i

Restorative neurologyEssay

In NeuropsychiatrySubmitted for partial fulfillment of Master Degree

BySamy 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

Faculty of Medicine-Ain Shams University

Dr. Haitham Hamdy salem

Lecturer of Neuropsychiatry

Faculty of Medicine-Ain Shams University

Faculty of Medicine

Ain Shams University

2011

i

CCoonntteennttss

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

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

8. Stem cell therapy Huntington chorea

References

Summary

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.

1

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).

5

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 trans- differentiation

which describes the conversion of a cell of one

6

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

10

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).

13

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).

15

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 hiddenamong ordinary adult cells. Here, human embryonic stem cells arerecognized 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 andEbert, 2008).

24

(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).

25

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

26

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).

27

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

28

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).

29

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

30

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

31

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

32

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

33

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 DescriptionBrian

Bone marrow

Endothelium

Skeletal muscle

Skin

Digestivesystem

Pancreas

Liver

Stem cells of the brain can differentiate into the three kinds ofnervous tissue-astrocytes, oligodendrocytes, and neurons-and insome cases, blood-cell precursor.

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

These stem cells are called hemangioblasts and are known todifferentiate into blood vessels and cardiomyocytes. They mayoriginate in bone marrow, but this is uncertain.

These stem cells may be isolated from muscle or bone marrow. Theymediate muscle growth and may proliferate in response to injury orexercise.

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

Located in intestinal crypts, or invaginations. These stem cells areresponsible for renewing the epithelial lining of the gut.

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

The identity of liver stem cells is still unclear. Stem cells from bonemarrow may repair some liver damage, but most repairs seems to becarried out by the hepatocytes (liver cells) themselves.

Table (1): Sources of adult stem cells (Limbert et al., 2008).

34

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

35

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).

36

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.

37

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

38

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).

39

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 revealingnumerous cells in the stroke border zone and the hippocampus ipsilateral tothe stroke (A). Inset shows doublecortin bromodeoxyuridine labeled cells.The VCAM-1 (arrows) is highly expressed in the stroke affectedhemisphere 48 hours after stroke (B). DCX = doublecortin; BrdU =bromodeoxyuridine; DAPI = 4'6-diamidino-2-phenylindole (Guzman et al.,2008).

40

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).

41

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

42

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–

43

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

44

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).

45

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

46

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

47

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).

48

Figure (15): Neuronal Pathways that degenerate in Parkinson's disease.Signals that control body movements travel along neurons that project fromthe substantia nigra to the caudate nucleus and putamen (collectively calledthe 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

49

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

50

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

51

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 aParkinson’s patient before and after fetal tissue transplantation, the imagetaken before surgery (left) shows uptake of a radioactive form of dopamine(red) only in the caudate nucleus, indicating that dopamine neurons havedegenerated. Twelve months after surgery, an image from the same patient(right) reveals increased dopamine function, especially in the putamen(Winkler et al., 2005).

52

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

53

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

54

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).

55

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 progressivedeath of DA neurons in the substantia nigra and decreased DA innervationof 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

56

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

57

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 stemcells (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

58

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).

59

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

60

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

61

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).

62

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

63

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 thecortex and neural stem cell proliferation in the subventricular zone anddentate gyrus; these hypothetical sections of brain illustrate the ischemictissue and the neurogenic regions. A: Drawing showing the subventricularzone of the lateral ventricles. B: Drawing showing the dentate gyrus in thehippocampus. Note the migration of cells from the subventricular zonetoward the infarcted area. Red dots represent proliferating and/or migratingneural stem and/or progenitor cells (Kalluri and Dempsey, 2008).

64

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

65

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).

66

Figure (20): Schematic drawing showing proliferation and differentiationof neural stem cells. These cells proliferate in response to mitogens anddifferentiate into neurons, astrocytes, and oligodendrocytes on exposure tovarious 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).

67

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).

68

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-1increases the migration of cells away from neurogenic regions toward theischemic tissue. The stem cells leaving the neurogenic regions exit the cellcycle, during which time TGFb1 promotes the differentiation of stem cellsinto neuroblasts (migrating immature neurons). Alternately, if proliferatingcells in the neurogenic region are exposed to TGFb1, it will inhibit theprocess, thus slowing down the proliferation of cells (Kondziolka andWechsler, 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).

69

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

70

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

71

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

72

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

73

based and immunotherapy based approaches in spinal cord

injury. In 2006 Ziv and colleagues described a two stage process

of repair. In the first stage, myelin specific T cells induce

transplanted NPC migration to the injury site and also activate

microglia to adopt a neuroprotective phenotype; in the second

stage, NPC interaction with T cells and microglia leads to better

tissue preservation, increased endogenous neurogenesis, and

improved functional recovery.

Although basic differences between the post ischemic brain

and the post injury spinal cord might preclude the translation of

this approach to stroke, the common themes of a limited

therapeutic window to promote a more neurogenic

microenvironment beckon a closer look. Indeed, studies by

these same authors as well as others have revealed how

modulation and/or suppression of inflammation after stroke can

promote neuroprotection and neurogenesis. This fascinating

cross talk between neural and immune cells reveals additional

therapeutic opportunities for attenuating post ischemic

inflammatory damage and providing a

more hospitable environment for the

persistence of both endogenous and transplanted NPCs (Shyu et

al., 2006).

Stem cell induced neuroplasticity:

The ability of cell based therapies to enhance injury induced

plasticity has been demonstrated with other somatic stem cell

types. Human cord blood cells in the ischemic cortex increase

nerve fiber sprouting from the contralateral to the ischemic

74

hemisphere. Neural progenitor cells were first reported to alter

plasticity in models of epilepsy. Transplanted NPCs have also

been shown to scavenge inhibitory molecules that limit the

reorganization of the injured brain. We have found that NPCs

possess the potential to alter synaptogenesis by secreting

thrombospondins, which are a family of large extracellular

matrix proteins. Thrombospondins 1 and 2, secreted by

immature astrocytes, are necessary and sufficient for the

development of ultrastructurally normal synapses (Xiao et al.,

2007).

Possible ways to monitor cell treatment:

The clinical application of cell transplantation therapy for

stroke requires a means of noninvasive monitoring these cells in

patients. Several imaging modalities, including MRI,

bioluminescence imaging, PET, and in vivo fluorescence

microscopy, have been used to track stem cells in vivo. Of these

methods, however, only MRI and PET are practicable for

clinical use. MRI is a high spatial resolution method for tracking

cells in real time. Cells are preloaded with molecules that

substantially alter proton resonance, such as superparamagnetic

iron oxide (SPIO) and ultra SPIO (USPIO) particles. In vivo,

SPIO labeled cells appear as hypointense areas in tissues,

because of a susceptibility artifact most pronounced in iron

sensitive T2- and T2*-weighted images. With high resolution

MR imaging, even single mammalian cells have been depicted

after SPIO labeling. Several studies have shown the ability of

75

MR imaging to longitudinally track transplanted iron labeled

cells in different animal models, including stroke. Using clinical

grade human fetal derived NSCs (Guzman et al., 2008).

Future imaging modalities may involve a combination of the

high spatial resolution and excellent anatomical detail of MRI

with the high sensitivity of PET. An alternative direction for

stem cell imaging is the development of MR reporter genes

(Hass et al., 2005).

One great advantage of this technique is that no external

contrast agent needs to be administered for imaging. However,

the application of MR reporter genes for in vivo studies has

been extremely limited, mainly because of the low sensitivity of

the technique. In addition to tracking the transplanted stem cells

after stroke, surveillance of the microenvironment and

monitoring the therapeutic effect of the stem cells will play a

major role in the assessment of clinical efficacy. Several

preclinical studies have demonstrated the possibility of

monitoring the inflammatory reaction after stroke by using

USPIO particles (Kelly et al., 2004).

76

Stem Cell Therapy forDemyelinating Diseases

Multiple sclerosis (MS) is an autoimmune inflammatory

demyelinating disease of the central nervous system (CNS) that

is a leading cause of disability in young adults. This disease is

notorious for making a patient totally bed bound, not only

resulting in bed sores but also inviting all kinds of infections to

the disabled, depressed patient. Although intensive therapy has a

key role in managing this complex disease, even in this era of

advancement, the slowing accumulation of neurological damage

in MS has remained mostly irreversible. Along with therapies

that improve blood circulation, mitochondrial energy

production, and anti-inflammatory, antimicrobial,and

antioxidant factors, there is increased optimism among scientists

about slowing and perhaps reversing the disease with a

comprehensive program that includes stem cell therapies (Olek,

2010).

Precursor cells in the adult CNS are capable of regenerating

oligoden-drocytes and myelin:

In different experimental models of focal demyelination, it

has been shown that endogenous cells in the adult rodent CNS

have the potential for regenerating oligodendrocytes and myelin.

Several studies have investigated the nature of the cells that

remyelinate the adult CNS after induction of demyelinated

lesions. The lack of spontaneous remyelination in

77

experimentally chemical demyelinated lesions that were X-

irradiated to kill proliferating cells, suggests that cell division is

an absolute prerequisite for myelin regeneration. Although

differentiated oligodendrocytes may survive within such lesions,

they are unable to rebuild myelin sheaths (Charron et al.,

2009).

Myelin regeneration fails in MS:

Attempts to regenerate myelin can be also recognized

pathologically in brains of MS patients by the existence of

shadow plaques, which are partially remyelinated lesions. The

process of spontaneous functional remyelination is often

incomplete and limited, leading to permanent axonal loss and

fixed neurologic disability. Failure of remyelination could stem

either from insufficiency of endogenous remyelinating cells or

from lack of environmental support for this process (johnson,

2011).

Data from experimental models of demyelination and from

human brain tissue indeed suggest that several factors may have

a role in limiting myelin regeneration in the adult brain and its

subsequent failure. In experimental focal demyelination, it has

been shown that only a subpopulation of local progenitor cells

react to injury and generate new oligodendrocytes and myelin.

Although the existence of progenitor cells was demonstrable in

acute and chronic MS lesions, they did not exhibit reactive

increase in cell number as compared with normal white matter

(Battler and Leor, 2006).

78

This suggests that the response of the progenitor cell

population to the demyelinating process in the human brain is

deficient. It has also been suggested that repeated demyelinating

episodes in chronic and relapsing MS causes depletion in the

endogenous pool of progenitor cell transplantation for diseases

of myelin cells. Although progenitor cells decrease in number

after experimental focal demyelination, this was not observed in

pathological specimens of chronic MS lesions (Charles and

Alfredo, 2005).

In some patients, there was progressive loss of

oligodendrocytes and myelin without reactive remyelination,

whereas in others, who exhibited strong T cell and macrophage

activity, there was robust remyelination, indicating the

important role of tissue support to the remyelinating response.

Cell migration seems to be another limiting factor in myelin

regeneration. It has been shown that only progenitor cells that

reside at the margins of experimental lesions migrate into the

lesion core and remyelinate it, whereas long distance migration

of progenitor cells does not occur in the brain parenchyma

(Crosta, 2010).

Both environmental factorsand basic properties of

endogenous adult progenitor cells limit the degree of

spontaneous remyelination. The apparent linkage between the

acute inflammatory phase and myelin regeneration and the

necessity to remyelinate before axonal damage occurs, may

define a narrow time window when remyelination is feasible.

79

Although this time window may be too narrow for adequate

endogenous progenitor cell mobilization, it may also determine

the window of opportunity for therapeutic cell transplantation

(Olek, 2010).

Rationale for cell transplantation into the CNS to repair

myelin:

Basically, there are two therapeutic approaches to induce

remyelination in MS and both have shown promising results in

experimental animals:

1. Promotion of endogenous remyelination by growth factors

therapy.In acquired demyelinating diseases, such as MS, growth

factor therapy has proven effective in enhancing the endogenous

brain’s capacity for repair. Insulin like growth factor-1 (IGF-1)

and glial growth factor-2 (GGF2) are neurotrophic factors that

promote survival and proliferation in the oligodendrocyte

lineage. Treatment with these factors was beneficial clinically

and pathologically in animals with EAE.

2. Transplantation of myelin forming cells is a mode of

delivering the entire “cell factory” that manufactures myelin.

This approach may be advantageous over other modes of gene

therapy, in which targeting the gene to specific cells and tissues

and controlling its degree of expression may be problematic or

even detrimental (johnson, 2011).

Experimental transplantation has been performed in several

animal models of myelin disease. Transplanted myelin forming

cells remyelinated focal lesions in the optic nerve and spinal

80

cord and restored normal conduction properties, indicating fully

functional regenerated myelin (Crosta, 2010).

Remyelination by various myelin forming cells:

The traditional primary condition for any cell type to be a

candidate for therapeutic cell transplantation in myelin disorders

has been its ability to remyelinate and to restore nerve function.

Neural transplantation for remyelination has been studied for

more than 25 years, following early work by several

researchers. Various cell populations were shown to myelinate

efficiently after transplantation into experimental animals

(Einestien and Ben-Hurr, 2008).

The oligodendrocyte lineage:

Studies designed to identify the oligodendrocyte lineage

cells that had best myelinating potential showed that when

focally injected to chemically induce demyelinated lesions,

postmitotic oligodendrocytes had poor remyelinating capacity,

whereas OPCs showed greater mitotic, migratory, and

regenerative properties. Interestingly, transplanted OPCs were

found to be more efficient in remyelination than endogenous

OPCs (Charles and Alfredo, 2005).

Schwann cells:

Remyelination of CNS axons has been achieved by other

myelin forming cells as well. Schwann cells, the peripheral

myelin forming cells, have excellent myelinating properties in

the CNS. They produce thick and compact myelin after

transplantation into the CNS and can restore normal conduction

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velocity in the dorsal columns of the spinal cord, indicating

functional recovery. The main advantage of using these

peripheral nervous system myelin forming cells is that Schwann

cells can be isolated from a sural nerve biopsy of patients

affected with a myelin disorder of the CNS, cultured and

expanded in vitro under appropriate conditions, cryopreserved,

and finally serve for autologous transplantation into

demyelinated CNS areas. Another potential advantage of

transplanted autologous Schwann cells is that they might escape

the autoimmune attack in MS that is directed against central

myelin antigens (Battler and Leor, 2006).

Olfactory nerve ensheathing cells:

Olfactory nerve ensheathing cells (OECs) display properties

of both astrocytes and Schwann cells. These cells are unique in

that they continue to develop in the olfactory epithelium

throughout life from which they migrate to the olfactory bulb.

Studies have shown that although these cells do not normally

make myelin, they are able to do so when transplanted to areas

of demyelination in the brain or spinal cord. These cells have a

capacity to grow in vitro and to remyelinate large axons with a

Schwann cell like pattern of myelin and improve conduction

properties after transplantation into the demyelinated adult rat

CNS. These cells seem also to promote axonal growth and

secrete neurotrophic molecules (Emerson et al., 2008).

Thus, the relative availability of these cells, their apparent

myelinating properties, and their trophic effect on axonal

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growth make them another promising candidate for autologous

therapeutic transplantation. However, it is not yet clear whether

OECs can be expanded in sufficient amounts for human

transplantation and whether they will need to be isolated only

from the olfactory bulb, located intracranially, or also more

easily from the olfactory mucosa, situated at the back of the

nose outside the cranium (Einestien and Ben-Hurr, 2008).

Neural stem cells:

The main advantage of stem cells is that they are non

transformed precursors that are potentially able to self renew

indefinitely, allowing their expansion in large quantities. NSCs

can be expanded in vitro, maintain their capacity for self

renewal, and generate a progeny of the three neural cell lineages

(Charron et al., 2009).

NSCs retain their functional plasticity after in vitro

passaging and after several freezing thawing cycles and they can

still be modulated in vitro by exposure to different growth

factors. These uncommitted NSCs can integrate and repair the

damaged CNS and thus might represent a renewable source of

cells that can be used for transplantation procedures. Stem cells

can adapt their lineage fate and function according to

environmental needs (Crosta, 2010).

Transplanted NSCs of various origins improved the clinical

outcome in experimental models of stroke, spinal cord trauma,

and proved to have good myelinating properties as well.

Intraventricularly transplanted clonal NSCs in the newborn shi

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mouse disseminated in widespread brain areas and participated

in myelination. Some recipient animals showed a decrease in

their symptomatic tremor. In adult animals with traumatic spinal

cord injury, rat NSCs migrated in the spinal cord and

differentiated into myelinating oligodendrocytes (Charles and

Alfredo, 2005).

Recently, it was also shown that intraventricular, as well as

intravenous, transplantation of stem cells into mice with chronic

EAE, resulted in clinical improvement. This was correlated by

graft derived and endogenous remyelination and by reduction in

axonal pathology. These data suggest that cell transplantation in

de- and dysmyelinated human disease may have therapeutic

potential for functional restoration. In the process of neural

development, there is continuous functional and lineage

potential specification of the stem cell, before entering the

neuronal, oligodendroglial, or astroglial lineage (Olek, 2010).

Although early NSCs in the developing cortical plate expand

very rapidly, they are not migratory cells. In the SVZ of the

CNS reside stem cells that generate mainly glia progeny. Donor

derived myelin was observed after SVZ precursor cells were

propagated in culture with epidermal growth factor (EGF) and

transplanted into the spinal cords of md rat and sh pup and to

the retinas of young mice (Battler and Leor, 2006).

Embryonic stem cells

ES cell lines can actually be established from virtually all

mammals and can be banked and propagated in vitro almost

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indefinitely, with maintenance of a normal karyotype and

totipotency, as was shown by the culturing of mouse ES cell

lines in the presence of leukemia inhibitory factor. Mouse ES

cells can be induced to differentiate in vitro into neurons

(David, 2009).

The sequential use of growth factors, such as fibroblast

growth factor-2 (FGF2), EGF, and platelet derived growth

factor (PDGF), in a program that mimics embryonic

development, has been successful to derive glial precursors

from mouse ES cells. The myelinogenic potential of mouse ES-

derived OPCs, that were expanded in vitro, was demonstrated in

the embryonic md rat brains, when these cells extensively

myelinated the brain and spinal cord. When transplanted in a

rodent model of chemically induced demyelination and in spinal

cords of shi mice, mouse ES-derived progenitor’s cells were

also able to differentiate into glial cells and remyelinate

demyelinated axons in vivo (Goldman and Windrem, 2006).

Since the isolation of human ES cells, it has been possible to

generate an endless source of transplantable human ES-derived

neural precursors. Transplanted human ES-derived neural

progenitors into newborn mice differentiated into all three

neural lineages, including oligodendrocytes. The most important

potential hazard in stem cell (and especially ES cell)

transplantation is the risk of tumor formation. By definition, ES

cells are capable of forming teratomas after transplantation.

These teratomas contain cells of the three embryonic layers, i.e.,

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ectoderm, mesoderm, and endoderm. When undifferentiated

mouse ES cells were transplanted into the brain, they could

generate brain cells, but at the expense of teratoma formation

(Svendsen and Ebert, 2008).

Clearly, the commitment to a restricted neural lineage should

be complete before transplantation in order to eliminate this

problem. Indeed, transplantation of mouse and human ES-

derived neural precursors, obtained after multiple in vitro

passages and exposure to various growth factors, did not result

in teratoma formation. Another problem in stem cell

transplantation is the possibility of graft rejection. In most

studies of xeno-transplantation of human cells into rodents, graft

rejection did not pose a significant problem. However, there is

no long term systematic follow up of the survival of these cells

in the brain (Einestien and Ben-Hurr, 2008).

Bone marrow stromal cells:

A central issue in the field of stem cell biology is the

suggestion that plasticity of stem cells is marked to the degree

of promiscuity, where stem cells of one tissue may generate

cells of other tissues. It has been shown that adult mouse and

human bone marrow stromal cells (BMSCs) can differentiate in

vitro into other cell types, including muscle, skin, liver, lung,

and neural cells. In humans affected by hematologic

malignancies, peripherally injected BMSCs enter the brain and

produce new neurons and microglia. Recent reports suggest that

these cells could contribute to the generation of new neural cells

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in the adult brain by a direct conversion of transplanted BMSCs

into neurons or oligodendroglial cells, referred to as

transdifferentiation and/or assimilation of transplanted cells or

their progeny into existing neurons and formation of

heterokaryons, referred to as cell fusion (Crosta, 2010).

In rats with a demyelinated lesion of the spinal cord,

intravenous or brain injection of isolated mononuclear BMSCs

resulted in varying degrees of remyelination. In addition, bone

marrow derived stromal cells from transgenic green fluorescent

protein (GFP) mice that were injected directly into the

demyelinated spinal cord of immunosuppressed rats produced

myelin and improved axonal conduction velocity. These

observations stress the notion that it may not be mandatory to

introduce glial committed cells for remyelination in vivo, as the

developing and acutely demyelinated CNS may instruct other

cells to differentiate into the required lineage. Accordingly,

BMSCs might be useful as a therapeutic tool for brain repair by

autologous transplantation (David, 2009).

Problematic issues in cell transplantation for demyelinating

diseases:

Most experimental data on cell transplantation for

remyelination has been obtained from genetic dysmyelinating

models, where transplanted cells integrate into the normal

developmental program of the CNS, or in models of acquired

focal demyelination. However, the chronic and multifocal

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nature of MS raises several crucial issues that need to be

considered in order to bring the therapeutic cell transplantation

approach closer to clinical reality (Battler and Leor, 2006).

Route of cell delivery:

Another key issue for cell transplantation in MS is the route

of cell delivery. Because MS is a multifocal disease, it is

impossible to introduce regenerating cells into all foci of

disease. Moreover, it is often difficult to determine which of the

multiple foci observed in the brain by magnetic resonance

imaging (MRI) is most important clinically. Also, current

neuroimaging techniques do not identify the specific

pathological pattern of the lesion, and whether it is amenable for

effective remyelination (Svendsen and Ebert, 2008).

Therefore, it is necessary to contemplate the optimal route of

cell delivery that will promote efficient targeted migration of

transplanted cells into multiple lesions for repair. Because most

white matter tracts that are involved in MS are in close

proximity to ventricular and spinal subarachnoid spaces, then

intraventricular and intrathecal transplantation may serve as an

efficient route of delivering remyelinatingcells. After

intracerebro-ventricular injection, transplanted cells may

disseminate throughout the neuroaxis without a separating

barrier from the CNS white matter. Intraventricular

transplantation of OPCs and stem cells led to widespread

myelination in the genetic dysmyelinating models of the shi

mouse and the md rat (Goldman and Windrem, 2006).

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Migration of transplanted cells in the brain:

A crucial feature of transplanted cells is their ability to

migrate into inflamed brain areas, integrate, and differentiate.

The regenerative potential of transplanted cells is dependent on

their ability to arrive to the active inflammatory demyelinated

lesions. Cell migration is a major limiting factor in

remyelination. In the lesioned CNS, spontaneous remyelination

is a local event because of the limited migration of endogenous

remyelinating cells. Whereas transplanted multipotential NSCs

migrate and integrate in the embryonic and newborn rodent

CNS and adopt cellular identity according to local and temporal

cues, the normal adult brain does not permit large distance

migration and does not support transplanted neural cell survival.

Transplanted precursors were found to possess superior

migratory capabilities (figure 22) (Battler and Leor, 2006).

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Figure (22): a MRI of an MS patient showing multiple lesions, mainly inthe periventricular area. b Green fluorescent protein (GFP)+, PSA-NCAM+neurospheres were transplanted into the ventricles of chronic EAE mice. cand d Magnetically labeled mouse PSA-NCAM+ cells can be observed inthe brain in vivo as hypointense signals on MRI. Coronal (C) and transverse(D) MRIs show the cells (arrows) migrating in the corpus callosum andfimbria (Battler and Leor, 2006).

After intraventricular transplantation of spheres, cells

migrated almost exclusively into inflamed periventricular white

matter tracts, but not into gray matter. There was a general

correlation between the severity of the inflammatory response

and the degree of transplanted cell migration into the brain.

After transplantation into EAE rats, themajority of the

precursor cells differentiate into glia cells (30%

oligodendrocytes, 25% astrocytes).

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Because MS is a multifocal relapsing disease, these findings

exemplified the potential use of an intraventricular

transplantation as a route of cell delivery in MS, bringing cells

close to white matter tracts and enabling their inflammation-

induced targeted migration. These findings suggest a linkage

between parenchymal inflammation and setting regenerative

mechanisms in motion. As such, the inflammatory process that

develops in disease foci during clinical relapses of

demyelinating diseases may serve to attract remyelinating cells.

Thus, the brain inflammatory process may have a dual,

contrasting action in inflicting brain injury and recruiting the

regenerative process simultaneously (Olek, 2010).

This stresses the notion that combination of cell

transplantation and immunomodulation for MS in the future will

need to be developed as nonreciprocally antagonistic modes of

treatments. To this end, it is important to dissect the pro

regenerative components in the inflammatory process and target

the immunomodulatory treatment without inhibiting

regenerative processes. Tracking transplanted cells can be

performed non-invasively to develop successful clinical (stem)

cell based therapies, it will be important to develop methods that

can assess the fate and distribution of cells non-invasively. It is

obvious that traditional histopathological methods for cell

detection used in animal studies, which requires the removal of

tissue, cannot be applied to patients in most cases (Charles and

Alfredo, 2005).

Stem Cell Therapy for Amyotrophic

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), first described by

Charcot in the nineteenth century is a relentlessly progressive

neurodegenerative disorder of motor neurons in cerebral cortex,

brain stem and spinal cord that causes muscle weakness,

disability, and eventually death, with a median survival of three

to five years. ALS is also known by the eponym "Lou Gehrig's

Disease," after the famous baseball player who was affected

with the disease. Motor neuron disease (MND) is the preferred

term in the United Kingdom, but in the United States ALS and

MND are sometimes used interchangeably (Maragakis et al.,

2010).

Cell transplant based neuroprotection:

A variety of different cell types have been examined with

the intent of modulating the multifactorial cascade of events

contributing to the pathological interplay between and within

microglia, astrocytes, and neurons at the local neuronal

microenvironment. These attempts to affect neuronal survival

have predominantly incorporated cell types known to directly or

indirectly serve a physiological support role, including olfactory

ensheathing cells, Sertoli cells, ESCs, and neural precursor cells

at varying levels of lineage restriction and differentiation.

Finally, the modification of local immunological function has

been alternately attempted through the use of both bone marrow

stromal cells and umbilical cord blood (Crosta, 2010).

Physiological support of cell engraftment:

Both olfactory ensheathing cells and Sertoli cells have been

exploited in mutant superoxide dismutase 1 (mSOD1) ALS

animal models because of their known in vivo physiological

support roles. Olfactory ensheathing cells provide trophic

support and guidance to advancing axons of the primary

olfactory receptor neurons of the nasal mucosa as they traverse

the cribriform plate before synaptic connection with projection

neurons of the olfactory bulb. In vitro studies have indicated

OEC production of mRNA encoding nerve growth factor,

BDNF, GDNF, and CNTF, whereas alternate studies have

corroborated the production of corresponding neurotrophic

proteins (Svendsen and Ebert, 2008).

Approaches to glial cell replacement:

The supplementation or replacement of endogenous astroglia

has been attempted with a variety of different cell types of

varied lineage restriction and differentiation potential from

embryonic, fetal, or adult tissues. The first successful attempts

to promote indefinite ESC survival in an in vitro culture utilized

murine cells supplemented with media containing a basal feeder

layer of inactivated mouse embryonic fibroblasts either with or

without leukemia inhibitory factor (LIF) supplementation.

Removal of LIF or the basal feeder layer, when present, resulted

in the initial differentiation of these cells, as evidenced by

embryoid body (EB) formation. Subsequent discoveries have

demonstrated that LIF application does not prevent EB

formation, as does the use of a basal feeder layer, when applied

to hESCs (Lindvall and Kokaia, 2010).

Cell transplant based neurorestoration:

The restoration of lost neurological function is a goal

relevant to the repair of damage due to a wide variety of causes,

including neurodegenera-tive conditions such as ALS.

However, this process requires successful negotiation of

multiple complex issues, including ability for transplanted

neuronal precursor cells to survive transplantation, terminally

differentiate, send peripheral axonal projections, generate

synaptic connections at the neuromuscular junction, and result

in functional incorporation (Charron et al., 2009).

Approaches to Motor Neuron Production:

Achieving the appropriate culture conditions required to

reproducibly generate cell types of a neuronal or neuronal

subtype lineage has required the application of detailed

knowledge of the microenvironment encountered by ESCs

residing within the developing neural tube. In the developing

embryo, growth promoting chemicals called “morphogens”

provide directional cues along both dorsoventral and

rostrocaudal planes, guiding neuronal growth. Therefore,

identification of the appropriate morphogens followed by their

subsequent application at empirically derived concentrations

and time points represents a means to recapitulate the in vivo

conditions promoting terminal differentiation into cell types of

defined lineages. The application of retinoic acid as a

caudalizing factor and sonic hedgehog as a ventralizing factor to

age appropriate embryonic cells can be used to preferentially

differentiate this pluripotent cell type into a terminally

differentiated motor neuron. Enhancement of neurite outgrowth

has been extensively studied in vitro and is affected by a variety

of substances such as cAMP analogs, fatty acid derivatives,

phosphodiesterase inhibitors, and Rho kinase inhibitors (Jodi et

al., 2004).

Role of Stem Cell Therapies in ALS:

Stem cells could help patients with ALS in several ways.

Ideally, they could be induced to differentiate into lower motor

neurons in order to replace those neurons that die because of

ALS. Perhaps stem cells could rescue dying motor neurons by

reconnecting these neurons to partly denervated muscle before it

has died completely. Better yet, they could be induced to

differentiate into upper motor neurons in the cortex and connect

to the lower motor neurons (Metwally, 2009).

Stem cells also play a supportive role in maintaining the

viability of or extending the function of surviving motor

neurons. The stem cells could be induced to differentiate into

supporting cells, glia, or interneurons that might produce factors

that would support motor neurons, or perhaps the stem cells

themselves might produce such factors (Svendsen and

Langston, 2004).

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Stem Cell Therapy for Muscular

Dystrophy

The muscular dystrophies are an inherited group of

progressive myopathic disorders resulting from defects in a

number of genes required for normal muscle function. The

Duchenne and Becker muscular dystrophies are caused by

mutations of the dystrophin gene and are therefore named

dystrophinopathies. Weakness is the principal symptom as

muscle fiber degeneration is the primary pathologic process.

Muscular dystrophies, include X-linked recessive as in

Duchenne MD, autosomal recessive as in limb-girdle MD type

2, or autosomal dominant as in Facioscapulohumeral MD,

myotonic dystrophy and limb girdle MD type 1 (Darras, 2010).

The dystrophinopathies have varying clinical characteristics:

• Duchenne muscular dystrophy (DMD) is associated with themost severe clinical symptoms

• Becker muscular dystrophy (BMD) has a similar presentationto DMD, but a relatively milder clinical course

• An intermediate group of patients, known as "outliers," maybe classified clinically as having either mild DMD or severeBMD (Gussoni et al., 2002).

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Cell therapy:

The concept of a cell based therapy to promote muscle

regeneration, in particular in recessive types of MD, originated

with the observation of the intrinsic ability of myofibers to fuse

to each other. Thus, functional correction could be obtained by

generating hybrid muscle fibers, where the donor nuclei provide

the missing gene product. Based on this premise, several

investigators have assessed the ability of adult myoblasts to treat

DMD, which is characterized by the lack of dystrophin (David,

2009).

Murine models for muscular dystrophy include the naturally

occurring mdx mice. The mdx mice are dystrophin deficient due

to a stop codon point mutation in exon 23 of the dystrophin

gene. Although initial results in mdx mice were encouraging,

early clinical trials failed due especially to the poor survival and

limited migratory ability of injected myoblasts (Gabellini et al.,

2006).

The lesson learned from these early studies is that a more

primitive cell population endowed with self renewal and

differential potential would be preferable for therapeutic

applications. Accordingly, a number of studies involving

different sources of stem cells, including muscle satellite cells,

vessel derived stem cells (mesoangioblasts) and embryonic stem

cells have provided better outcomes with wider distribution of

transplanted cells, higherlevels of engraftment, and

importantly, improvement in the contractile properties of

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muscles from transplanted dystrophic mice (Cerletti et al.,

2008).

Because all these studies were performed in recessive forms

of MD, it is unknown to what extent dominant forms of MD

could benefit from stem cell based therapy. Since it takes fewer

nuclei to add back a missing product than it takes to dilute out a

deleterious product, the bar is expected to be higher for effective

treatment of a dominant disease, however this will depend on

the specific disease mechanism (Sampaolesi et al., 2006).

In experimental studies, engraftment and ameliorate the

phenotype in a mouse model would produced for a dominant

neuromuscular disorder. For these studies, we have chosen mice

over expressing FRG1, which have been proposed as a mouse

model for Facioscapulohumeral dystrophy (FSHD). Transgenic

mice over expressing FRG1 (medium and high expressers)

develop a myopathy and phenotypic abnormalities that are

typical of MD, including abnormal spinal curvature, skeletal

muscle atrophy, increased variability in fiber size, fiber

necrosis, centrally located nuclei and fibrosis (D’Antona et al.,

2007).

Although there is some controversy in the field as to the

relationship of this pathophysiology to human FSHD, for the

purposes of this study, the salient feature is that it is dominantly

inherited. A total of 24 FRG1-medium mice at 12 weeks of age

were used for these experiments. All mice were treated with an

immunosuppressive agent (tacrolimus; Sigma) to prevent

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rejection of nonisogenic cells at a daily dose of 5mg/kg

intraperitoneally (IP) from the day before cell injection until the

time of euthanasia (30 days) (David, 2009).

EBs were subjected to Pax3 induction (doxycycline was

added to the EB culture medium) from day 2 to day 5 of EB

differentiation, at which point early myogenic progenitors were

purified by FACS using antibodies to PDGFαR and Flk-1.

PDGFαR+Flk-1- cells were expanded in the same medium

containing doxycycline for 7-10 days and then transplanted into

FRG1 transgenic mice In the first series of experiments, female

FRG1 mice were transplanted with Pax3-induced ES derived

myogenic progenitors through intramuscular (i.m.; n=4) and

intravenous (i.v.; n=4) injections. 24 hours before

transplantation, 15 μl of cardiotoxin (10μM, Sigma) was

injected into the tibialis anterior (TA) muscle of each mouse

(both legs) to induce muscle injury (Sampaolesi et al., 2006).

In the intramuscular group, mice received 1 × 106 cells by

direct intramuscular injection into the left tibialis anterior (TA)

muscle whereas the right TA muscle received only PBS

(control). In the systemic i.v. group, mice received 5 × 105 cells

through tail vein injection while the control group (n=4)

received PBS. After 30 days, muscles were harvested and

analyzed by immunofluorescence to assess engraftment by the

presence of GFP (donor cells) and MHC, a marker of terminal

muscle differentiation. In a second set of experiments, female

and male FRG1 mice were transplanted with Pax3-induced ES-

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derived myogenic progenitors through intramuscular injection

(a total of 6 mice in each group), as described above, and

muscle function was assessed 30 days following the

transplantation. Muscle cryosections were also evaluated for

engraftment (Deasy et al., 2007).

Muscles were frozen in isopentane cooled in liquid nitrogen.

Tissue cryosections (6-8 mm) were fixed with cold acetone for

5 min., permeabilized with 0.5% Triton X-100 (Sigma) for 20

min., blocked with 10% goat serum (or 3% BSA) for 1 hr, and

then incubated with primary antibodies: anti-MHC (1:20

overnight 4°C) and chicken anti-GFP (1:500, 1hr RT; Abcam).

For secondary staining, appropriate secondary alexa fluor

antibodies were used (Invitrogen). As observed, whereas PBS-

injected FRG1 mice were negative for GFP, transplantation of

Pax3-induced ES-derived myogenic progenitors, independent of

the route of administration, resulted in substantial engraftment

as evidenced by the presence of GFP+MHC+ myofibers. In the

case of intramuscular injection, transplanted cells migrated and

engrafted well around the site of injection. As observed

previously, these cells were able to home to the muscle

following their systemic injection. TA muscles from these mice

contained about 14.5 ± 1.07 % of GFP+ myofibers (D’Antona

et al., 2007).

The measurement of muscle contractile properties was

performed as previously described. Interestingly we observed

gender differences in regard to the contractile ability of TA

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muscles from FGR1 mice. Whereas male FRG1 mice showed

significant reduction in their contractility ability when compared

to the B/6 control group, female mice demonstrated no

functional deficit. Accordingly, cell transplantation had no

effect in this latter group, while the transplantation of Pax3-

induced ES-derived myogenic progenitors in male FRG1

resulted in improvement of maximal isometric tetanic and

specific force. It is important to note that engraftment levels

were similar among female and male groups. CSA and weight

were not affected by cell transplantation. According to our

previous results, while systemic delivery of ES-derived

myogenic progenitors into mdx mice reduced these parameters

close to wild-type mice, intramuscular transplantation did not

affect CSA and weight, as observed here. However it is

important to note that mdx and FRG1 mice present quite distinct

phenotypes. While mdx mice are hypertrophic due to the

multiple rounds of degeneration and regeneration, FRG1 mice

are atrophic (David, 2009).

Gender differences in skeletal muscle have been

demonstrated with energy metabolism, hormonal status, and

muscle fiber types. For instance male skeletal muscles generally

contain more fast fibers than female muscles. Conversely,

during repeated contractions, female muscles are generally more

fatigue resistant and recover faster. It has been shown in FRG1

mice that fast fibers are preferentially involved in the pathology

(D’Antona et al., 2007).

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Accordingly, staining for slow and fast isoforms of MHC in

male and female FRG1 mice showed gender specific

differences. Female FRG1 mice contained significantly more

slow myofibers than males, as evidenced by NADH tetrazolium

reductase staining, while immunohistochemistry for a fast

isoform of MHC revealed a lower frequency of fast myofibers.

This may explain, at least in part, the results observed here.

Consistently, female FRG1 mice presented much less fibrosis

than their male counterparts, as evidenced by Mason’s

Trichrome staining, which corroborates their milder phenotype.

It is important to note that cell transplantation did not affect

fibrosis or the ratio of fast and slow myofibers in male or female

FRG1 mice. Moreover, it has recently been pointed out that

gender also affects stem cell mediated skeletal muscle

regeneration, with female muscle derived stem cells (MDSCs)

having higher potential for muscle regeneration than their male

counterparts. Although these studies were performed in mdx

mice through transplantation experiments, one could

hypothesize that resident MDSCs in female FRG1 mice are

better able to respond to the damage caused by overexpression

of FRG1 (Deasy et al., 2007).

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Stem Cell Therapy for Huntington's

Disease

Huntington disease (HD) is an inherited progressive

neurodegenerative disorder characterized by choreiform

movements, psychiatric problems, and dementia. The disease is

named after the American doctor George Huntington, who first

described the condition in detail in 1872. It is caused by a

trinucleotide (CAG) expansion in the Huntington gene on

chromosome 4p and inherited in an autosomal dominant pattern.

The pathophysiology of HD is not fully understood, although it

is thought to be related to toxicity of the mutant huntingtin

protein (Sutton and Suchowersky 2010).

Current work in HD cell therapy is broadly classified into 1

of 3 aims:

1) To harness the ability of the brain to self repair through the

upregulation of endogenous stem cells/neurogenesis;

2) To replace dead and/or dying neurons through fetal or stem

cell transplantation; and

3) To protect neurons vulnerable to disease progression through

the administration of neuroprotective trophic factors via cell or

viral delivery.

Clinical trials of fetal derived cell transplantation in the

striatum have been performed since the 1990s in patients with

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HD. However, a more cogent understanding of the pathological

processes that give rise to HD and the behavior of both

endogenous and exogenous cells in the diseased brain will

contribute to not only additional or improved therapeutic

strategies, but also a better context through which the success of

therapeutic manipulations can be analyzed and understood

(figure 23) (Claire et al., 2008).

Figure (23): Drawing illustrating therapies in HD. Sites of celldegeneration (caudate and putamen) in HD appear in red, and ongoing adultneurogenesis in the human brain appears in green. Target of fetal derivedcell transplantation into the diseased striatum is represented by the syringe(Claire et al., 2008).

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Restoring or manipulating neurogenesis as endogenous cell

therapy in HD:

One focus of cell therapy has been to understand the deficits

in endogenous stem cell regulation in HD, primarily in the adult

hippocampal, dentate gyrus (DG) and SVZ, with the aim that

restoring or upregulating endogenous neurogenesis may

partially ameliorate disease symptoms and repair the damaged

brain. Neurogenesis occurs in the hippocampus and SVZ of

adult mammals, including humans (figure 24) (Johann et al.,

2007).

Figure (24): Drawing depicting sites of neurogenesis in the rodent brain. A:Newborn hippocampal neurons (blue) are born in the subgranular zone ofthe hippocampus and migrate into the granule cell layer of the DG as theymature and integrate. B: Neurons (blue) originate in the SVZ lining thelateral ventricles and migrate via the rostral migratory stream (RMS) to theolfactory bulb. CA1 = cornu ammonis 1 of the hippocampus proper; CA3 =cornu ammonis 3 of the hippocampus proper; GCL = granule cell layer; GL= glomerular layer (Johann et al., 2007).

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Neural stem cells in the SVZ olfactory bulb and DG can

differentiate into all lineages of the adult CNS including

neurons. Furthermore, endogenous NSCs outside the 2 standard

neurogenic regions have been shown, in some instances, to have

the capacity to differentiate into neurons and integrate

appropriately in regions undergoing cell death (Chen et al.,

2004).

Neurodegeneration or injury can upregulate proliferation and

promote the migration of newborn cells to the site of damage.

However, it is not clear if this increase in cell genesis is

neuroprotective and/or linked to functional recovery, if the

increase is of a magnitude to be of therapeutic value, or if this

upregulation in proliferation represents pathological network

reorgani-zation (Johann et al., 2007).

Neural stem cells in the SVZ and hippocampal subgranular

zone develop into electrically mature and fully integrated

neurons. In the subgranular zone ofthe hippocampus,

progenitor cells become neurons predominantly and

functionally integrate into the DG. Neural precursor cells

originating in the SVZ of the lateral ventricle migrate

tangentially through the rostral migratory stream, guided by

astrocytes to the olfactory bulb where they become fully

differentiated and electrically competent. Neurogenesis has

been implicated in learning and memory and may be necessary

for the functional integrity of the circuits into which new cells

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generate. The rate of neurogenesis in adult mammals is

dynamically regulated by a variety of physiological and

pathological factors (Jin et al., 2005).

The microenvironment plays a key role in maintaining the

neurogenic potential of the adult brain; it also plays a role in the

fate restriction of endogenous NSCs in neurogenic regions.

Local conditions of the micro-environment, including factors

released by astrocytes, play a crucial role in neurogenesis.

Dysregulation of this environment in the degenerating brain can

result in a non permissive environment for neurogenesis

(Svendsen and Ebert, 2008).

Despite the expression of mutant the huntingtin gene (htt),

the proliferation, longevity, differentiation, and survival of

NPCs derived from the hippocampus and SVZ of HD transgenic

mice are similar to those of wild type derived NPCs in vitro,

suggesting that abnormalities in neuro-genesis in vivo are due to

the microenvironment in which the NPC resides and not the

intrinsic properties of the precursor itself. Although there is no

change in basal SVZ NPC proliferation or maturation in a

rodent model of HD, the absence of increased proliferation of

SVZ NPCs in response to quinolinic acid indicates deficits in

the SVZ microenvironments (Phillips et al., 2005).

Although little work has been done on the effect of HD on

adult human hippocampal neurogenesis, the SVZ in adult

humans with HD has been well characterized, increased cell

proliferation in, and the thickness of, the SVZ correlate with

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pathological grade and CAG repeat length. It is unclear whether

this increase in proliferation is a protective response by the CNS

to replace or protect damaged cells in the striatum or whether

the upregulation in neurogenesis contributes to pathology in the

striatum, Most of the cells born in the diseased SVZ exhibit

phenotypes of glial cells (progenitors or mature glial fibrillary

acidic protein positive astrocytes) with fewer neurons.

Furthermore, this effect is region specific there is increased

proliferation in the central and ventral regions of the SVZ,

whereas striatal atrophy occurs mostly in the dorsal region

(Battler and Leor, 2006).

The birth site of new neurons in proximity to the

degenerating striatum as well as the upregulation of

neurogenesis in this region in response to damage in HD raises

the possibility that the brain can at least partially compensate for

neurodegeneration. Indeed, in a rat lesion model of HD, SVZ

progenitor cells have been shown to migrate from the site of

their inception toward the lesion. A better understanding of the

mechanisms underlying adult neurogenesis within the context of

HD would contribute to our ability to harness the therapeutic

potential of these cells (Svendsen and Ebert, 2008).

Strikingly, the beneficial impact on cognitive and motor

behavioral measures through the restoration of adult

neurogenesis in rodent models of HD has revealed the

therapeutic potential of endogenous NPCs. The induction of

neurogenesis through trophic factors or antidepressant treatment

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has alleviated motor and cognitive deficits in rodent models of

HD. In a mouse model of HD, treatment with fibroblast growth

factor–2, a neuroprotective growth factor, increased cell

proliferation by 150% in the SVZ (compared with 30% in wild

type littermate controls) and was accompanied by an increase in

immature doublecortin positive neurons and a decrease in

aggregations formed by mutant huntingtin protein. These cells

migrated to the striatum (the primary site of pathology) and

formed projections. Improved motor performance on a test for

motor coordination (rotarod) and extended lifespan were also

observed (Jin et al., 2005).

Cognitive and hippocampal neurogenic deficits in HD

transgenic mice can be rescued (that is, return to normal or near

normal levels of behavior) by chronic administration of the

antidepressant fluoxetine without affecting motor deficits

characteristic of HD and thus suggesting that in addition to

striatal pathology, secondary abnormalities in adult

hippocampal neuro-genesis play a role in the affective

symptoms of HD. This finding is in line with the dependency of

behavioral outcomes on hippocampal neurogenesis following

antidepressant treatment. Finally, the forced induction of

neostriatal neurogenesis also slows disease progression and

alleviates motor and cognitive impairments in a mouse model of

HD By correcting abnormalitiesin adult hippocampal

neurogenesis or manipulating endogenous neurogenic regions to

contribute to the repair of degenerating neuronal populations, it

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is theoretically possible to nonsurgically treat both the

underlying pathologies and the cognitive and motor symptoms

of HD (Chen et al., 2004).

Transplantation of fetal derived cells as exogenous cell

therapy in HD:

One of the most studied areas of HD cell therapy is the

transplantation of fetal derived cells into the brain of a

symptomatic patient with HD. Extensive work on the safety and

efficacy of the transplantation of fetal striatal allografts into a

diseased adult brain has been completed in both rodent and

primate models, and several clinical trials have been performed

since the 1990s. Although full recovery has not been observed

following fetal allografts in the striatum of humans with HD,

some suggestions of delayed disease progression indicate

positive functional outcomes. The contribution of these grafts to

functional recovery is enhanced by the fact that implanted cells,

lacking the disease causing gene, do not themselves appear

vulnerable to neurodegenerative processes, an effect that is also

seen in transplants in patients with PD (Bachoud-Lévi et al.,

2006).

All clinical trials to date have been focused on the

transplantation of fetal derived cells into the diseased striatum.

Because HD pathophysiology involves several brain regions,

including nonstriatal sites, cell implantation into the striatum

alone may limit the ability of grafts to address or reverse HD

symptoms. Whereas grafted regions are relatively spared from

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neurodegeneration, surrounding brain regions continue to

degenerate, which may limit the functional outcome of

transplantation. Furthermore, the timing of transplantation may

be crucial to the functional benefits of the graft; the majority of

patients involved in clinical transplantation trials have had mild

to moderate HD, and the impact of the disease stage on

transplant efficacy remains to be measured (Claire et al., 2008).

Much evidence has accumulated about the efficacy of fetal

derived cell transplantations in animal models of HD. All

striatal cell types from the allo and xenotransplantation of fetal

or embryonic derived striatal tissue have been shown to survive,

grow, and establish functional afferent and efferent connections

to host tissue and display appropriate electrophysiolo-gical

properties in both rodent and primate models of HD. authors

have demonstrated that behavioral motor deficits are improved

by the transplantation of striatal tissue into the degenerate

striatum of rodents and primates. Both pre and postoperative

motor training in animal models of HD have also had a

significant impact on functional recovery, presumably by

enhancing the plasticity of grafts and host circuitry (Svendsen

and Ebert, 2008).

In primates, striatal allografts and xenografts survive,

differentiate into mature dopamine and cyclic adenosine

monophosphate regulated phosphoprotein positive striatal cells,

and receive dopaminergic innervation. These changes are

correlated with improvements in both motor performance,

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including recovery in a test of skilled motor performance, and

cognitive function. Cellular and behavioral improvements

following fetal derived striatal cell transplantation are thought to

occur through circuit reconstruction, the normalization of

neurotransmitter release, and/or the production of trophic

factors (Claire et al., 2008).

In this respect, van Dellen and colleagues have demonstrated

the potential therapeutic benefit of targeting the host cortex as a

site of transplantation. Wild type fetal cortical cells were

transplanted into the anterior cingulate cortex of neonatal HD

transgenic mice. This therapy delayed the onset of an overt

motor deficit but did not enhance motor coordination. This

study highlights the potential benefit of targeting the widespread

pathophysiology of HD, not just neurodegeneration in the

striatal regions, as it may allow preservation of striatal function

through the delivery of neuroprotective trophic factors, such as

brain derived neurotrophic factor (BDNF), from spared

nonstriatal regions that project to the striatum (Bachoud-Lévi et

al., 2006).

Clinical trials involving the transplantation of fetal striatal

tissue in humans were begun in the late 1990s. As in animal

models of HD, most transplanted tissue was derived from the

human fetal striatum (whole or partial ganglionic eminence) and

transplanted into the host striatum. The amount and cellular

composition of the transplanted cells must be regulated, as

transplants with, 30% striatal content are ineffective, and the

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selective dissection of striatal primordial tissue (that is, the

lateral region of the lateral ganglionic eminence) has proven

disappointing from a functional perspective. As in PD, the

safety of allografts of fetal striatal neural tissue has been

demonstrated in HD. Similar to its successful use in cell therapy

for PD, hibernation medium used to store harvested cells for up

to 8 days has allowed the collection of adequate amounts of

tissue and the flexibility to prepare tissue at sites and times other

than the site and time of transplantation surgery (figure 24)

(Claire et al., 2008).

Figure (25): Flair MRI obtained in a patient with HD who had received agraft of fetal derived striatal tissue into the caudate and putamen, showingthe patient’s status 3 (A), 8 (B), 13 (C), and 32 months (D) aftertransplantation. The origin of the apparent persistent signal along the impacttract is unknown but may represent surviving donor tissue or host glialreaction (Claire et al., 2008).

Graft survival has been reported in several clinical trials.

MRI has been successfully used to locate and monitor the

growth of grafted tissue, and metabolic assessment has also

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been effective in evaluating the efficacy of transplantation.

Tissue at the site of grafting has been shown on MRI to increase

in growth by 6 months post transplantation, without overgrowth.

Furthermore, inferences regarding the health of the graft, based

on MR spectroscopy and PET, suggest that graft sites are

populated by adult neurons that are metabolically active. For

example, the maturation of transplanted cells led to the

stabilization of brain striatal and cortical metabolism in 3 of 5

patients receiving bilateral fetal striatal grafts in an open label

study. In the remaining 2 patients, striatal and cortical hypo

metabolism progressed (Johann et al., 2007).

However, reductions in metabolic activity, including a

significant loss of dopamine receptor binding, have been

documented in patients 2 years following intrastriatal

transplantation of fetal derived cells. A 7% decline in striatal

metabolism was observed on fluorine-18 labeled

fluorodeoxyglucose PET over a 6 year period in patients who

had received functional benefit from the transplants; although

not significantly different, this decline was lower than the

average for patients with HD who did not receive implants. On

average, dopamine receptor (D2) binding declines by 5-15% in

control patients with HD as measured on PET. In general,

increases in hypometabolism in patients with HD who have

received transplants are consistent with those in control patients

with HD, although 1 patient showing clinical improvement after

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transplantation also showed improvement on striatal raclopride

PET scans (Claire et al., 2008).

In addition, postoperative FLAIR imaging showed a

persistent signal that could represent grafted tissue, host reaction

(gliosis), or both. Postmortem histological evidence for the

survival and integration of fetal derived cell striatal

transplantation has also been reported in 3 patients who died

between 18 and 79 months after the procedure. Fetal lateral

ganglionic eminence was bilaterally grafted into striata with

HD. Autopsy results revealed that grafts were clearly

demarcated and grew to 5-10% of the normal human caudate

putamen tissue volume after 18 months in 1 patient. Grafted

cells displayed morphological features characteristic of both the

developing and the mature striatum and were innervated by

appropriate host target regions, although the presence of

efferents was very limited in the tissue from patients surviving

6 years after transplantation, despite minimal gliosis within the

grafted tissue (Keene et al., 2007).

Notably, grafted fetal striatal cells survive, develop, and

integrate into host tissue without being affected by the disease

process itself; that is, no mutant huntingtin aggregates were

observed within the transplanted region, and the graft showed

no signs of immunological rejection. The sparing of grafted

tissue from disease progression has also been reported in other

transplant cases, including in patients with PD. The success of

allografts of fetal derived striatal tissue in patients with HD

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hinges on motor and cognitive improvements following

transplantation (Chen et al., 2004).

Improvements on the Unified Huntington’s Disease Rating

Scale motor score have been reported as early as 1 month after

unilateral grafting of fetal derived striatal tissue and have

stabilized between 6 months and 2 years after transplantation.

Patients assessed up to 6 years after bilateral transplantation

have demonstrated that immediate improvements in motor

scores (between 6 months and 2 years) may not persist long

term. However, the rate of motor and cognitive decline may be

reduced in some patients who have undergone transplantation.

Of those patients exhibiting initial functional and metabolic

benefits from transplantation in the clinical trial, clinical

improvement reached a plateau after 2 years and declined

variably from 4 to 6 years. Dystonia progressed more rapidly

and consistently than other motor symptoms, but transplantation

of fetal derived striatal tissue appeared to alleviate choreatic

symptoms (Bachoud-Lévi et al., 2006).

Cognitive performance was also stabilized on non timed

tests in these patients. The remaining 2 patients who showed

neither metabolic nor functional improvement at the initial 6

month follow up continued to decline over the 6 year course of

study at a rate similar to that seen in patients with HD who had

not received transplants (Rosser et al., 2002).

Clinical trials have included patients in earlier stages of the

disease, which reduces the risks of this complication.

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Complications from immunosuppressive drug treatment

following transplantation have been generally predictable with

largely reversible side effects and thus have not been a major

safety concern. However, the issues of whether

immunosuppressive therapy is needed for intracerebral

transplantation and the optimal duration of immunosuppressive

treatment have not been resolved (Claire et al., 2008).

The use of fetal derived striatal cells for transplantation has

shown that grafts into the degenerative adult brain are safe and

at least partially efficacious in terms of some immediate

functional response. However, this approach seems to be less

viable over the long term. In addition to the ethical and practical

considerations regarding the source of cells for human fetus

derived allografts and the limited repair potential of striatal only

grafts, many groups have turned to alternative strategies for

obtaining cells for therapy in HD, the most notable of which has

been the utilization of stem cells (Keene et al., 2007).

Stem Cell Based Cell Therapy in HD

Stem cell therapy in the adult brain with HD has received

increasing attention for its potential to mitigate pathological

neurodegeneration by replacing lost neuronal and/ornon

neuronal populations, by triggering endogenous repair

mechanisms, or by protecting existing cells primarily through

trophic support. Stem cell transplantation is a promising field in

cell therapy for HD as stem cells are relatively easy to obtain

compared with primary fetal tissue and have the potential to be

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manipulated to eliminate possible problems of host rejection.

Neural stem cells can be isolated from the fetal, neonatal, and

adult brain and propagated in culture (McBride et al., 2004).

Grafted NSCs can develop both morphological and electrical

properties of mature neurons and integrate into host circuitry.

Transplanted immature neurons or embryo derived NPCs have

been shown to migrate toward the site of degeneration,

differentiate, and form synaptic connections in several models

of neurodegeneration. Transplanted ESCs can differentiate into

neurons in the HD affected striatum and appear to migrate to

nearby cortical regions where they have been found to express

markers of immature neurons. One of the first challenges to

stem cell therapy in HD is to determine which source of stem

cells is most efficacious, and many sources have been

examined. In addition to human ESCs, stem cells derived from

mesenchyme in adults have been investigated as a readily

available source of stem cells in HD (Song et al., 2007).

Whereas few neurons have been formed from mesenchyme

derived stem cell grafts, transplantation of these grafts has

elicited some behavioral recovery. Similarly,

xenotransplantation of adult peripheral precursor cells

originating from porcine Sertoli cells of the testes has shown the

rescue of locomotor impairments in an HD lesion model in rats,

probably acting through neuroprotection given the effectiveness

after only a short time (McBride et al., 2004).

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Neural stem cell treatments have resulted in either full or

partial functional recovery in tests of lesion induced

impairment. Transplanted human and mouse derived NPCs in

an excitotoxic HD lesion model not only have survived in the

striatal site of implantation, but also have migrated and stained

for markers of immature neurons in cortical regions. Following

transplantation into a mouse model of HD, murine ESC derived

NPCs, genetically modified to promote neuronal differentiation,

formed GABAergic neurons with appropriate outgrowth. The

grafts were also functionally beneficial given that the animals

showed improvement in rotational behavior. Human NSCs

appear to behave similarly to murine derived NSCs in rodent

models of HD. Intravenously transplanted human NSCs migrate

to the striatum, reduce striatal atrophy, and contribute to

functional improvement in a rodent lesion model of HD (Song

et al., 2007).

The differentiation of stem cells or treatment with growth

factors in vitro prior to implantation may facilitate fate

determination while mitigating the risk of tumor formation

posed by stem cells. Additionally, stem cell therapy has the

potential to alleviate cognitive deficits in degenerative disease,

such as memory impairment, through the replacement of

degenerating cells and the restoration or preservation of proper

network function (Claire et al., 2008).

Although the gold standard in stem cell treatment has been

the production of neurons, the differentiation of transplanted

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cells into non neuronal lineages, such as astrocytes, may serve

neuroprotective or regenerative roles in degenerative

environments. Additionally, there is at least corollary evidence

that diseased glial cells contribute to HD pathophysiology.

Endogenous or transplant derived nonneuronal cells must be

included in any consideration of the efficacy of stem cell

therapy in HD. In neurogenic regions, astrocytes interact with

NSCs and NPCs to promote proliferation and fate determination

and may stimulate the maturation and integration of newly

formed neurons (Song et al., 2007).

The contribution by non neuronal cells may be particularly

relevant in regions affected by HD in which it is hypothesized

that abnormalities in the microenvironment, rather than neuron

intrinsic properties, primarily contribute to the degenerative

environment. Taken together; results from initial stem cell

therapy investigations have indicated that stem cells are viable

cell sources for transplantation in animal models of HD (Claire

et al., 2008).

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Stem cell therapy for Alzheimer's

Disease

One hundred years ago, 1906, the German psychiatrist Alois

Alzheimer presented the case of a 54 years old woman who

suffered from progressive dementia. He described the clinical

and pathological symptoms for the first time, now known as

Alzheimer’s disease (AD) (Ermini, 2006).

AD is a neurodegenerative disorder characterized by

progressive dementia with memory loss as the major clinical

manifestation. Impairment of short term memory is often

prominent, but remote memory loss also appears to be affected

over time. Other important features of AD are an altered

behavior including paranoia, delusions, depression, impairments

of attention, perception, reasoning and comportment, and a

progressive decline in language function (Grabowski, 2010).

The main neuropathologic findings in the AD brain are

amyloid plaques which are multicellular lesions containing

extracellular deposits of amyloid β-protein (Aβ) consisting of

amyloid fibrils (7-10 nm) intermixed with non fibrillar forms of

the Aβ peptide. Neuronal and synaptic loss, neurofibrillary

tangles, and deposits of β amyloid protein in senile plaques

involve the basal forebrain cholinergic system, amygdala,

hippocampus, and cortical areas. The plaques are surrounded by

dystrophic neuritis, another form of Aß deposition appears as

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diffuse or pre amyloid plaques where Aβ occurs in a non-

fibrillar, less dense and amorphous form in the neuropil,

cerebral amyloid angiopathy (CAA) is detected in 30 % of the

general population, compared to 80% in AD patients (Ermini,

2006).

Stem cell based therapies for AD:

The situation for neuronal replacement aiming at functional

restoration in AD is extremely complex because the stem cells

would have to be pre differentiated in vitro to many different

types of neuroblasts for subsequent implantation in a large

number of brain areas. Since acetylcholinesterase inhibitors,

which enhance cholinergic function, induce some temporary

improvement in AD patients, the cognitive decline could

hypothetically be improved by transplantation of stem cell

derived basal forebrain cholinergic neurons (figure 26) (Lindvall

and Kokaia, 2010).

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Figure (26): Stem cell based therapies for AD. AD leads to neuronal loss inthe basal forebrain cholinergic system, amygdala, hippocampus, andcortical areas of the brain; formation of neurofibrillary tangles; and amyloidprotein accumulation in senile plaques. Stem cell based therapy could beused to prevent progression of the disease by transplanting stem cellsmodified to release growth factors (Lindvall and Kokaia, 2010).

Experimental evidence indicates that it should be possible to

generate such cells from stem cells. However, to give long

lasting symptomatic benefit, a cholinergic cell replacement

approach would require intact target cells, host neurons that the

new cholinergic neurons can act on, and they are probably

damaged in AD. Stem cell based cell replacement strategies are

very far from clinical application in AD. The disease symptoms

in AD could partly be due to impaired formation of new

hippocampal neurons from endogenous NSCs in the subgranular

zone of the dentate gyrus, which is believed to contribute to

mood regulation, learning, and memory. Mouse models of AD

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have provided equivocal data, with studies demonstrating both

increased and decreased hippocampal neurogenesis, one

important factor being disease severity, with a compensatory

increase in progenitor proliferation in the early stages and

decreased proliferation and survival with advanced pathology

(Zhao et al., 2008).

Formation of immature hippocampal neurons has been

reported to increase in senile AD patients, while in another

study, neurogenesis was not altered in pre senile cases. Later

studies have indicated deficient maturation of new neurons in

AD brains. As a result, approaches to enhance neurogenesis

and/or maturation could be considered potential stem cell based

therapies for AD. Clearance of brain β-amyloid has been

proposed to be of value in halting disease progression in AD.

Active β-amyloid vaccination in young AD mice, using as

antigen a sequence of the β-amyloid peptide, decreased β-

amyloid burden and increased hippocampal neurogenesis.

Moreover, passive β-amyloid immunotherapy with an antibody

specific for aggregated β-amyloid restored neurogenesis and

morphological maturation of new hippocampal neurons in aged

transgenic mice with β-amyloid related impairments of

neurogenesis (Charles and Alfredo, 2005).

Stem cell based gene therapy could deliver factors

modifying the course of AD and may be advantageous because

of the capacity of stem cells to migrate and reach large areas of

the brain. Preclinical studies that provide a rationale for this

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approach include one demonstrating that basal forebrain grafts

of fibroblasts producing nerve growth factor (NGF), which

counteracts cholinergic neuronal death, stimulate cell function

and improve memory in animal models of AD (Zhao et al.,

2008).

Stem cells could also be engineered to carry other genes,

such as that encoding BDNF, which has substantial

neuroprotective effects in AD models. Transplantation of

fibroblasts producing the β-amyloid degradingprotease

neprilysin have been shown to reduce plaque burden in AD

mice, although this may not lead to reduction in the number of

pathogenic β-amyloid oligomers or prevent cognitive deficits

(Lindvall and Kokaia (2010).

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Stem Cell Therapy for Cerebral palsy

Cerebral palsy (CP) consists of a heterogeneous group of

non progressive clinical syndromes that are characterized by

motor and postural dysfunction, it is caused by abnormal

development or damage in one or more parts of the brain that

control muscle tone and motor activity. The resulting

impairments first appear early in life, usually in infancy or early

childhood. Infants with cerebral palsy are usually slow to reach

developmental milestones such as rolling over, sitting, crawling,

and walking. Although the disorder itself is not progressive, the

appearance of neuropathologic lesions and their clinical

expression may change over time as the brain matures (Miller,

2010).

Stem cell implantation:

In clinical study, more than 88 cases are already treated by

stem cells with clinical improvement in around 70 % cases.

Cells of highest purity, viability and integrity from world quality

laboratory were used. As it has been proved that more

transplantation cycles gives better and better results. Any

perinatal injury to infant brain or hypoxic damage to cerebral

cortex is mainly responsible for cerebral palsy, few factors,

which needed to be thought over. When we are thinking about

treatment of cerebral palsy:

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1. Bone marrow transplantation is autologous, so there is no fear

of rejection or mismatch or need for immunosuppressant

required in other transplantation surgeries.

2. No confirmed documentary evidence that autologous stem

cells are dangerous (except embryonic stem cells). In

combination with BMSCT, We also use cord stem cells which

we are using do not have any surface antigen, so they have no

immune or rejection from body. Cord stem cells also have much

better regeneration potential. Combination therapy compliments

each other as bone marrow stem cell produces much favorable

environment for cord stem cell to act.

3. Good results are documented in otherwise dead end patients.

Other treatment modalities are only supportive and empirical.

4. Many of cases have shown definite clinical improvement till

to date (Battler and Leor, 2006).

Stem cell therapy is used to induce regeneration in various

neurological disorders, including cerebral palsy. During the

treatment authors give 6 doses of stem cells for transplantation

cycles, 3 autologous bone marrow related and 3 cord

mesenchymal cells in high doses. As it has been proved that

more transplantation cycles gives better and better results.

Treatment lasts for 17 days and Patient will need admission for

6 days and 11 days gap in between (Cohen et al., 2009).

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Treatment results:

Follow up statistics from 50 cerebral palsy patients showthat close to 67% experienced improvements after stem celltherapy. The type of improvements reported include: decreasedspasticity; better coordination; improved motor function,improved posture stability; better cognition resulting incommunication improvements; gaining the ability to sit, standor even walk unassisted. Improved speech & I.Q. was observedin 56% of patients. 43% reported a decrease or even absence ofepileptic seizures following treatment. About 20% showedimproved cognition (Battler and Leor 2006).

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Batten DiseaseBatten disease is a rare but fatal neurodegenerative disease

that affects children and for which no treatment exists, other

than possible future stem cell research treatments. The disease is

named after the British physician Frederick Batten, who first

identified and described the condition in 1903, it is also known

as Spielmeyer Vogt Sjogren Batten disease. Batten disease

manifests itself in children somewhere between the ages of 4

and 10 years. Early signs might include vision impairment, poor

circulation, hyperventilation, reduced communicative ability,

and behavioral changes. As a neurodegenerative disease, Batten

disease causes the patient to progressively lose motor skills,

communication skills, and brain functions (Svendsen and Ebert,

2008).

The results are distressing not just to the patients concerned

but also to their careers. Batten disease is a genetically inherited

disease that leads to a mutation in the patient that prevents the

elimination of toxins from the brain. It also leads to a build up

of lipofuscins in the body, and these combinations of proteins

and fats are the symptoms by which the disease is detected.

Since the 1990s, a team of scientists have discovered certain

elements of the X chromosome, resulting in the discovery of the

genetic causes of Batten disease, among other conditions. In the

years since, six genes have been discovered that are associated

with the onset of the disease, although it has not yet been

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determined what functions most of these genes possess (Hough

et al., 2010).

However, the determination of the cause of the disease has

led to an indication of how treatments could be created. These

treatments are based on injecting genetic material directly into

the affected area, the brain of an affected child. In the last few

years, several teams have been working with injecting fetal stem

cell material into the brains of children with Batten disease, and

there have been reports of positive outcomes, although it is too

soon to determine whether a permanent cure is possible or

whether the treatment is temporary in effect. Nevertheless,

patients have responded well to the treatment, and certain motor

skills and communication skills have been returned to them.

Parents who are able to talk to their children after years of them

being unable to speak find their quality of life to be greatly

improved, irrespective of whether that improvement will be

sustained permanently (Svendsen and Ebert, 2008).

The treatment is a proprietary preparation of human central

nervous system stem cells. To avoid patients rejecting the

foreign cells, the immune system must be suppressed to some

extent, and it is in this area that most problems are anticipated to

arise. However, tests show that this problem has been

successfully negotiated within the trials conducted (Miller,

2010).

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A further stream of research has been sparked by the

detection of mature onset Batten disease in a breed of dogs.

Testing shows that perhaps 5% of these dogs may suffer from

the disease. Dog owners and their associations have been

working in partnership with patients and their careers in sharing

information and jointly sponsoring and supporting research. It is

hoped that new forms of treatment might, in due course, arise

from this collaboration (Hough et al., 2010).

The source of most of the genetic material used to treat

Batten disease is aborted fetuses, which are the main source of

the stem cells required. However, fetal cells are currently the

only known source for the stem cells required, and the treatment

relieves terrible suffering. In the future, alternative sources of

stem cells might be identified, including, for example, children

who have died from natural causes or as the victims of road

traffic or other accidents. The use of organs from such sources is

much more widely accepted in most societies. A further ethical

issue concerns the use of children in medical experimentation.

Although only children suffer from Batten disease, there is still

the need to consider whether they are able to give informed

consent to a new and unproven form of treatment, especially

given the progressive damage to their brains. The basic principle

that applies is that consent is provided on behalf of the children

by their parents or other legally appointed guardian (Svendsen

and Ebert, 2008).

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Stem Cell Therapy for AutismAutism spectrum disorders (ASD) are a group of

biologically based neurodevelopmental disorders characterized

by impairments in three major domains: socialization,

communication, and behavior. These disorders include autistic

disorder (classic autism, sometimes called early infantile autism,

childhood autism, or Kanner's autism), Rett disorder, childhood

disintegrative disorder, pervasive developmental disorder not

otherwise specified (PDD-NOS), and Asperger disorder (also

known as Asperger syndrome) (Augustyn et al., 2011).

Hypoperfusion of brain in autism:

Children with autism have been consistently shown to have

impaired, or subnormal CNS circulation, as well as resulting

hypoxia. Defects include basal hypoperfusion, and decreased

perfusion in response to stimuli that under normal circumstances

upregulates perfusion. In numerous studies the areas affected by

hypoperfusion seem to correlate with regions of the brain that

are responsible for functionalities that are abnormal in autism.

For example, specific temporal lobe areas associated with face

recognition, social interaction, and language comprehension,

have been demonstrated to be hypoperfusedn in autistic but not

control children (Augustyn et al., 2011).

Immune deregulation in autism:

The fundamental interplay between the nervous system and

the immune system cannot be understated. Philosophically, the

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characteristics of self/non self recognition, specificity, and

memory are only shared by the immune system and the nervous

system. Physically, every immune organ is innervated and bi-

directional communication between neural and immune system

cells has been established in numerous physiological systems. In

autism, several immunological abnormalities have been detected

both in the peripheral and the central nervous systems.

Astrocytes surround various portions of the cerebral

endothelium and play a critical role in regulating perfusion, and

blood brain barrier function (Vargas et al., 2005).

Physiologically, astrocytes play an important protective role

against infection, generating inflammatory cytokines. Through

secretion of various chemokines, astrocytes play an important

role in shaping adaptive immune responses in the CNS.

Astrocyte secretion of a soluble neurotoxic substance has been

demonstrated to be involved in disease progression. Astrocyte

hyperactivation has been demonstrated in this disease by

imaging, as well as autopsy studies. In multiple sclerosis,

astrocytes play a key role in maintaining autoreactive responses

and pathological plaque formation (Ichim et al., 2007).

Treatment of hypoperfusion defect by umbilical cord blood

CD34+ stem cell administration:

Therapeutic angiogenesis, the induction of new blood

vessels from preexisting arteries for overcoming ischemia, has

been experimentally demonstrated in peripheral artery disease,

myocardial ischemia, and stroke. Angiogenesis is induced

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through the formation of collateral vessels and has been

observed in hypoperfused tissues. This process is believed to be

coordinated by the oxygen sensing transcription factor hypoxia

inducible factor-1 (HIF-1). During conditions of low oxygen

tension, various components of the transcription factor dimerize

and coordinately translocate into the nucleus causing

upregulation of numerous cytokines and proteins associated

with angiogenesis (Ichim et al., 2007).

The potency of tissue ischemia stimulating angiogenesis is

seen in patients after myocardial infarction in which bone

marrow angiogenic stem cells mobilize into systemic circulating

in response to ischemia induced chemotactic factors. The

association between neural angiogenesis and neurogenesis after

brain damage is not only temporally linked but also connected

by common mediators, for example, SDF-1 secreted in response

to hypoxia also induces migration of neural progenitors.

Angiogenic factors such as vascular endothelial growth factor

(VEGF) and angiopoietin have been implicated in post ischemia

neurogenesis (Ishibashi et al., 2009).

Methods to enhance angiogenesis and as a result

neurogenesis are numerous and have utilized approaches that

upregulate endogenous production of reparative factors, as well

as administration of exogenous agents. For example,

administration of exogenous cytokines such as fibroblast growth

factor-2 (FGF-2), erythropoietin, and G-CSF, has been

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performed clinically to accelerate healing with varying degrees

of success (Kalluri and Dempsey, 2008).

A promising method of increasing angiogenesis in situations

of ischemia is administration of cells with potential to produce

angiogenic factors and the capacity to differentiate into

endothelial cells themselves. Accordingly, the use of CD34+

stem cells has been previously proposed as an alternative to

growth factor administration. Therapeutic administration of

bone marrow derived CD34+ cells has produced promising

results in the treatment of end stage myocardial ischemia, as

well as a type of advanced peripheral artery disease called

critical limb ischemia. Additionally, autologous peripheral

blood CD34+ cells have also been used clinically with induction

of therapeutic angiogenesis (Augustyn et al., 2011).

Cord blood has been used successfully for stimulation of

angiogenesis in various models of ischemia. In one report, the

CD34+, CD11b+ fraction, which is approximately less than half

of the CD34+ fraction of cord blood, was demonstrated to

possess the ability to differentiate into endothelial cells. In

another report, VEGF-R3+, CD34+ cells demonstrated the

ability to differentiate into endothelial cells and were able to be

expanded 40-fold expansion. The concentration of this potential

endothelial progenitor fraction in cord blood CD34+ cells is

approximately ten fold higher as compared to bone marrow

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CD34+ cells (1.9% +/- 0.8% compared to 0.2% +/- 0.1%)

(Vargas et al., 2005).

Given the potency of cord blood CD34+ cells to induce

angiogenesis in areas of cerebral hypoperfusion, we propose

that this cell type may be particularly useful for the treatment of

autism, in which ischemia is milder than stroke induced

ischemia, and as a result the level of angiogenesis needed is

theoretically lower. However at face value, several

considerations have to be dealt with. Firstly, cord blood contains

a relatively low number of CD34+ cells for clinical use.

Secondly, very few patients have access to autologous cord

blood; therefore allogeneic cord blood CD34+ cells are needed

if this therapy is to be made available for widespread use. There

is a belief that allogeneic cord blood cells can not be used

without immune suppression to avoid host versus graft

destruction of the cells (Ichim et al., 2007).

Safety concerns regarding allogeneic CD34+ cells are

divided into fears of graft versus host reactions, as well as host

versus graft. The authors of the current paper have published a

detailed rationale for why administration of cord blood cells is

feasible in absence of immune suppression. Essentially, GVHD

occurs in the context of lymphopenia caused by bone marrow

ablation. Administration of cord blood has been reported in over

500 patients without a single one suffering GVHD if no immune

suppression was used. Although host versus graft may

conceptually cause immune mediated clearing of cord blood

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cells, efficacy of cord blood cells in absence of immune

suppression has also been reported. Accordingly, we believe

that systemic administration of expanded cord blood derived

CD34+ cells may be a potent tool for generation of neo

angiogenesis in the autistic brain (Vargas et al., 2005).

Immune modulation by mesenchymal stem cells:

The treatment of immune deregulation in autism is expected

to not only cause amelioration of intestinal and systemic

symptomology, but also to profoundly influence neurological

function. Reports exist of temporary neurological improvement

by decreasing intestinal inflammation through either antibiotic

administration or dietary changes. Although, some anti

inflammatory treatments have yielded beneficial effects, no

clinical agent has been developed that can profoundly suppress

inflammation at the level of the fundamental immune

abnormality. We believe mesenchymal stem cell administration

may be used for this purpose. This cell type, in allogeneic form,

is currently in Phase III clinical studies for Crohn's disease and

Phase II results have demonstrated profound improvement

(Svendsen and Ebert, 2008).

An important characteristic of MSCs is their ability to

constitutively secrete immune inhibitory factors such as IL-10

and TGF-b while maintaining ability to present antigens to T

cells. This is believed to further allow inhibition of immunity in

an antigen specific manner, as well as to allow the use of such

cells in an allogeneic fashion without fear of immune mediated

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rejection. Antigen specific immune suppression is believed to

allow these cells to shut off autoimmune processes (Keilhoff et

al., 2006).

Further understanding of the immune inhibitory effects of

MSCs comes from the fact that during T cell activation, two

general signals are required for the T cell in order to mount a

productive immune response, the first signal is recognition of

antigen, and the second is recognition of costimulatory or

coinhibitory signals. MSCs present antigens to T cells but

provide a coinhibitory signal instead of a co stimulatory signal,

thus specifically inhibiting T cells that recognize them, and

other cells expressing similar antigens. Supporting this concept,

it was demonstrated in a murine model that MSCs

transplantation leads to permanentdonor specific

immunotolerance in allogeneic hosts and results in long-term

allogeneic skin graft acceptance (Augustyn et al., 2011).

Other studies have shown that MSCs are inherently immuno-

suppressive through production of PGE-2, interleukin-10 and

expression of the tryptophan catabolizing enzyme indoleamine

2, 3, dioxygenase as well as Galectin-1. These stem cells also

have the ability to non specifically modulate the immune

response through the suppression of dendritic cell maturation

and antigen presenting abilities. Immune suppressive activity is

not dependent on prolonged culture of MSCs since functional

induction of allogeneic T cell apoptosis was also demonstrated

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using freshly isolated, irradiated MSCs (Svendsen and Ebert,

2008).

Others have also demonstrated that MSCs have the ability to

preferentially induce expansion of antigen specific T regulatory

cells with the CD4+ CD25+ phenotype. Supporting the potential

clinical utility of such cells, it was previously demonstrated that

administration of MSCs inhibits antigen specificT cell

responses in the murine model of multiple sclerosis,

experimental autoimmune encephalomyelitis, leading to

prevention and/or regression of pathology. Safety of infusing

MSCs was illustrated in studies administering 1–2.2 × 106

cells/kg in order to enhance engraftment of autologous bone

marrow cell. No adverse events were associated with infusion,

although level of engraftment remained to be analyzed in

randomized trials. The ability of MSCs on one hand to suppress

pathological immune responses but on the other hand to

stimulate hematopoiesis leads to the possibility that these cells

may also be useful for treatment of the defect in T cell numbers

associated with autism (Ichim et al., 2007).

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Stem cell therapy for Retinal

Degeneration

In the back of the eye exists a thin layer of neural cells that

convert external light into neural signals, known as the retina; In

comparison with other CNS targets, the retina offers some

unique advantages that make it considerably more favorable for

the development of transplantation and stem cell therapies. The

neural sensory retina is a unique structure of the CNS, This

consists of many neuronal types, but the rods are mainly

responsible for detecting low levels of light and the cones can

detect different wave lengths (color) (Diana and Gabriel,

2008).

Other types of neuron connect back to the brain (retinal

ganglion cells) and transmit signals from the rods and cones into

the processing region of the cortex, and finally the visual cortex

where the information is converted into an image. Other

important layers in the eye include the pigment epithelial layer,

which is a pigmented single layer of cells that supports rods and

cones through taking up waste material generated during the

process of detecting light. Loss of function in any of these cells

can result in diminished or lost sight. Retinal degeneration can

occur early or late in life (Svendsen and Ebert, 2008).

The most common cause of blindness in people older than

their 60s is macular degeneration, the degeneration of

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photoreceptors and epithelial layers. The macula is the part of

the retina that handles detailed vision. Macular degeneration

arises when the retinal pigment epithelial cells, the layer of cells

under the retina, become dysfunctional. This failure leads to

degeneration of the macula, the center of the retina, and

diminishing central vision. The patient slowly loses the ability

to read, recognize faces, and see fine details. There are two

forms of macular degeneration. Dry degeneration comes from

the atrophy of the retinal pigment epithelial layer, which

generates a loss of rod and cone function and, eventually,

blindness. Wet macular degeneration arises from abnormal

growth of blood vessels, which produces a leakage of blood and

protein below the macula, producing irreversible damage to the

photoreceptors (Aoki et al., 2007).

Dry degeneration occurs in 90 % of the age-related cases.

Diabetic retinopathy, which damages blood vessels, is another

leading cause of vision loss in the world, and retinitis

pigmentosa is a hereditary genetic disease that affects the rods

and cones. Many researchers are working on retinal stem cells

of one type or another. It is clear that during development there

are retinal stem cells that can be isolated and grown in culture

for many passages. These seem to lose their ability to make rods

and cones very quickly, however, embryonic stem cells have

been used as a source of retinal neural stem cells, these cells are

more plastic and can give rise to many retinal cell types,

although the exact cocktails of factors needed to produce retinal

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cells is the subject of intense investigation right now (Diana

and Gabriel, 2008).

The goal is to be able to generate large numbers of retinal

cells that will be useful for both potential transplant therapies

and drug screening, the adult cilliary margin of the human and

mouse eye has been shown to harbor adult retinal stem cells that

appear to grow in culture fairly easily. With regards for treating

eye diseases with stem cells there have been no trials to date

using either fetal or embryonic tissue, although there may be

some plans to do this in the near future. Interestingly, there have

been many pre clinical studies where various types of stem cell

have been injected into the eye and shown to have functional

effects, which of these eventually make it to the clinic remains

speculative at this time (Ofri and Narfström, 2007).

There have been 2 general approaches to the transplantation

of stem cells or stem cell derived cells to rescue the

degenerating retina:

1) Photoreceptor survival promoted by restoring the supportive

functions of the retinal pigment epithelial cells (RPECs) through

subretinal grafts of stem cell derived RPE like cells.

2) Directly replacing lost photoreceptors with transplanted stem

cells and retinal precursor cells coaxed to differentiate and

integrate into the outer nuclear layer of the degenerating retina

(Charles and Alfredo, 2005).

In contrast to therapies for Huntington disease and ALS,

which have already entered clinical trials, and Parkinson

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disease, for which graft viability and host integration have been

investigated and verified, the potential of these cell based

therapies for retinal degenerative disorders is still being

determined in animal models. A study of subretinal injections of

human ESC derived RPECs rat model of retinal degeneration

(caused by inhibition of RPEC phagocytosis of photoreceptor

outer segments due to mutation in the receptor tyrosine kinase

gene) reported rescue of visual response and acuity assessed

using electroretinography, optomotor acuity thresholds, and

luminance threshold recordings in the superior colliculus (Lund

et al., 2007).

However, there is no direct evidence that the observed

functional improvements were due to rescue of RPEC

phagocytosis rather than a general neuroprotective effect

brought on by the transplanted cells. Indeed, a variety of cell

types have been shown to provide temporary rescuing effects

through secretion of neurotrophic factors when transplanted into

the retina, and the human ESC derived RPECs have been shown

to produce pigment endothelium derived factor, a trophic factor

with protective and morphogenetic effects on photoreceptors.

Furthermore, the grafted cells were observed to aggregate at the

injection site instead of integrating into the host retina with poor

long term cell retention that parallels the decline in rescue

effects at 4 weeks after transplantation (Svendsen and Ebert,

2008).

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Attempting to rescue the degenerating retina by cellular

replacement of lost photoreceptor neurons presents its own

unique set of challenges. In cases of severe retinal degeneration,

the retina may have already undergone substantial postreceptor

remodeling (in the early stages of these diseases the retina

beyond the photoreceptor layer remains relatively anatomically

and functionally intact). Therefore, grafted stem cells would

need to differentiate into functional photoreceptor neurons and

establish the intricate functional connections with bipolar and

horizontal cells in order to restore the neural retinal circuitry.

Subretinal injections of photoreceptor precursor cells (isolated

from the retina during rod photoreceptor genesis) into mouse

models of retinal degeneration demonstrated cell integration

into the retina, expression of the synapse protein bassoon,

formation of synaptic contacts with bipolar cells identified by

immunostaining with phosphokinase C, positive rod

photoreceptor differentiation based on immunostaining for

proteins involved in the phototransduction pathway (phosducin

and rhodopsin), and improved visual responses assessed with

extracellular field potentials of the retinal ganglion cell layer

and papillary reflex to light stimulation (MacLaren et al.,

2006).

Furthermore, advances in therapeutic cloning could lead to

successful generation of stem cells from adult somatic cells to

facilitate transplantation of autologous cells for treatment of

patients with retinal degeneration. In addition to treating

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pathological entities that primarily involve the death of

photoreceptors in the outer retina, stem cell therapies are also

being investigated for treating degeneration of the inner retina

specifically, diseases that cause the degeneration of the optic

nerve and retinal ganglion cell (RGCs) (for example, glaucoma,

Leber hereditary optic neuropathy, and ischemic optic

neuropathy) for which there are currently no treatments

available to improve vision once vision loss has occurred.

Regeneration of the optic nerve after injury or disease is

difficult for a number of reasons (Ofri and Narfström 2007).

Optic nerve degeneration is usually accompanied by RGC

apoptosis, whether it is due to elevated intraocular pressure, as

in glaucoma, or excitotoxicity, as in retinal ischemia and

reperfusion injury. Furthermore, as is the case with most CNS

neurons, RGCs in the adult mammalian retina do not have the

ability to reinitiate axonal growth after injury on their own

despite having this capacity during development. Finally,

similar to other areas of the CNS, reactive gliosis and

upregulation of factors such as myelin-associated protein in the

local environment following injury inhibit regrowth of axons

that need to reestablish synaptic connections with the lateral

geniculate nucleus. Overcoming these challenges by using stem

cell therapies would likely require a combination of cell

transplantation and neuroprotective strategies (Lund et al.,

2007).

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One major advantage of the inner retina and optic nerve is

that molecularly the mechanisms that ultimately result in RGC

apoptosis and optic nerve loss are the same classic mechanisms

described for neurodegenerative events in other parts of the

CNS. As such, both basic and clinical stem cell therapy

investigations that target the inner retina and optic nerve might

provide results that translate to or, at least guide, further

research into similar therapies for other neurodegenerative

disorders. In a different and intriguing line of research

investigators are attempting to use ESCs to essentially grow

eyelike structures rather than replace a lost cell type or provide a

neuroprotective environment (Aoki et al., 2007).

Most of these neurons in N-methyl.D-aspartate treated retina

went on to express ganglion cell specific markers. However,

functional assessment in a more physiologically relevant in vivo

model remains to be done, and the clinical impact of this

approach, if any, remains to be determined. Finally, the adult

retina offers another unique potential source of transplantable

cells (MacLaren et al., 2006).

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Stem cell therapy for spinal cord

injury

Spinal cord injury is defined as damage to the spinal cord

that results in partial or complete loss of function, whether

temporary or permanent. The three most common causes of

spinal cord injury are direct trauma, compression by disk

herniation, and damage caused by occluded spinal arteries.

Although the range of symptoms that result from a spinal cord

injury is largely determined by the size and location of the

lesion, most patients will at least experience chronic pain

accompanied by lifelong heart and lung complications. Current

therapies focus on physical rehabilitation and counseling to deal

with the emotional frustration of disability, but nothing can be

done to regenerate the spinal cord. As such, research for

treatments targets four main concepts: limiting the damage,

neuroreconstru-ction, stimulating regrowth of neurons, and

retraining neural circuits to restore body functions. Stem cells

have shown great promise and potential for restoration of the

damaged spine (Eftekharpour et al., 2008).

Recent advances in cell replacement therapy for SCI:

Cell death is inevitable after SCI. The adult spinal cord

harbors endogenous stem/progenitor cells, collectively referred

to as NPCs that might be responsible for normal turnover of the

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cells. However, the proliferative activity of endogenous NPCs is

too limited to support significant self repair after SCI. Thus,

various cellular transplantation strategies have been adopted in

models of SCI. Cell replacement approaches in the setting of

SCI can be used to achieve 2 broad goals: 1) regeneration,

which seeks to replace lost or damaged neurons and induce

axonal regeneration or plasticity; and 2) repair, which seeks to

replace supportive cells such as oligodendrocytes in order to

induce remyelination and prevent progressive myelin loss (Ziv

et al., 2006).

Spinal cord injury leads to interruption of ascending and

descending axonal pathways, loss of neurons and glial cells,

inflammation, and demyelination. Stem cell based therapies

could be used to treat individuals with spinal cord injury in

several ways. First, transplanting stem cell derived spinal

neuroblasts could lead to the replacement of damaged or dead

motor and other neurons. Second, transplanting stem cell

derived OPCs could promote remyelination. Last, transplanting

stem cells modified to release different factors could counteract

detrimental inflammation (figure 27) (Lindvall and Kokaia,

2010).

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Figure (27): Stem cell based therapies for spinal cord injury (Lindvall andKokaia, 2010).

Human pluripotent stem cells:

The derivation of HESCs in 1998 with their pluripotential

ability to produce all cell types of the human body has sparked

significant interest in their use as therapeutic tools. Considering

the complexity of the nervous tissue and the importance of non

neuronal elements, the pluripotentiality of HESCs makes them

an optimal candidate that can potentially differentiate into

different cell types for the reformation of cellular networks.

Using an excitotoxicity brain injury models, it has been shown

that transplanted hESCs express immunological labeling for

vascular endothelial cells, glia, and different neuronal subtypes

with morphologically normal synapses (Eftekharpour et al.,

2008).

The generation of autologous pluripotent stem cells from

skin cells has been reported. By using viral gene transfer into

somatic cells of adult humans, pluripotent cells can be generated

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that have similar immunological characteristics to the donor.

These pluripotent cells are similar to ESCs in that they retain the

developmental potential to differentiate into any cell type. This

discovery will obviate the ethical issues surrounding the source

of ESCs and eliminate the need for immunological matching

between the host and donor. However, several issues including

the potential mutation of these cells after integration of the

carrier viral gene into the host DNA must be resolved before

these cells can be used in clinical trials (Lindvall and Kokaia,

2010).

Fetal stem cells:

Fetal tissue grafts were tested in human patients with CNS

disorders and Parkinson disease as early as the 1970s. Some

reports have suggested that fetal derived NPCs can differentiate

into neurons and oligodendrocytes after transplantation into a

contusion SCI model with some improved behavioral outcome.

The transplantation of olfactory ensheathing cell (OECs) from

fetal sources, has gained considerable media attention; however,

the limited scientific data from this study severely limit

enthusiasm for this procedure as a therapeutic option in patients

(Raisman and Li, 2007).

Banking of umbilical cord blood cells, another type of fetal

stem cells, for the cure of immunological and hematological

disorders as well as a potential source for stem cell based

therapeutic approaches, has expanded rapidly. The

multipotentiality of these cells to produce the main cell types of

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nervous tissue has been recently reported. These cells have also

been used in experimental SCI models and were shown to

improve functional recovery due to neurotrophic function or

increased myelination. However, further work must be done to

fully characterize the extent of functional recovery induced by

umbilical cord blood cells and to further delineate their

mechanisms of action (Lindvall and Kokaia, 2010).

Adult derived stem and progenitor cells:

Adult derived stem/progenitor cells have been identified in

many tissues including those from skin, eye, pancreas, brain,

and spinal cord. These somatic cells have an extensive capacity

for self renewal and the multipotentiality to produce all the

major cell types of their specialized tissue of origin. However,

experimental evidence has suggested the possibility of

transdifferentiation of these cells into other cell types, when

transplanted ectopically into other tissues such as the spinal

cord. Although this topic is the subject of active research, in this

review we focus on selected reports that have shown some

beneficial outcome when adult cells were transplanted into an

SCI model (Yiu and He, 2006).

Schwann cells and OECs:

The PNS myelinating Schwann cells would seem to be an

obvious substitute when considering a cell based remyelination

strategy. Interestingly, infiltration of endogenous Schwann cells

at the site of injury and their contribution to spontaneous

myelination after SCI is well documented, although the impact

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of such schwannosis which is associated with increased CSPG

formation and may have deleterious effects on regeneration is

unclear and could even promote neuropathic pain. Schwann

cells can be easily harvested from the patient’s peripheral

nerves, and a relatively short time is required to produce

clinically suitable material for autologous transplantation (Rishi

et al., 2009).

After PNS injury, Schwann cells provide a growth

promoting environment for regenerating axons through

secretion of neurotrophic factors1 and extracellular matrix

elements and ultimately myelinate the axons. Several groups

have studied transplantation of Schwann cells after SCI and

have shown successful integration of these cells in the host

tissue with some regeneration of sensory axons and myelination

of these axons associated with modest improvement of hind

limb function. A poor survival rate has been reported for these

cells when transplanted into the epicenter cavity, although the

endogenous Schwann cells will fill up the cavity (Raisman and

Li, 2007).

The therapeutic effects of Schwann cells have been shown to

be enhanced when these cells are transplanted with their

phenotypically similar cells, OECs. These are peripheral glial

cells that support the growth of newly generated axons from the

olfactory neurons to the olfactory bulb but do not myelinate the

olfactory axons in their native environment. However, some

authors believe that OECs are not only involved in axonal

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regeneration but also in remyelination after SCI or in a

demyelinating disease. In contrast, others have demonstrated a

lack of myelination in axons associated with OECs transplanted

after SCI. The controversial myelinating ability of OECs is

affected by several experimental variables including the age of

the donor and the isolation methods used (Yiu and He, 2006).

Mesenchymal stem cells:

The ability of these cells to differentiate into neural cells is

controversial; therefore, it has been suggested that the functional

improvement might be effected through neuroprotective

pathways. The in vivo expression of neural tissue markers

nestin and GFAP in MSCs or their neuron like morphological

characteristics under certain tissueculture conditions as

evidence of the transdifferentiation ability of MSCs to neural

tissue cells. Nonetheless, nestin and GFAP are not specific

markers for nervous tissue and can be found in mesodermal

tissues such as muscle and cartilage (Ziv et al., 2006).

The reported neuron like morphological characterristics of

MSCs after tissue culture manipulation can best be described as

tissue culture artifact and has not been supported by functional

properties associated with normal neurons such as

electrophysiological assessment. The possibility of MSC

transdifferentiation into neuronal progeny remains to be

investigated. Alternatively the functional benefits of MSC

transplantation in CNS injuries can be explained by their ability

to provide the host tissue with growth factors or modulate the

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host immune system. These cells can be used in combinatorial

therapies as tools for growth factor delivery. Transplantation of

bone marrow MSCs, engineered to deliver neurotrophins, can

induce axonal growth through the glial scar in a model of

chronic SCI in rats; however, it seems that these approaches do

not result in functional recovery after chronic SCI, indicating

the lack of appropriate synapse formation by the regenerated

axons with the host tissue (Lindvall and Kokaia, 2010).

Adult derived NPCs:

Adult NPCs contribute to neurogenesis and gliogenesis in

some regions of the CNS in various species including humans.

These tissue specific somatic stem cells are located in the

periventricular regions and in white matter tissue throughout the

brain and spinal cord and can differentiate into all neural cell

types under defined conditions. Using selective in vitro

expansion and cell sorting based on antigenic properties and

molecular techniques, these cells can be directly derived from

the adult tissue; however, so far no specific marker has been

identified for human adult NPCs (Eftekharpour et al., 2008).

Recently, using a combined therapeutic strategy, we have

shown the beneficial effects of adult NPC transplantation in a

clinically relevant model of SCI in rats. This compressive model

consists of a modified aneurysm clip that delivers a calibrated

closing force. The 23-g clip used in our studies produces a

moderately severe SCI with central cavitation and loss of 80%

of axons in the spinal cord white matter, demyelination of the

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surviving axons in the residual subpial rim, and spastic

paraparesis. Therefore, this model is of translational relevance

to the majority of patients with chronic SCI (Lindvall and

Kokaia, 2010).

The adult NPCs were harvested from the subventricular zone

of adult mice that express the yellow fluorescence protein (YFP)

gene, allowing for easy tracking of transplanted cells. Cells

were clonally driven in vitro in the presence of growth factors.

These free floating colonies (called neurospheres) were

composed of 1% neural stem cells and 99% progenitor cells.

The dissociated adult NPCs transplanted into the areas rostral

and caudal to the injury epicenter in a delayed fashion at 2 or 8

weeks post injury to represent 2 clinically relevant phases in

SCI pathophysiology, the subacute and chronic stages,

respectively. We adopted a novel post transplantation delivery

of epidermal growth factor, basic fibroblast growth factor, and

platelet derived growth factor AA approach aimed at:

1) Promoting the survival of the grafted adult NPCs.

2) In vivo differentiation of undifferentiated NPCs towards

an oligodendroglial lineage (Rishi et al., 2009).

Transplantation of adult NPCs into the subacutely

injured spinal cord:

When transplantation was delayed to 2 weeks after injury, a

substantial survival rate of 40% of the transplanted cells was

observed (figure 28).

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Figure (28): Photomicrographs demonstrating that transplanted adult NPCssuccessfully survive, migrate, and integrate with the injured spinal cord.Confocal images from a longitudinal section of injured spinal cord takenfrom the dorsal spinal cord of an injured rat that received cell transplants 2weeks post injury. The low magnification image (A) shows a substantialsurvival of YFP-NPCs within the injured spinal cord at 8 weeks posttransplantation. Grafted YFP-NPCs (green) were dispersed along therostrocaudal axis of the spinal cord approximately 5 mm from theimplantation sites (asterisks). The YFP-NPCs also migrated to thecontralateral side of the spinal cord to a lesser extent. Double labeling withthe neuronal marker bIII tubulin (Tuj1) showed that YFP-NPCs residepredominantly in the white matter (WM) area. GM = gray matter(Eftekharpour et al., 2008).

The multipotent capability of the subventricularly derived

adult NPCs to differentiate into the 3 main cell types of the

nervous tissue has been shown in our experiments as well as in

previous reports in vitro, but the transplanted NPCs were mainly

driven towards a glial fate. The lack of neuronal differentiation

156

by engrafted NPCs in the adult spinal cord has been reported

repeatedly by different groups of authors (Ziv et al., 2006).

The tendency of NPCs toward glial differentiation after

transplantation may also reflect the intrinsic properties of the

spinal cord that favor glial differentiation. This may be

attributed to potential inhibitory or lack of promoting factors for

neurogenesis by NPCs in different CNS tissues and needs to be

taken into consideration when cell therapy strategies are

designed for neuronal replacement. Because loss of

oligodendrocytes and myelin is a major contributor to white

matter damage after trauma, it is rather intriguing to speculate

that the propensity of adult NPCs to form oligodendrocytes after

transplantation into the adult injured spinal cord may reflect the

influence of the host tissue to replenish a particular cell type to

maximize repair. However, whether demyelinated axons exert

such effects on NPC integration and differentiation remains to

be investigated (Eftekharpour et al., 2008).

Myelin is critical to the precise molecular organization of

axons. The nodal distribution of Na+ channels, paranodal

preference of contactin associated protein, and juxtaparanodal

adherence of K+ channels is closely related to myelin and is

disrupted following demyelination (Sasaki et al., 2007).

However, the potential contribution of endogenous

myelinating cells in these models cannot be ruled out unless

mutant dysmyelinated models such as neonatal myelin deficient

rats or shiverer mice are used. Beneficial structural and

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functional effects of neural stem cells in genetic mutant models

indicate that long term molecular abnormalities can be

successfully reversed in the adult dysmyelinated CNS axons.

Although neuroanatomical assessment of myelination is a

common approach determining the success of cell therapy

approaches, direct physiological assessment to investigate the

functionality of the new myelin has been overlooked in

literature. Using in vivo spinal cord evoked potential recordings,

we observed that adult NPC induced myelination promoted

enhanced spinal cord axonal conduction in the shiverer mice, as

evidenced by increased amplitude, reduced latency, and

enhanced estimated conduction velocity.Although

remyelination has been shown to be involved in adult NPC

mediated recovery after SCI, we cannot exclude other potential

protective/trophic effects of adult NPCs. It is possible that the

transplanted adult NPCs may increase axonal sprouting and

plasticity that could be responsible for some of the observed

locomotor recovery (Rishi et al., 2009).

Functional locomotor recovery following subacute

transplantation of adult NPCs has been also reported by other

investigators. However, recent reports have noted an increase in

pain sensation, known as allodynia, which was observed when

NPCs chiefly differentiated into astrocytes. Allodynia was

diminished after genetic modification of the naive adult NPCs to

enhance their differentiation towards oligodendrocyte lineage.

Of note, there are no observed changes in pain sensation in

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experiments, which can be explained by low rates of astrocytic

differentiation by the transplanted adult NPCs in our

transplantation protocol. However, these reports suggest that the

therapeutic evaluation of any intervention for the repair of SCI

should include detailed sensory and motor assessments

(Lindvall and Kokaia, 2010).

Transplantation of NPCs into chronic spinal cord lesions:

Chronic SCI continues to pose a major challenge for

patients, physicians and translational scientists. To date, limited

success has been achieved in applying regenerative technologies

to the complex pathobiology of chronic SCI. This may be

attributed to the environmental restrictions present in

chronically injured spinal cord including glial scarring and

syrinx formation. In our experience with adult NPC therapy for

the repair of SCI, we have learned that the chronically injured

spinal cord environment is not as hospitable as the subacutely

injured tissue for the transplanted cells. Grafted adult NPCs

mostly failed to survive after transplantation into the chronically

injured spinal cord (Chen et al., 2004).

These results suggest the presence of inhibitory components

or the lack of promoters such as growth factors in the

environment of chronically injured spinal cord. Considering the

complexity of chronic SCI pathobiology, it is important to adopt

a multifactorial approach to overcome the environmental

restrictions present in the chronically injured spinal cord. Such

159

strategies will need to overcome the inhibitory effects for the

glial scar, endogenous inhibitory molecules such as Nogo and

Rho, and the presence of post traumatic cyst (Sasaki et al.,

2007).

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Stem cell therapy for Peripheral nerves

Injuries

Peripheral nerves Injuries are common and debilitating,

affecting 2.8% of trauma patients and resulting in considerable

long term disability. Although peripheral nerves do exhibit the

potential to regenerate axons and re innervate the end organ,

outcome following severe nerve injury, even after repair,

remains relatively poor. This is likely because of the extensive

injury zone that prevents axon outgrowth. Even if outgrowth

does occur, a relatively slow growth rate of regeneration results

in prolonged denervation of the distal nerve (Metwally, 2009).

Whereas denervated Schwann cells (SCs) are key players in

the early regenerative success of peripheral nerves, protracted

loss of axonal contact renders Schwann cells unreceptive for

axonal regeneration. The assumption has been that peripheral

nerve injuries recover, given the observation of spontaneous

axonal regeneration following insult. While this capacity for

regeneration is higher than that of the central nervous system,

complete recovery is fairly infrequent, misdirected, or

associated with debilitating neuropathic pain. In fact,

satisfactory results only tend to occur following relatively minor

injuries, such as neurapraxia or axonotmesis (Keilhoff et al.,

2006).

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Nerve transection is associated with notoriously poor

outgrowth compared with other injuries, particularly when the

distance between injury and target is long. Poor outcome from

peripheral nerve injury is especially evident when repair is

performed after a temporal delay, occurring frequently in

clinical practice. Due to the nature of most nerve injuries where

the nerve is left in physical continuity, the propensity for

spontaneous recovery is not immediately known. As such,

surgical repair is significantly delayed in a great number of

cases. Even patients undergoing immediate nerve repair are

subject to a lengthy denervation of the distal nerve as a result of

the low rate of regeneration (~ 1 mm/day in humans) and the

long regeneration distances required to reach the end organ

(Metwally, 2009).

Elongation of regenerating axons is initially supported by

resident Schwann cells that undergo a phenotypic change from

myelinating to growth supportive following initial denervation.

Proliferating Schwann cells are a rich source of neurotrophins,

cell adhesion molecules, and cytokines that support axonal

regeneration and recruit further cells into the injury site. Unless

axonal contact is reestablished in a timely fashion, however, this

growth supportive environment is not maintained (Cui et al.,

2008).

Denervated Schwann cells progressively lose their ability to

express regeneration assisting genes and in effect become

"turned off." As the capacity of the denervated distal nerve to

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support axonal regeneration is highly dependent on proliferating

Schwann cells within the basal lamina tubes that guide

elongating axons to their denervated target, this loss of vitality

and functionality in distal Schwann cells directly translates to

poor muscle reinnervation outcomes. Although placement of

interposed autologous nerve grafts offers a cell rich material

through which axons can regenerate, their use is not ideal

because of donor site morbidity, lack of donor tissue

availability, and nonspecific regeneration. New advances in

tissue engineering have introduced synthetic nerve guide

conduits that are capable of bridging small defects in peripheral

nerves (up to ~ 3 cm in humans), but their relatively inert

microenvironment reduces their value for larger or more chronic

injuries (Bellamkonda, 2006).

It appears that combined approaches with cells or trophic

factors within synthetic tubes may extend their functionality.

Indeed, delivery of SCs in a variety of repair paradigms has

been successful in promoting regeneration and remyelination of

the injured spinal cord and peripheral nerve. However, human

SCs must be derived from invasive nerve biopsies in sufficient

numbers for regeneration and are only available after a lengthy

expansion time in vitro. Therefore, several groups have turned

their attention to identifying more accessible sources of SCs like

cells for transplant therapies (Metwally, 2009).

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Considerations for optimizing stem cell therapy for

peripheral nerve repair:

Number and method of stem cell delivery:

Although often not reported, the number of cells delivered to

nerve injuries in animal models varies considerably between

studies. While some have used as few as 4 × 103 cells, others

have transplanted 2 × 107 cells, but there has often been little

explanation for the selection of cell numbers in these studies. It

is admittedly difficult to compare the number of cells delivered

in widely different repair paradigms, but it is fair to state that

there are likely an ideal number of cells that should be

determined for each cell type or repair scenario. Just as too few

cells may not translate to a therapeutic effect, delivery of too

many cells may also have detrimental results. This was

exemplified beautifully by a study using transplanted Schwann

cells delivered in 10 mm nerve gap (Cui et al., 2008).

Optimization strategies should take the number of delivered

cells into account. Similarly, the way in which cells are

delivered to the injury site has varied between studies, ranging

from direct microinjection, suspension within artificial tubes,

and seeding within devitalized muscle or nerve grafts. Although

the choice of stem cell delivery method may depend on the type

and extent of nerve injury in question, it may be optimized by

providing transplanted cells an environment that will favor their

survival and integration, such as within structured fibers or

biomatrices (Keilhoff et al., 2006).

164

Differentiation state of delivered stem cells:

Part of the appeal of using precursor or stem cells for

supplementing peripheral nerve repair is their capacity for self

renewal, such that it is possible to deliver large numbers of

dividing cells to the injury site. By delivering stem cells into the

injured nerve in a naive state, this proliferative capacity is

maintained, and it is expected that cells will be prompted by the

microenvironment to differentiate into the required cell type. In

vitro studies have demonstrated that neural stem/progenitor cells

in coculture with cells from the nervous system will take on a

phenotype similar to their partner tissue’s origin: dorsal root

ganglion cultures will induce a peripheral neuron/Schwann

cell/smooth muscle phenotype, and a cerebellar feeder layer will

induce differentiation into CNS neurons (Brannvall et al.,

2008).

Nevertheless, incidence of differentiation from naive

precursor cells within the peripheral nerve is rather low in many

cases. Choosing to pre-differentiate stem cells toward a desired

phenotype prior to delivery into the repair site may be an

effective strategy to ensure a more precise and complete

therapeutic effect. It may be that cells at later developmental

stages (vs embryonic stem cells, for example) possess more

mature intrinsic molecular programs to direct them to their

target destination (Bellamkonda, 2006).

165

Because it is well known that mature SCs survive

denervation events by secreting autocrine factors such as insulin

like growth factor, neurotrophin-3, and plant derived growth

factor BB, might an appropriately differentiated stem cell also

possess similar machinery for self preservation and thus be an

ideal candidate for supplementing the injured peripheral nerve?

We have found that SKPCs, when injected as naive sphere

forming cells, do differentiate into GFAP positive SCs in

response to cues found in the local environment of the injured

peripheral nerve. However, long term survival and maintenance

of SCs markers is greatly improved by pre-differentiating the

cells to SCs phenotype prior to delivery. On the other hand,

others have reported that allowing stem cells to differentiate

before delivery accelerates post transplant cell death, perhaps

owing to increased expression of major histocompatibility

complex antigens or reduced proliferation rates (Svendsen and

Ebert, 2008).

In addition to survival of stem cells, their effect on

surrounding tissues may be modified based on their level of

differentiation prior to transplantation. For example, when naive

adult neural stem cells were injected into a lesioned spinal cord,

the resulting aberrant sprouting resulted in profound allodynia.

If gliogenesis in these cells was suppressed by prior treatment

with neurogenin-2, there was an overall greater functional

improvement (Brannvall et al., 2008).

166

Improving survival of transplanted stem cells:

Whether due to technical challenges or oversight, it is an

unfortunate reality that survival of stem cells delivered to nerve

injury sites is reported only infrequently. When quantified,

precursor cells have shown between 0.5 and 38% survival,

depending on evaluation time point and cell type. When naive

SKPCs delivered into an acutely injured nerve, survival after 2

weeks was ~ 10.5%, whereas when delivered into a nerve that

had been previously chronically denervated, the number of

detected skin derived precursor cells (SKPCs) decreased to

5.8%. Seeing that 78% of the surviving stem cells in the chronic

model had differentiated into GFAP-positive Schwann cells, we

next used pre-differentiated (Schwann cell like) SKPCs and

found that we could increase survival to ≥ 8% (Pan et al.,

2007).

Without quantification of survival in stem cell

transplantation experiments, it is difficult to determine whether

they are being retained long enough and in enough numbers to

confer a sufficient benefit to regeneration. The danger of

exogenous cell therapy is of course cell death caused by

immune system attack. Although some authors have reported

considerable phagocytosis of transplanted stem cells, this may

be due to species/strain mismatching of donor and recipient, as

many others have not observed this trend. In fact, we have

observed a highly interesting pattern of surviving transplanted

167

SKPCs that are spatially separate from phagocytic ED-1

positive macrophages (Keilhoff et al., 2006).

If a minimum survival time of stem cells is indeed required

to observe a therapeutic effect, strategies should be devised to

increase the amount of time cells remain in grafted regions.

Survival and effectiveness of transplanted cells can be improved

by ex vivo genetic manipulation or concomitant delivery of

protective agents or trophic factors. Pan and colleagues found

that administration of granulocyte colony stimulating factor to

animals receiving transplants of amniotic fluid mesenchymal

stem cells not only improved survival of transplanted cells but

also augmented nerve regeneration over that of a primarily cell

based approach. Additionally, differences in the material in

which stem cells are delivered have demonstrated varying

capacities to support long term cell survival. Finally,

immunosuppressive regimens, especially in the light of

interspecific transplants may protect stem cells from premature

clearance from the nerve injury site (figure 29) (Metwally,

2009).

168

Figure (29): Confocal image. The SKPC Schwann cells are not

immediately cleared by host immune system. Eight weeks following

injection into an initially decellularized (by repetitive freeze-thawing) nerve

graft bridging a 12-mm defect created in the rodent sciatic nerve, SKPC

Schwann cells (red), and ED-1-positive macrophages (green) are spatially

segregated within the longitudinal extent of the nerve graft. The finding that

there is very little colabeling of SKPC–Schwann cells with ED-1 positive

macrophages suggests that transplanted cells are not phagocytosed in any

large quantity within the host nerve, Original magnification ×400

(Metwally, 2009).

Influence of final stem cell phenotype on regenerative

success:

As with survival, differentiation of stem cells within the

injury site has demonstrated a mixed correlation to therapeutic

effect. Some studies have demonstrated a need for

differentiation to glial phenotype to observe adequate

regeneration of neural tissue, and others have shown

improvement with little to no differentiation of stem cells at the

169

assessment end point. Furthermore, the glial differentiation of

transplanted stem cells within the injured peripheral nerve has

tended to vary between studies, the difference in repair

paradigms (devitalized muscle graft versus crush injured nerve)

may explain the disparity in the ability of these cells to

differentiate, outlining the need for careful consideration of the

method of delivering stem cells to the injury site (Keilhoff et al.,

2006).

In the cases in which adequate regeneration and

improvement of outcomes occurs without SCs differentia-tion

of transplanted precursors, it may be that the cells are

supporting axonal growth by additional mechanisms such as the

production of cytokines or harnessing the inflammatory

response. Although C17.2 neural stem cells show little

differentiation into SCs phenotype in the chronically denervated

peripheral nerve, their secretion of various matrix

metalloproteinases, capable of breaking down growth inhibiting

chondroitin sulfate proteoglycans, likely underlies their ability

to elicit superior regeneration (Pan et al., 2007).

Careful examination of ultimate cell fate with correlation to

functional outcome is strongly recommended for future

precursor transplant studies and will be required to fully answer

this question for each cell type and repair strategy. It may be

that, at least for some precursor cell types, there is a minimum

level of differentiation to S100β/MBP/GFAP-positive SCs that

is required for acceptable regeneration outcomes. If this is the

170

case, effectiveness of precursor transplantation could be

improved using technology that exists to directly alter the

regenerative microenvironment by continuous delivery of

neuregulins, forskolin, or other differentiation-promoting factors

(Svendsen and Ebert, 2008).

Methods for tracking fate of transplanted stem cells:

Stem cell transplantation for peripheral nerve repair should

give careful thought on strategies to track the fate of

transplanted cells over time. There is often little importance

placed on prelabeling cells prior to delivery into the injured

nerve, and as such authors cannot comment on the mechanism

of any advantage conferred by cell therapy. Others have used

labeling techniques that are not sufficiently robust or long

lasting to be detected at the study end points. Chemical markers

such as bisbenzimide and PKH26 have been used to label SCs

delivered to peripheral nerve injuries, but their usefulness is

limited to the short term and may in fact affect the viability and

phenotype of transplanted cells (Bellamkonda, 2006).

Genetic labeling with either fluorescent protein such as GFP

is increasingly popular and appears to be a relatively long

lasting method that is not deleterious to transduced cells. We

have used the lipophilic carbocyanine derivative Cell T racker

CM-DiI (Molecular Probes) to reliably label SKPCs within a

variety of nerve injury models with no dilution or loss of signal

for ≥ 10 weeks following transplantation. These dyes have the

171

advantage of being technically simple to use, rapid, and resistant

to leakage to nearby cells. Emerging technologies such as

quantum dots offer an exciting alternative to traditional cell

labeling methods. These nanoparticles are available in a wide

range of photostable colors and are resistant to chemical and

metabolic degradation, making them ideal for use in long-term

fate tracking of transplanted stem cells (Brannvall et al., 2008).

172

Summary

Stem cells are emerging as one of the most exciting new

areas of neuroscience, not only in terms of revealing insights

into normal development, but also as a therapeutic agent for a

range of neurological diseases. In both of these aspects, they

will affect neurological practice by providing insights into

mechanisms of disease as well as curative cell therapies.

Cell therapy is in fact, a type of organ transplant which has

also been referred to as "xenotransplant therapy", "cellular

suspensions", "glandular therapy" or "fresh cell therapy". The

procedure involves the injection of either whole fetal xenogenic

(animal) cells (e.g. from sheep, cows, pigs, and sharks) or cell

extracts from human tissue. The latter is known as autologous

cell therapy if the cells are extracted from and transplanted back

into the same patient. Several different types of cells can be

administrated simultaneously as embryonic, fetal (derived from

fetal tissues), umbilical cord and adult (somatic) stem cells

including haemopoietic, mesenchymal, hepatic, neuronal,

epidermal and pancreatic stem cells.

The types of cells that are administered correspond in some

way with the organ or tissue in the patient that is failing. No one

knows exactly how cell therapy works, but it was claimed that

the injected cells travel to the similar organ from which they

were taken to revitalize and stimulate that organ's function and

173

regenerate its cellular structure. In other words, the cells are not

species specific, but only organ specific.

Replacing the worn out or injured cells by functional cells to

restore the normal function of the tissues or organs is the

underlying principle of CRT, otherwise also called as

regenerative or restorative medicine. It is hoped that the organs

or tissues treated by this approach can perform their normal

function more efficiently than the ones treated by conventional

therapies like transplantation and pharmocological therapy.

Cell therapy has been used successfully to rebuild damaged

cartilage in joints, repair spinal cord injuries, strengthen a

weakened immune system, treat autoimmune diseases, Acquired

Immunodeficiency Syndrome (AIDS) and help patients with

neurological disorders such as Parkinson's disease (PD), stroke

and Alzheimer's disease and further uses have shown positive

results in the treatment of a wide range of chronic conditions

such as arteriosclerosis, congenital defects, and, myocardial

infarction, PD , diabetes which are due to the progressive (acute

/ chronic) loss of functional cells due to disease or injury to the

cells or ageing.

Cell therapy for PD has significantly advanced in recent

years. Open label clinical trials have provided proof of principle

that transplantation of fetal DA neurons can improve patients’

neurological symptoms. Stem cell based approaches could be

used to provide therapeutic benefits by implanting modified

stem cells to release growth factors which protect existing

174

neurons and transplanting stem cell derived DA neuron

precursors into the putamen.

Compared with neurodegenerative disease, stroke possesses

special conditions that impact the potential success of

transplantation to enhance neurological recovery including; the

anatomy, time of the stroke, the vascular supply, site of

implantation and type of patients enrolled in clinical trials.

Another disease of interest is demyelinating disease such as

multiple sclerosis which presents particular and serious

problems to those attempting to develop cell-based therapies:

the occurrence of innumerable lesions scattered throughout the

CNS, axon loss, astrocytosis, and a continuing inflammatory

process, to name but a few. Nevertheless, the limited and

relatively focused nature of damage to oligodendrocytes and

myelin, at least in early disease, the large body of available

knowledge concerning the biology of oligodendrocytes, and the

success of experimental myelin repair, have allowed cautious

optimism that therapies may be possible.

Amyotrophic lateral sclerosis is characterized by progressive

dysfunction and degeneration of motor neurons in cerebral

cortex, brain stem and spinal cord. To have long-term value,

stem cell therapy must restore function of both upper and lower

motor neurons. Successful replacement of cortical motor

neurons requires not only re-establishment of spinal cord

connections but also precise functional integration of the new

neurons into cortical circuitries.

175

Also, there have been several different experimental

approaches to cell therapy for Muscular dystrophies which are a

heterogeneous group of inherited disorders clinically manifested

by progressive muscle weakness and wasting and, in some

cases, death. Although much of the molecular genetics of

muscular dystrophies have been elucidated, there is still no

effective cure for any of them.

Huntington’s disease is an autosomal dominant

neurodegenerative disorder associated with progressive cell loss

and atrophy predominantly in the striatum and neocortex, Cell

therapy in Huntington's disease aims at restoring brain function

by replacing these neurons.

Alzheimer’s disease is a neurodegenerative disorder

characterized by progressive dementia with memory loss and

altered behavior including paranoia, delusions, depression, and

impairments of attention, perception, reasoning and

comportment, Stem cell-based therapy could be used to prevent

progression of the disease by transplanting stem cells modified

to release growth factors. Alternatively, compounds and/or

antibodies could be infused to restore impaired hippocampal

neurogenesis.

Cerebral palsy consists of a heterogeneous group of non

progressive clinical syndromes that are characterized by motor

and postural dysfunction, Stem cell therapy is used to induce

neuronal regeneration, The type of improvements reported

include: decreased spasticity; better coordination; improved

176

motor function, improved posture stability; better cognition

resulting in communication improvements.

Autism spectrum disorders are a group of biologically based

neurodevelopmental disorders characterized by impairments in

three major domains: socialization, communication, and

behavior, stem cells induce angiogenesis and neurogenesis in

areas of cerebral hypoperfusion, their ability to constitutively

secrete immune inhibitory factors leads to Immune modulation,

(in autism, several immunological abnormalities have been

detected both in the peripheral and the central nervous systems).

Neural retinal degeneration is a common cause of blindness

in people older than 60 years, stem cell transplantation rescue

the degenerating retina by supportive functions of the retinal

pigment epithelial cells and directly replacing lost

photoreceptors and retinal precursor cells.

Spinal cord injury leads to interruption of ascending and

descending axonal pathways, loss of neurons and glial cells,

inflammation, and demyelination. Stem cell-based therapies

could be used to treat individuals with spinal cord injury in

several ways. First, transplanting stem cell-derived spinal

neuroblasts could lead to the replacement of damaged or dead

motor and other neurons. Second, transplanting stem cell-

derived OPCs could promote remyelination. Last, transplanting

stem cells modified to release different factors could counteract

detrimental inflammation.

177

Peripheral nerves Injuries are common and debilitating,

affecting 2.8% of trauma patients and resulting in considerable

long term disability, stem cells delivery in a variety of repair

paradigms has been successful in promoting regeneration and

remyelination of the injured peripheral nerve.

Despite the fact that there is much basic work left to do and

many fundamental questions still to be answered, researchers

are hopeful that effective repair for once hopeless clinical

problems may eventually be achieved. Whether through

developing replacement cells or activating the body's own stem

cells in vivo, research on the use of stem cells for

neurodegenerative disorders is a rapidly advancing and

promising field.

178

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