
as immune rejection is a difficult problem that can only be
circumvented with immunosuppressive drugs. Embryonic stem
cells from a donor introduced into a patient could cause
transplant rejection, however, whether the recipient would reject
donor embryonic stem cells has not been determined in human
experiments (Sonja et al., 2006).

Figure (13): Sources of stem cells (Limbert et al., 2008).

35

Types of Stem Cell transplantation:
Stem cell transplantation can be classified according to the
genetic relation between the donor and recipient into 4 classes:
1- Autograft: In which the donor and recipient is the same
individual.
2- Isograft or syngenic graft: In which the donor and
recipient are genetically identical (e.g., monozygotic
twins).
3- Allograft or homograft: In which the donor and recipient
are genetically unrelated but belong to the same species.
4- Xenograft or heterograft: In which the donor and
recipient belong to different species (David, 2009).
Application of stem cells:
1) Basic science application:
Stem cells are ideally suited to allow for the study of complex
processes that direct early unspecialized cells to differentiate
and develop into the more than two hundred cell types in the
human body (Bianco et al., 2005).
2) Medical research applications:
Stem cell studies may allow researchers to follow the processes
by which diseases and impairments caused by genetic
abnormalities first manifest themselves biochemical or
structurally in cells and tissues. Using stem cells to produce
large numbers of genetically uniform cultures of organ tissues
for example, liver, muscle, or neural would allow controlled
comparison of the effects of drugs or chemical on these tissues.

36

Alternatively, testing drugs against stem cell tissues varying
genetic makeup could allow tissue specific stem cell may
provide a constant in vitro source of such cellular material
(Bianco et al., 2005).
The site of stem cell implantation:
The transplantation can be described as orthotropic or
heterotropic:
1- Neurologic transplantation: Refers to donor tissue
implantation in the anatomically correct position in the
recipient.
2- Heterotropic transplantation: Refers to the relocation of
the implant in the recipient at a site different from the
normal anatomy (David, 2009).
Route of stem cell delivery:
Reports have indicated that after stereotactic
intraparenchymal, intracerebro-ventricular, intravenous and
intraarterial transplantation, stem cells can home to sites of
injury in the CNS and induce functional recovery. Of these
various transplantation techniques, those that depend on
intravascular delivery of stem cells for stroke are particularly
attractive.
Intravascular delivery:
In addition to its minimal invasive nature, intravascular
delivery may allow stem cells to have a superior interaction
with injured tissue. A comparative study revealed that direct
intracerebral transplantations resulted in the largest number of

37

cells at the lesion site, followed by intracerebro-ventricular and
intravenous transplantations (Guzman et al., 2008).
However, researchers in that study only assessed the
absolute number of cells in the perilesional area and took no
account of whether these cells were therapeutically distributing
to all injured areas of brain parenchyma on a microscopic level.
Many believe that intravascular delivery of stem cells may lead
to a wider distribution of cells around the lesion as compared
with focal perilesional transplants, thereby leading to superior
stem cell–injured tissue interactions (Xiao et al., 2007).
Mechanism of wide distribution:
The cells travel in the blood stream and follow a chemo
attractant gradient generated by inflammation in the injured
brain. Unfortunately, intravenously delivered cells pass through
the systemic and pulmonary circulation systems and home to
other organs as well, which significantly reduces cell homing to
the injured brain. Intravenous injection of human MSCs into
rats 24 hours after stroke showed that only 4% of the cells
entered the brain, the number of cells entering the brain
increased over time and peaked at Day 21 post-stroke. At Day
56, 60% of these surviving cells differentiated into glia, and
20% into neurons. Despite the fact that the number of cells
entering the brain was limited, functional recovery was
enhanced by intravenous delivery (Pluchino et al., 2005).

38

Intracarotid injection:
Another route of intravascular delivery is intra arterial,
which would circumvent body circulation. The first pass of stem
cells injected into the carotid artery would be the brain, this
route of delivery have demonstrated functional recovery after
stroke and traumatic brain injury. In 2006 Shen and colleagues
injected donor rat BMSCs into the internal carotid artery of rats
24 hours post-stroke and successfully induced functional
recovery. In another study, the same group injected donor rat
BMSCs into rats 24 hours after stroke and observed that
injected cells localized around the infarction area in the brain
and very few were found in the heart, lungs, liver, spleen, and
kidney (figure 14) ( Guzman et al., 2008).

Figure (14): Confocal laser scanning microscopy images revealing
numerous cells in the stroke border zone and the hippocampus ipsilateral to
the stroke (A). Inset shows doublecortin bromodeoxyuridine labeled cells.
The VCAM-1 (arrows) is highly expressed in the stroke affected
hemisphere 48 hours after stroke (B). DCX = doublecortin; BrdU =
bromodeoxyuridine; DAPI = 4'6-diamidino-2-phenylindole (Guzman et al.,
2008).

39

The debate over the best delivery route is further
complicated by the fact that there is still a great deal of
controversy concerning the mechanism by which stem cells lead
to enhanced functional recovery in patients who have
experienced stroke. The 2 most discussed mechanisms are as
follows: 1) cellular replacement, by way of the functional
integration of stem cells; and 2) secretion of neurotrophic and
angiogenic factors. If the mechanism of recovery is cellular
replacement, then transendothelial migration is necessary and
the methods that allow the highest concentrations of stem cells
in the injured brain areas ought to be pursued; however, there is
significant evidence that stem cells may provide their benefits
by secreting various neuroprotective factors (Guzman et al.,
2008).
In summary, the best route of human stem cell delivery has
not been determined, but the intravascular route is particularly
attractive because of its ease of administration, minimal
invasiveness, and potential for widespread cell distribution
together with widespread secretion of neuroprotective,
proangiogenic, and immunomodulatory factors. Intuitively, the
intraarterial route of delivery seems better than the intravenous,
given that injected cells first pass the target organ that is, the
brain prior to being redistributed in the systemic circulation
(Pluchino et al., 2005).

40

Timing of transplantation
Undoubtedly the fate and function of transplanted cells will
depend on any or all of these alterations, and the optimal time of
transplantation is unknown. The timing of transplantation
depends mainly on the goal of treatment, for example,
neuroprotection, which should happen early after the insult, or
neuroregeneration/cell replacement, which can be done once a
lesion has stabilized. We can envision a future in which we will
rely on multimodal stem cell treatment, depending on a
combination of early and late administrations of different cell
types (Guzman et al., 2008).
Early intravascular cell delivery
In animal models with a neuro inflammatory component
such as stroke, traumatic brain injury, spinal cord injury, and
multiple sclerosis, therapeutic somatic stem cells (for example,
BMSCs, umbilical cord blood stem cells, MSCs, and NPCs)
target inflamed CNS areas where they persist for months and
promote recovery through neuroprotective mechanisms. It is
thought that the process of transendothelial migration of somatic
stem cells may be regulated in a manner similar to that of
inflammatory cells. As early as 30 minutes after stroke, the
infiltration of leukocytes, both polymorphonuclear leukocytes
and monocytes/macrophages, can be observed (Goldman and
Windrem, 2006).
Chemoattraction, adhesion, and transendothelial migration
of inflammatory cells is regulated by specific inflammatory

41

mediators, which have been identified in experimental and
human stroke. The temporal expression profile of adhesion
molecules, cytokines, and chemokines after stroke has been well
described. Vascular cell adhesion molecule–1 (VCAM-1) has
been shown to reach a peak level 24 hours after experimental
stroke. At the bedside, soluble VCAM-1 concentration in
plasma is increased in patients with acute stroke. Intercellular
adhesion molecule–1 levels have been elevated as early as 4
hours after stroke with sustained levels for up to 1 week (Zhang
and Lodish, 2005).
Monocyte chemo-attractant protein–1, a key chemoattractant
factor for the recruitment of circulating peripheral cells to the
stroke area and an important factor for stem cell migration, is
upregulated 3 days after stroke and then returns to baseline after
1 week. Similarly, stromal-derived factor–1(SDF-1) is known to
be a potent chemoattractant for inflammatory as well as stem
cells (including BMSCs and NSCs) and is expressed early after
stroke. Anatomically, adhesion molecule upregulation as well as
chemokine expression has been shown to be highest in the
stroke-affected penumbral region. Blocking the different
pathways of chemoattraction and cell adhesion in stroke-
affected rodents reduced the number of infiltrating
inflammatory cells (Belmadani et al., 2006).
In mice lacking intercellular adhesion molecule–1 (ICAM-
1), a significant reduction in inflammatory cellular infiltrate and
a reduction in lesion size were noted. Treatment with anti–

42

ICAM-1 antibodies was a successful neuroprotective means of
reducing lesion size and apoptosis in experimental stroke.
However, a clinical trial exploring the feasibility of using an
ICAM-1 blocking antibody failed to demonstrate any beneficial
effects in the patients. There is some evidence that
intravascularly administered stem cells undergo the same
process as inflammatory cells, including chemoattraction,
adhesion, and transendo-thelial migration after stroke,
potentially making this route an ideal way of cell delivery (Hill
et al., 2004).
Late intraparenchymal cell transplantation
In contrast to the acute intravascular cell treatment, the
intraparenchymal approach has been hindered by poor outcomes
if the stem cells are transplanted too early after stroke.
Excitotoxicity, oxidative stress, and inflammation post ischemia
make the ischemic brain a hostile environment for
intracerebrally transplanted cells. In fact, we have found a
negative correlation between graft survival and inflammation.
Additionally, we demonstrated that human NSCs transplanted
too close or into the stroke area have very limited survival at
days after stroke (Belmadani et al., 2006).
Transplanting cells 3 weeks after stroke, when there is a
significant decrease in inflammation, led to greater graft
survival than transplanting 5–7 days after stroke. Taken
together, early intravascular cell therapy might benefit from the
processes tied to post-stroke inflammation but might be

43

detrimental to cells directly transplanted intra-parenchymally.
Therefore, intra-parenchymal cell replacement therapy might be
useful as a second line or delayed stem cell treatment strategy
(Grabowski, 2010).

44

Stem cell therapy in Parkinsonism

Parkinson’s disease (PD) otherwise known as ‘’paralysis
agitans’’ or ‘’shaking palsy’’ was classically described by James
Parkinson in 1817. His description of “Involuntary tremulous
motion with lessened muscular power, in parts not in action and
even when supported, with a propensity to bend the trunk
forward and to pass from a walking to a running pace, the
senses and intellect being uninjured” has stood the test of time.
PD is also defined as a debilitating neurodegenerative disorder
of insidious onset in middle or late age characterized by the
selective loss of nigrostriatal dopaminergic neurons and loss of
dopamine in the striatum (Abayomi, 2002).
Parkinson’s disease is second only to Alzheimer’s disease
with a prevalence of 1 in 10,000. Although it is uncommon in
people under age 40 years, the incidence of PD greatly increases
with age, affecting approximately 1% of individuals older than
60 years (Lane et al., 2008).
Pathology:
The basic pathology is cell degeneration and loss of
pigmented neurons in the pars compacta of the substantia nigra
and locus ceruleus with atrophy and glial scarring. The
degenerated pigmented neurons contain Lewy bodies which are
intracytoplasmic eosinophilic hyaline inclusions composed of
protein filaments (ubiquitin & synuclein), and do not have the
electronic microscropic appearance of any known viral or

45

infective agent (fig. 15). Lewy bodies are characteristic of
Parkinson’s disease except in post-encephalitic Parkinson’s and
parkengene mutants. However, they could be found in 4% of
brain without parkinsonian features and these are likely cases of
subclinical Parkinson’s as 80% of the zona compacta cells must
degenerate before clinical symptoms become apparent
(Abayomi, 2002).
The pars compacta contains 450,000 dopaminergic neurons.
With the loss of dopaminergic neurons at those sites, there is
deficiency of dopamine in the basal ganglia, chiefly the striatum
(caudate nucleus and putamen). Furthermore, the enzymes
required for dopamine synthesis, DOPA decarboxylase and the
rate limiting enzyme tyrosine hydroxylase are reduced. In
addition, there is deficiency of neurotropic factors such as glial
and brain derived neurotrophic factors. However, neurons in the
striatum with dopamine receptors remain intact and are
responsible for the therapeutic effects of levodopa. In the
Parkinsonism unresponsive to levodopa, striatal neurons are
degenerated. Genetic and environmental factors are important in
the mechanism of neuronal deaths due to neuronal necrosis or
apoptosis. In neuronal necrosis there is disintegration of cell and
organelles and subsequent removal by phagocytic and
inflammatory response with increased cellular permeability. In
apoptosis on the other hand, there is rapid programmed cell
death in response to a toxic stimuli. There is chromatin
condensation, DNA fragmentation and cell shrinkage, with

46

relative sparing of organelles without inflammatory changes or
increased cellular permeability (Golbe, 2003).
Among the factors that have been implicated in neuronal
degeneration in Parkinson’s disease are mitochondrial
dysfunction, oxidative stress, and the actions of excitotoxins,
deficient neurotrophic support and immune mechanisms. HLA-
DR positive reactive microglial cells and cytokines such as
interleukin 1 (IL-1) and tumor necrosis factor-a play significant
role in the pathogenesis of Parkinson’s disease. Oxidative stress
with excess reactive oxygen species and free radical damage
involving one or more unpaired electrons react with nucleic
acids, proteins and lipids, this metabolic derangement results in
generation of toxic byproducts and increased oxidative stress
with resultant cellular damage (figure 15) (Lane et al., 2008).

47

Figure (15): Neuronal Pathways that degenerate in Parkinson's disease.
Signals that control body movements travel along neurons that project from
the substantia nigra to the caudate nucleus and putamen (collectively called
the striatum) (Lane et al., 2008).

Cell replacement therapy:
The idea of growing dopamine cells in the laboratory to treat
Parkinson’s is the most recent step in the long history of cell or
tissue transplantation to reverse this devastating disease. The
concept was, and still is, straightforward: implant cells into the
brain that can replace the lost dopamine releasing neurons.
Although conceptually straight forward, this is not an easy task.
Fully developed and differentiated dopamine neurons do not
survive transplantation, so direct transplantation of fully
developed brain tissue from cadavers, for example, is not an

48

option. Moreover, full functional recovery depends on more
than cell survival and dopamine release; transplanted cells must
also make appropriate connections with their normal target
neurons in the striatum (Lindvall and Kokaia, 2010).
Under the basic principle of restoring dopamine producing
neurons via neural grafts, extensive studies have been done to
bring this to fruition. One of the first attempts at using cell
transplantation in humans was tried in the 1980s. This surgical
approach involved the transplantation of dopamine producing
cells found in the adrenal glands, which sit atop the kidneys in
the abdomen dramatic improvement in Parkinson’s patients by
transplanting dopamine producing chromaffin cells from several
patients’ own adrenal glands to the nigrostriatal area of their
brains; it showed dramatic improvement in Parkinson’s patients.
Another strategy was previously attempted in the 1970s, in
which cells derived from fetal tissue from the mouse substantia
nigra was transplanted into the adult rat eye and found to
develop into mature dopamine neurons (Panchision, 2006).
The functional integration of dopamine neuron grafts prove
the efficacy of the cell replacement principle, but in reality, this
clinical outcome is extremely inconsistent with respect to the
percentage of cells that survive the grafting procedure and the
amount of dopamine produced by the new neurons. In fact,
average functional improvement of patients in the experiments
only rises about 20%. Across the board, subjects achieve
functioning levels less than or equal to that of patients

49

undergoing deep brain stimulation, which carries a lower
morbidity risk (Lane et al., 2008).
Cell transplantation:
Transplantation of primary ventral mesencephalic tissue into
the striatum aims to restore brain circuitry and function lost as a
result of PD. The main objective of primary tissue
transplantation has been to provide proof of principle that
grafted dopaminergic neurons can i) survive and restore
regulated dopamine release, ii) integrate with the host brain to
reinstate frontal cortical connections and activation, and iii) lead
to measurable clinical beneﬁts together with improved quality
of life. Preclinical work in animal models of PD has shown that
grafted dopaminergic neurons, extracted from the developing
ventral mesencephalon (VM) can survive, reinnervate the
lesioned striatum, and improve motor function (Winkler et al
2005).
Over the past two decades, a series of open label clinical
trials have provided convincing evidence to show that human
embryonic nigral neurons taken at a stage of development when
they are committed to a dopaminergic phenotype can survive,
integrate and function over a long time in the human brain.
There is good evidence of graft survival, with grafted neurons
developing afferent and efferent projections with the host
neurons. Long term survival of dopaminergic grafts is possible
up to 10 years after transplantation, and there have been no

50

reported cases of overt immunorejection even after several years
of withdrawal from immuno-suppression (Olanow et al 2003).
Evidence from Positron Emission Tomography (PET)
scanning has revealed signiﬁcant increases in activation in the
areas reinnervated by the grafted cells, and longitudinal clinical
assessments indicate signiﬁcant functional recovery for motor
control, in some cases for more than 10 years, in the most
successful cases, patients have either reduced dependency for or
completely withdrawn from L-dopa treatment. Post mortem
studies similarly show good survival of transplanted neurons
and well integrated grafts (figure 16) (Winkler et al., 2005).

Figure (16): Dopamine Neuron Transplantation: PET images from a
Parkinson’s patient before and after fetal tissue transplantation, the image
taken before surgery (left) shows uptake of a radioactive form of dopamine
(red) only in the caudate nucleus, indicating that dopamine neurons have
degenerated. Twelve months after surgery, an image from the same patient
(right) reveals increased dopamine function, especially in the putamen
(Winkler et al., 2005).

51

The precise mechanism responsible for these dyskinesia
remains unknown but it does not appear to be related to graft
overgrowth resulting in excessive dopamine release. One
possibility surrounds the quality of dissected tissue. Successful
trials have used either freshly dissected tissue or tissue that has
been stored in culture for only a few days. One of the trials
reporting cases of severe dyskinesias used tissue stored in
culture for up to four weeks and it may be that holding tissue in
this way reduces its dopaminergic composition (Olanow et al.,
2003).
A further issue concerns the identiﬁcation of dense
hyperdopaminergic areas within the graft of some patients with
graft induced dyskinesias. This may have caused uneven striatal
innervation and excessive dopamine release into non
reinnervated areas. It is also possible that variable side effects of
graft induced dyskinesias are related to patient selection.
Greater functional improvement is associated with younger
patients, and in patients with less advanced disease. This is most
likely because the neuropathology is relatively conﬁned to the
nigrostriatal pathway and may have better trophic support
compared to patients with more advanced disease (Winkler et
al., 2005).
Stem cells:
Stem cells could provide one such source and would
overcome the issue of limited availability of fresh primary fetal
cells. A wide range of stem cells are being investigated as

52

potential sources of dopaminergic neurons for transplantation,
stem cells can be obtained from various sources (Morizane et
al., 2009).
The majority of research thus far with respect to the
formation of dopaminergic neurons for the treatment of PD is in
embryonic stem cells and neural stem cells. Dopaminergic
neurons are more easily obtained from neural stem cells in the
developing ventral mesencephalon (VM) than other parts of the
developing central nervous system but the number of
dopaminergic cells produced is still very low. Despite genetic
manipulation and the addition of various growth and
differentiation factors, generating large numbers of
dopaminergic cells from this cell type has had mixed results
(Panchision, 2006).
However, greater success has been achieved with the more
complex ES cells. Derived from blastocysts donated following
in vitro fertilization these cells are truly pluripotent. Promising
data have been obtained with dopaminergic neurons derived
from mouse ES cells, signiﬁcantly improving motor function in
a rat model of PD. However, directing the differentiation of
human ES cells has proved complex and while 50% of cells
spontaneously differentiate into neurons upon leukemia
inhibitory factor (LIF) withdrawal, few are dopaminergic. Thus,
there is the need to develop protocols to ‘direct’ differentiation.
The most successful published protocols describe multiple
culture stages in which different transcription and growth

53

factors are added at controlled time points (Goldman and
Windrem, 2006).
However, despite good yield of dopaminergic neurons in
vitro, clinically relevant long term survival and behavioral
recovery in animal models rivaling that of primary tissue has yet
to be convincingly demonstrated. Neuronal stem cells unlike
embryonic stem cells, which are only derived from the
embryonic blastocyst, neural stem cells can be found both in
embryonic neural tissue and also in specific neurogenic regions
of the adult brain. If the in vivo survival of neural stem cells can
be improved they hold the potential to provide autologous
transplantation as patients provide the cells for their own
recovery (Morizane et al., 2009).
Interestingly, stem cells may not just be useful as dopamine
factories in the striatum. Some studies in both rodent and
primate models have shown significant behavioral recovery
following transplantation with neural stem cells. In addition to
the generation of a small population of dopaminergic neurons
other cells within the graft were found to be releasing growth
factors which are purportedto exert neuro-protective or
neuroregenerative influences. While more evidence needs to be
accumulated on the longevity of this effect, it broadens the
potential of neural stem cells from simple dopamine
replacement to preserving and enhancing remaining
dopaminergic neurons (Svendsen and Langston, 2004).

54

Stem cell based approaches could be used to provide
therapeutic benefits in two ways: first, by implanting stem cells
modified to release growth factors, which would protect existing
neurons and/or neurons derived from other stem cell treatments;
and second, by transplanting stem cell derived DA neuron
precursors/neuroblasts into the putamen, where they would
generate new neurons to ameliorate disease-induced motor
impairments (figure 17) (Lindvall and Kokaia, 2010).

Figure (17): Stem cell based therapies for PD. PD leads to the progressive
death of DA neurons in the substantia nigra and decreased DA innervation
of the striatum, primarily the putamen (Lindvall and Kokaia, 2010).

Neurogenesis:
As mentioned above endogenous stem cells are present in
speciﬁc regions of the brain. While the occurrence of
neurogenesis in the striatum and substantia nigra is debated, one

55

indisputable neurogenic region is the subventricular zone (SVZ)
lying adjacent to the striatum. The cells in the region are an
assortment of stem and progenitor cells that have the potential to
be mobilized and induced to differentiate by the presence of
growth factors or other small molecules. In the normal condition
75%–99% or the cells differentiate into granular GABAergic
neurons, with the rest forming periglomular neurons expressing
either tyrosine hydroxylase or GABA. The control of
proliferation and mobilisation of these cells may be
dopaminergic as both MPTP and 6-OHDA mediated dopamine
depletion reportedly decreases proliferation in this zone (Zhao
et al., 2008).
An additional source of endogenous source of new
dopaminergic neurons may be described presence of tyrosine
hydroxylase positive cell bodies in the striatum, which increase
in quantity with dopaminergic denervation. As yet there are no
imminent therapeutic strategies heading towards the clinic that
manipulate these endogenous systems but their potential is
waiting to be harnessed. Therapeutic strategies to increase
striatal dopamine could involve recruiting newly produced
neurons in the SVZ and encouraging them to migrate into the
striatum and differentiate into dopaminergic neurones or to
stimulate cells resident in the striatum. In order for this to be
achieved understanding more about these two processes of
neurogenesis and phenotypic switching in the striatum is
necessary, determining the intrinsic or extrinsic factors

56

responsible may provide an alternative set of mechanisms that
could be utilized to treat PD (Lane et al., 2008).
Graft standardization:
Grafting methods for the past 20 years have differed in
everything from procurement process to tissue composition to
implantation technique. To add even more variability, multiple
donors are needed to create a graft large enough to carry some
promise of efficacy. This, undoubtedly, plays an important role
in determining survival, growth, and integration of the
transplant (Morizane et al., 2009).
As stem cells can theoretically provide an endless source of
quality consistent neurons, standardization of the transplant
tissue will enhance the reliability of the procedure and its
results. One promising study has shown that implantation of
undifferentiated human neural stem cells (hNSCs) in
Parkinsonian primate brains can restore functionality.
Furthermore, the repair process not only reestablishes the gross
anatomical structure of the organ but does so with appropriate
proportions of neuron types. Guided by signals of the
microenvironment of the damaged brain, uncommitted hNSCs
are induced to differentiate into dopaminergic neurons, as well
as other cells that mediate neuroprotection (Lane et al., 2008).
Patient standardization:
With standardization of transplant material, patients must
likewise be evaluated for variables in their presentation of the
disease. Specifically, the distribution of the damaged neurons

57

should be taken into account before and after graft implantation.
In earlier studies, patient selections overlooked the preoperative
magnitude of the lesions, making it difficult to evaluate the
extent of the graft incorporation. Similarly, it was also unknown
whether continued postoperative degeneration of non grafted
regions would affect clinical response. Conversely, patients with
little or no postoperative damage showed the best functional
outcome. Because the decline of dopaminergic cells in areas
outside the nigrostriatal region seems to arise as PD progresses
to a more severe state, it may be that implantation during earlier
stages will exhibit a higher rate of success. This is mirrored in
the survival of transplanted tissue, which survives and integrates
better in younger patients. The reasons why this occurs have yet
to be fully determined, but it is known that neural growth factors
are expressed more in younger brains (Morizane et al., 2009).

58

Stem cell therapy in stroke
Stroke is defined as an abrupt focal loss of brain function
resulting from interference with the blood supply to part of the
central nervous system. It is one of the major causes of death
and disability among the adult population in the world. In spite
of the extensive research in the field of stroke biology, there is
little effective treatment for a completed stroke. Most strokes
fall into two main categories: ischemic (80%) or hemorrhagic
(20%) (Caplan, 2011).
The biology of cellular transplantation:
The transplantation of human neuronal cells is an approach
to reducing the functional deficits caused by CNS disease or
injury. Several investigators have evaluated the effects of
transplanted fetal tissue, rat striatum, or cellular implants into
small animal stroke models for the most part, clinical trial
designs using primary human fetal tissue into patients with
neurodegenerative diseases have lessened. The widespread
clinical use of primary human tissue is likely to be limited due
to the ethical and technical difficulties in obtaining large
quantities of fetal neurons at the same time; much effort has
been devoted to developing alternate sources of human neurons
for use in transplantation (Kondziolka and Wechsler, 2008).
When transplanted, these neuronal cells survived, extended
processes, expressed neurotransmitters, formed functional
synapses, and integrated with the host. During the retinoic acid

59

induction process, significant changes were seen in the neuron
precursor cells that resulted in the loss of neuroepithelial
markers and the appearance of neuronal markers. The final cell
product was a ≥ 95% pure population of human neuronal cells
that appeared virtually indistin-guishable from terminally
differentiated, post mitotic neurons. The cells were capable of
differentiation to express a variety of neuronal markers
characteristic of mature neurons, including all 3 neurofilament
proteins (L, M, and H); microtubule associated protein 2, the
somatic/dendritic protein, the axonal protein. Thus, the neuronal
phenotype made these cells a promising candidate for
replacement in patients with CNS disorders, as a virtually
unlimited supply of pure, post mitotic, and differentiated human
neuronal cells (Lindvall and Kokaia, 2010).
The concept of restoring function after a stroke by
transplanting human neuronal cells into the brain was conceived
in the mid 1990s. Research conducted in a rat model of transient
focal cerebral ischemia demonstrated that transplantation of
fetal tissue restored both cognitive and motor functions.
Ischemic stroke leads to the death of multiple neuronal types
and astrocytes, oligodendrocytes, and endothelial cells in the
cortex and subcortical regions. Stem cell based therapy could be
used to restore damaged neural circuitry by transplanting stem
cell derived neuron precursors/neuroblasts. Also, compounds
could be infused that would promote neurogenesis from
endogenous SVZ stem/progenitor cells, or stem cells could be

60

injected systemically for neuroprotection and modulation of
inflammation (Kondziolka and Wechsler, 2008).
Behavioral testing was conducted using a passive avoidance
learning and retention task and a motor asymmetry measure.
Animals that received transplants of neurons and treatment with
cyclosporine showed amelioration of ischemia induced
behavioral deficits throughout the 6 month observation period.
They demonstrated complete recovery in the passive avoidance
test, as well as normalization of motor function in the elevated
body swing test. In comparison, control groups receiving
transplants of rat fetal cerebellar cells, medium alone, or
cyclosporine failed to show significant behavioral improvement.
Subsequent studies have shown that these cells released glial
derived neurotrophic factor after transplantation into ischemic
rats (figure 18) (Lindvall and Kokaia, 2010).

Figure (18): Stem cell based therapies for stroke (Lindvall and Kokaia,
2010).

61

Embryonic stem cells:
Animal models have demonstrated that ESCs, when
transplanted into adult hosts, differentiate and develop into cells
and tissues and thus may be applicable for treating a variety of
conditions, including Parkinson’s disease, multiple sclerosis,
spinal injuries, stroke, and cancer. Transplanted ESCs are
exposed to immune reactions similar to those acting on organ
transplants; hence, immunosuppression of the recipient is
generally required. It is possible, however, to obtain ESCs that
are genetically identical to the patient’s own cells using
therapeutic cloning techniques (Kalluri and Dempsey, 2008).
Several studies showed that these cells are able to migrate in
response to damage, using MRI, that ESCs that were implanted
into the healthy hemisphere of rat brains 2 weeks after focal
cerebral ischemia (FCI), migrated along the corpus callosum to
the ventricular walls, and populated en masse at the border zone
of the damaged brain tissue (i.e., the hemisphere opposite to the
implantation sites). Another study showed that undifferentiated
ESCs xenotransplanted into the rat brain at the hemisphere
opposite to the ischemic injury migrated along the corpus
callosum toward the damaged tissue and differentiated into
neurons at the border zone of the lesion. In the homologous
mouse brain, the same murine ESCs did not migrate, but
produced highly malignant teratocarcinomas at the site of
implantation, independent of whether they were
predifferentiated in vitro to NPCs. These results imply that

62

ESCs might migrate to the damaged site. However, the
production of teratocarcinoma raises concerns about the safety
of ESC transplantation in patients with stroke (Battler and Leor,
2006).
Adult neural stem cells:
During the last century, the dogma existed that the adult
CNS was incapable of generating new neurons (neurogenesis).
Over the past decades, convincing evidence emerged that
neurogenesis in the adult CNS is a continuous physiological
process. Neurogenesis is present in two regions: the
subventricular zone (SVZ) and the subgranular zone of the
dentate gyrus (figure 19) (Kalluri and Dempsey, 2008).

Figure (19): Schematic drawings showing ischemia induced damage in the
cortex and neural stem cell proliferation in the subventricular zone and
dentate gyrus; these hypothetical sections of brain illustrate the ischemic
tissue and the neurogenic regions. A: Drawing showing the subventricular
zone of the lateral ventricles. B: Drawing showing the dentate gyrus in the
hippocampus. Note the migration of cells from the subventricular zone
toward the infarcted area. Red dots represent proliferating and/or migrating
neural stem and/or progenitor cells (Kalluri and Dempsey, 2008).

63

Additionally, other studies also indicated the existence of
NSCs in other regions of the CNS, namely the striatum, spinal
cord and neocortex. SVZ and dentate gyrus derived NSCs are
characterized by long term, self renewal capacity and
multipotency. Adult SVZ and dentate gyrus derived NSCs
persist throughout the life span of mammals including humans.
It is important to note that neurogenesis occurs in a
physiological mode or is exogenously modulated by external
signals or pathophysiological processes. External global
stimulants such as enriched environment, physical activity and
stress or application of defined molecules such as fibroblast
growth factor-2, vascular endothelial growth factor (VEGF),
brain derived neurotrophic factor (BDNF) and erythropoietin
differentially modulate adult neurogenesis. Finally, CNS disease
conditions such as seizures and traumatic brain injury
represented by respective animal models induce neurogenesis
(Panchision, 2006).
Role of neural stem cells:
Proliferation:
Neural stem cell proliferation involves the sequential
activation of several cell cycle dependent enzymes and proteins
to initiate either symmetrical (2 stem cells) or asymmetrical cell
division (1 stem and 1 progenitor cell). Erroneous activation of
cell cycle enzymes in differentiated cells like neurons, however,
can lead to either apoptotic death of the neurons or to the
formation of cancer cells. A plethora of growth factors are

64

expressed by the ischemic tissue, which may be responsible for
the ischemia induced neurogenesis. The increase in ischemia
induced neurogenesis could therefore be due to the upregulation
of growth factor content or their receptor expression (Hass et
al., 2005).
In addition, not all growth factors are stimulatory in
function. Although a variety of growth and trophic factors are
upregulated following ischemic injury, some are stimulators of
neural progenitor proliferation, whereas others block the self
renewal of cells. It is important to note that ischemia induced
migration of neuroblasts has been shown to be due to the
expression of monocyte chemoattractant protein (MCP-1),
which can attract the progenitor cells away from the neurogenic
niche, blocking their proliferation. Likewise, inhibition of
growth factor activity by its binding proteins may also interfere
with proliferation of cells. Interestingly, insulin like growth
factor–I (IGF-I) can stimulate the proliferation of progenitor
cells only in the presence of mitogens like fibroblast growth
factor–2 (FGF-2) and promotes differentiation following
mitogen (FGF-2) withdrawal (figure 20) (Kalluri and Dempsey,
2008).

65

Figure (20): Schematic drawing showing proliferation and differentiation
of neural stem cells. These cells proliferate in response to mitogens and
differentiate into neurons, astrocytes, and oligodendrocytes on exposure to
various growth factors (Kalluri and Dempsey, 2008).

Migration:
Several studies have shown that ischemia induces the
migration of neuroblasts into the striatum and cerebral cortex.
An enriched environment, however, increased the stroke
induced neurogenesis in the hippocampus, but decreased the cell
genesis and migration of neuroblasts into striatum. In addition,
erythropoietin has also been shown to be involved in the
proliferation of progenitor cells in the subventricular zone.
Hence, it appears that proliferation and migration of cells may
be interrelated, in the sense that they are elements of the same
process (Son et al., 2006).

66

It is thought that chemokines proteins released at the site of
injury by the inflammatory cells induce migration of the cells.
The expression of chemokine receptors on neural stem cells is
important for an efficient response to chemokines. The
expression of chemokine receptors is regulated by retinoic acid,
and that animals fed on a diet lacking retinoic acid have
decreased numbers of double cortin positive cells. Retinoic acid
has been shown to induce the differentiation of neurons in vitro.
Hence, it appears that lack of retinoic acid may have decreased
the neuronal differentiation and/ or the expression of chemokine
receptors, which results in decreased numbers of neuroblasts
(immature neuronal cells expressing double cortin) migrating
out of the neurogenic niche (figure 21) (Kondziolka and
Wechsler, 2008).

67

Figure (21): Schematic drawing showing ischemia induced neurogenesis.
Ischemia increases the expression of IGF-I, FGF- 2, TGFb1, and MCP-1.
The IGF-I and FGF-2 enhance the proliferation of cells, whereas MCP-1
increases the migration of cells away from neurogenic regions toward the
ischemic tissue. The stem cells leaving the neurogenic regions exit the cell
cycle, during which time TGFb1 promotes the differentiation of stem cells
into neuroblasts (migrating immature neurons). Alternately, if proliferating
cells in the neurogenic region are exposed to TGFb1, it will inhibit the
process, thus slowing down the proliferation of cells (Kondziolka and
Wechsler, 2008).

Differentiation:
The process of generating specialized cells from neural stem
cells is called differentiation. Neural stem cells can be
differentiated into neurons, astrocytes, and oligodendrocytes;
this involves interplay between intrinsic cellular programs and
extrinsic cues like growth factors provided by the surrounding
environment. Although neural progenitor cells proliferate in
response to ischemia, several immature neurons (double cortin
positive cells) were shown to be migrating toward striatum and
cortex (Battler and Leor, 2006).

68

Although IGF-I induces the proliferation and differentiation
of progenitor cells after ischemia, most of the cells that are
migrating toward the injury site are neuroblasts (immature
neurons) but not oligodendrocytes. Although speculative, a
neuronal differentiation factor like transforming growth factor
b1 (TGFb1), which is upregulated after ischemia, may be
responsible for the formation of neuroblasts during the post
ischemic neurogenesis. The differential response of IGF-I
toward proliferation and differentiation was shown to be
regulated by a mitogen activated protein kinase pathway.
Because matrix metalloproteinase (MMPs) play an important
role in both extracellular matrix (ECM) digestion and IGF
regulation, it is crucial to understand their direct (ECM
digestion) and indirect (IGF metabolism) effects on progenitor
cell differentiation. Hence it is possible that after stroke, cells
proliferate in response to mitogens and growth factors, some of
which exit the cell cycle due to the chemokine mediated
migration out of the neurogenic area, and differentiate into
neuroblasts in response to differentiation promoting factors such
as TGFb1. Thus, the final outcome depends on the spatial and
temporal expression of these factors following ischemia (Son et
al., 2006).
Potential mechanism involved in stem cell mediated
recovery after stroke:
Initial transplantation studies were focused on the potential
of NSCs to replace lost circuitry. Transplanted neural progenitor

69

cell (NPCs) in a rat model of global ischemia have been
reported to express synaptic proteins post transplantation.
However, only limited evidence demonstrates that transplanted
cells are able to sustain CNS repair through massive cell
replacement, especially to the extent that might be required after
stroke. Regardless of the characteristics of the experimental
disease, functional recovery achieved through NPC
transplantation does not correlate with absolute numbers of
transplant derived, newly generated, and terminally
differentiated neural cells (Emerson et al., 2008).
Stem cell induced neuroprotection:
Transplanted stem cells can provide neuroprotection and
reduce host cell death in the post stroke brain. Most authors
have reported functional recovery and a reduction in lesion size
when cells are transplanted within the first 24-48 hours after
stroke. The short timeframe in which NPCs affect recovery
cannot be explained by the regeneration of new neurons and
synapses, suggesting an important role for neuroprotection in
enhancing recovery. In fact, NPCs are known to exert direct
neuroprotection through the neutralization of free radicals,
inflammatory cytokines, excitotoxins, lipases, peroxidases, and
other toxic metabolites that are released following an ischemic
event (Ourednik et al., 2005).
The neuroprotective effects of transplanted NPCs are usually
accompanied by increased in vivo bioavailability of main
neurotrophins such as nerve growth factor, brain derived

70

neurotrophic factor (BDNF), ciliary neurotrophic factor,
vascular endothelial growth factor (VEGF), fibroblast growth
factor, and glial derived neurotrophic factor (GDNF).
Alternatively, one could argue that the cells did adhere to the
endothelium in the affected brain area and exerted their effect
through the secretion of GDNF but did not eventually engraft. A
different aspect of neuroprotection would be an effect on
endogenous neurogenesis after stroke. Endogenous
neurogenesis is increased after a stroke. Its exact function has
yet to be determined, but it may signify a natural repair
mechanism of the brain, which could be enhanced by
transplanted cells (Guzman et al., 2008).
Stem cell induced angiogenesis:
Transplanted NPCs can also enhance endogenous
angiogenesis. Increased vascularization in the penumbra within
a few days after stroke is associated with spontaneous functional
recovery. As early as 3 days after ischemic injury, new blood
vessels are observed in the stroke affected penumbra, and
growth continues to increase for at least 21 days. Transplanted
cell induced blood vessel formation has been reported with
BMSCs, NSCs, and cells from human cord blood or peripheral
blood. The ability of transplanted cells to increase endogenous
levels of angiogenic factors and chemoattractant factors (for
example, SDF-1) induces the proliferation of existing vascular
endothelial cells (angiogenesis) and mobilization and homing of

71

endogenous endothelial progenitors (vasculogenesis) (Shyu et
al., 2006).
The direct injection of recombinant stromal derived factor–1
(SDF-1) into the stroke affected rat brain resulted in the
increased recruitment of BMSCs and increased vascular density
in the ischemic brain. A similar effect was achieved by
treatment of stroke affected rats with granulocyte colony
stimulating factor (Ourednik et al., 2005).
Stem cell induced immunomodulation:
In addition to neuroprotection and enhancement of
angiogenesis, transplanted NPCscan also decrease post
ischemic inflammatory damage. A landmark study highlights
the remarkable ability of transplanted NPCs to promote
neuroprotection through an immunomodulatory strategy, once
within inflamed CNS areas, systemically injected NPCs persist
around the perivascular space where reactive astrocytes,
inflamed endothelial cells, and bloodborne infiltrating T cells
coreside. Adult NPCs can promote neuroprotection by releasing
antiinflammatory chemokines and by expressing
immunomodulatory molecules (Pluchino et al., 2005).
In animal models of stroke, decreased infiltration of
mononuclear cells has been demonstrated at the lesion borders
of ischemic areas in the CNS where NPCs accumulate. Working
under the paradigm of bidirectional communication between
transplanted cells and the inflammatory ischemic
microenvironment, one group has attempted to combine NPC

72

Thesis section: Restorative neurology

Thesis section: Restorative neurology

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