Faculty of science

4th Year Biochemistry special

AN ESSAY ON:
STEM CELLS AND IT’S APPLICATIONS

Submitted By:
Maha Mohammed Hosny EL-Brashy .
Supervised By:
Dr. Mohammed Abd EL-hady,
Lecturer of biochemistry.
 TABLE OF CONTENTS

1.Introduction .
1.1 Stem Cell Concepts.
1.1 a Stem Cell Self-Renewal And Differentiation .
1.1 b Stem Cell Potency , Plasticity.
1.2 Importance Of Stem Cells .
1.3 Stem Cell History.
2. Sources Of Stem Cells .
2.1 Adult (Somatic) Stem Cells (ASC) .
2.2 The Adult Stem Cell Niche .
2.3 Bone Marrow Stem Cells .
2.4 Haematopoietic Stem Cells (Hscs) .
2.5 Bone Marrow Stromal Stem Cells (Bmscs)/Mesenchymalstem Cells (Mscs) .
2.6 Multipotent Adult Stem Cells (Mapcs) .
2.7 Ascs From Other Tissues .
2.8 Cord Blood Stem Cells And Foetal Stem Cells .
2.9 Embryonic Stem Cells .
2.10 Epiblast Stem Cells .
Embryonic Stem Cells Vs. Adult Stem Cells .
Similarities And Differences Between Embryonic And Adult Stem Cells .
3. Stem Cells In Culture .
3.1 Xeno-Free Derivation And Culture Of Human Embryonic Stem Cells:
Current Status, Problems And Challenges .
4. Stem Cell Markers .
5.Stem Cells Applications .
5.a Uses In Research .
5.b Some Examples Of Treatments For Major Diseases .
5.c Stem Cells & Cancer .
6. Ethical Problems On Stem Cell Research .
6.a Moral Status On Human Stem Cells .
6.b Controversy On The Source Of The Stem Cell .

Introduction
For centuries, scientists have known that certain animals can regenerate missing parts of
their bodies. Humans actually share this ability with animals like the starfish and the
newt. Although we can’t replace a missing leg or a finger, our bodies are constantly
regenerating blood, skin, and other tissues. The identity of the powerful cells that allow
us to regenerate some tissues was first revealed when experiments with bone marrow in
the 1950s established the existence of stem cells in our bodies and led to the development
of bone marrow transplantation, a therapy now widely used in medicine. This discovery
raised hope in the medical potential of regeneration. For the first time in history, it became
possible for physi- cians to regenerate a damaged tissue with a new supply of healthy cells
by drawing on the unique ability of stem cells to create many of the body’s specialized
cell types .(1)

Once they had recognized the medical potential of regeneration through the success of
bone marrow transplants, scientists sought to identify similar cells within the embryo.
Early studies of human development had demonstrated that the cells of the embryo were
capable of producing every cell type in the human body.(1)

STEM CELL CONCEPTS

1) stem cell self-renewal and differentiation:

 A stem cell can be described as an immature, primal, or undifferentiated cell that is
usually capable of dividing to produce at least one daughter cell that is identical to the
mother cell, a process called self-renewal, which may be perpetuated over a number of
cell divisions or possibly even indefinitely(1-3).
some of the general concepts of self- renewal are illustrated in Fig. 1 may be relevant
and may, to some extent, occur in the same stem cell population, but overall asymmetric
division contributing to both self-renewal and differentiation is probably the
(4,5)
predominant mechanism.
The purpose of a stem cell is to give rise to specific differentiated cell types, enabling
our bodies to grow and function normally Over our lifetime, which may be several
decades, our body is required to constantly replenish and repair its tissues, thus it is also
incumbent on the stem cell to remain responsive and active for many years. With only
relatively small numbers of stem cells present in our body, it meets these demands
through the process of self-renewal.(6)

Possible basic mechanisms of stem cell self-renewal are represented in figures 1A–
1C. Of these asymmetric, replicating and differentiating divisions, 1-A is the classic self-
renewal process, with the stem cell dividing to give rise to two daughter cells, one
remaining identical to itself (i.e. self-renewal) and the other responding to subtle changes
in the local environment and going on to differentiate. However, the fact that our bodies
age, marked by a decreasing capacity to repair our tissues and the fact we are also prone
to developing tumors, indicates that processes 1- B and 1-C are also important. In effect,
stem cell division is likely to be a mosaic of several processes. Differentiation is also a
complex process but that depicted in figure1- D involving generation of progenitor /
transit amplifying cells, is one likely mechanism. The role of progenitors is to enter into
several rounds of division increasing cell numbers. With each division the progenitor
may become progressively more differentiated and eventually stops dividing, having
acquired the characteristics of a specific diferentiated cell type (7,8) .

The activity of progenitors, such as number and rate of cell divisions and range of
differentiated cell types formed, varies within different tissues.(9,10)

Evidence for symmetric differentiating division where a stem cell population is

reduced and eventually exhausted is supplied by the fact that our bodies age and our
capacity to repair tissues, where stem cells are known to be present likebone and skin
also diminishes with age. Evidence for symmetric, replicating division, where a stem
population expands and potentially overgrows the tissue is somewhat more controversial
but there is evidence to suggest that cancers may be the result of stem cell self-renewal
(11)
Figure.1. A general overview of the concept of stem cell self-renewal and differentiation.
2)stem cell potency, plasticity :

There are three basic measures of stem cell potency:

(i) Totipotent — can form all cell types that contribute to the formation of
an organism. This is restricted to the fertilised egg or zygote. (See Figure 2 ).
(ii) Pluripotent — can form most cell types of an organism including germ
cells but not the placental tissues (e.g. ES cells and EG cells).
(iii) Multipotent — can form most cells issues within a particular tissue or
tissues (e.g. “adult” stem cells — HSCs).
Figure 2 Embryonic and Adult Stem Cells

Edited from http://robby.nstemp.com/photo6.html

What determines stem cell potency is dependent to a large extent on the genetics of
the cell and whether it contains the appropriate active or activated genes and
programming to differentiate into particular cell type or range of different cell types. For
example, changes in local growth factor /cytokine/hormone etc. gradients, cell-cell and
cell-matrix contact are important in the switching “on” and “off” of genes and gene
pathways and possibly even re-programming of those pathways, thereby changing the
type and range of cells that are generated (12-16).

The process of differentiation where a cell acquires a particular set of characteristics

enabling it to perform a specific function (e.g. insulin production by pancreatic beta cells,
dopamine production by neural cells, bone formation by osteoblasts, etc.) usually
involves formation of an intermediate progenitor cell, sometimes also called a transit
amplifying cell. To some extent progenitor cells are similar to stem cells, particularly
“adult” stem cells, being able to divide a number of times, and may even have a very
limited capacity for self-renewal, but usually with each successive division the cells
become progressively more differentiated (17).

The majority of progenitor cells lie dormant or possess little activity in the tissue in
which they reside. They exhibit slow growth and their main role is to replace cells lost by
normal attrition. In case of tissue injury, damaged or dead cells, progenitor cells can be
activated. Growth factors or cytokines are two substances which trigger the progenitors to
mobilize towards the damaged tissue. At the same time, they start to differentiate into the
target cells. Not all progenitors are mobile and are situated near the tissue of their target
differentiation. When the cytokines, growth factors and other cell division enhancing
stimulators take on the progenitors, a higher rate of cell division is introduced. It leads to
the recovery of the tissue. (17)

Progenitor cells may also exhibit a degree of plasticity, ranging from being unipotent
and restricted to differentiating to one specific cell type through to being bi- or tripotent
and giving rise to several differentiated cell types. For example in skeletal tissue, cartilage
cells (chondrocytes), fat cells (adipocytes) and bone forming cells (osteoblasts) are
believed to arise from a common tri-potential progenitor and this progenitor is itself
derived from a stem cell located within the bone marrow microenvironment. Apoptosis
or programmed cell death is also an integral part of the process of cell proliferation and
differentiation, although it is not discussed here (18) .

Importance of stem cells

Stem cells have three important characteristics that distinguish them from other cells:
1. They are unspecialized cells that renew themselves for long periods through cell
division. They do not have any tissue-specific functions that allow them to perform
specialized functions. For example, a single stem cell cannot beat with another heart cell;
it cannot communicate with other cells, as nerves cells do (19)
2. Under certain conditions stem cells can be induced to become cells with special
functions, such as cells of the heart muscle or insulin-producing cells of the pancreas.
Unlike muscle, blood, or nerve cells, they can replicate themselves many times„ a process
called proliferation. A starting line of stem cells can proliferate in the laboratory for many
months and yield millions of cells. If this happens over a long period of time, the process
is called self-renewal (19) .
3. Stem cells give rise to specialized cells. When this occurs, the process is called
differentiation .signals from both inside and outside the cell may trigger this
differentiation genes control the internal signals. External signals include chemicals
secreted by other cells, physical contact with other cells, and certain molecules called
growth factors (19) .

Stem cell history

1900:The hematopoietic cell systems are highly researched for it was among the better
understood proliferation system and blood cells are marked in the peripheral blood and
other tissues by using Radioactive (s9fe) for newly produced red cells .also,blood cells
can be marked by utilizing tritiated Thymidine,showing the kinetics of cell production in
the bone marrow and distribution in the peripheral blood . These studies indicated an
orderly progression of proliferation and then differentiation in the marrow with release
to the blood (20)
1957: successful first attempt at intravenous infusion of bone marrow in patients
receiving Radiation and chemotherapy ,Mary Imogene Bassett Hospital,
Cooperstown,USA (20)

1959: Researchers at Jackson Laboratory,Maine,USA, identify a strain of mouse in

which testicular teratomas occur with an appreciable frequency (20)

1960: *Researchers made important discovery on bone marrows and nerve cells and they
found populations of hematopoietic stem cells ,which form all the types of blood cells in
the body and bone marrow stromal cells was discovered a few years later (20)
*Also, in 1960, the myth of rat’s brain having dividing cells was still in doubt . It was’t
until the 1990 ,scientists agreed that the adult brain does contain stem cells that are able to
generate the brain’s major cell types ( astrocytes and oligodendrocytes) which are non
neuronal cells(20)

1963: The first quantitative descriptions of the self-renewing activities of transplanted

mouse Bone marrow cells, At university of Toronto,Canada.(21)

1974: Researchers at the university of pennsy/vania ,USA ,demonstrate that mouse

Embryonic Stem cells (ES) can participate in the development of organisms aswellas
teratomas (21).

1978: First in vitro fertilization ( IVF ) baby born ,following fertilization of human eggs
outside the body by scientists at cambridge university , UK (21).

1981 : successful cultivation of mouse ES cells from explanted inner cell mass cells (21).

1998: Team lead by Dr. James Thompson at the University of Wisconsin isolates
embryonic stem cells for first time. Wisconsin Alumni Research Foundation receives
patents on embryonic stem cell work(22).

1999: Researchers remove 10-15 neural stem cells from a parkinson’s disease patient and
used them to reproduce 6 million dopaminergic neural stem cells (22).
These were introduced into the patient’s brain tissue ,producing a 62% increase in
dopamine uptake and a 40-50% improvement in certain motortasks(22).
2001: President Bush announces his plan to fund research on existing embryonic stem cell
lines(22).
2002 : *Study concludes bone marrow stromal cells are an accessible, expandable source
of cells that offer a promising future for spinal cord repair (22).
*Parkinson’s patient treated with own neural stem cells had symptoms reduced +80
percent(22).

. 2003: *Researchers inject a natural protein into the brains of 5 Parkinson’s patients; the
stimulated adult stem cells provided an average 61 percent improvement in motor
function (22).
* Researchers find that infusing bone marrow stem cells into patients after a heart attack
aided regeneration of the heart (22).

2004: * Researchers receive FDA approval for bone marrow stem cell transplants in
patients with severe heart failure; study shows that after 12 months, patients who were
treated had significant improvement (22).

2005: * Researchers perform the first ever pathology follow-up of one patient treated for
Parkinson’s disease; study showed that the protein stimulated sprouting of new neurons in
the brain (22).
*Scientists treat ten Parkinson’s patients with a protein to stimulate the patient’s own
brain stem cells and showed significant improvement in symptoms (22).
* Doctors find that patients with congestive heart failure improve greatly when treated
with their own adult stem cells (22).
* Cardiologist shows that a type of human bone marrow stem cell can turn into most
tissue types of the body(22).
* Islet cells can be donated from live donors for patients, opening transplant possibilities;
using technique, a mother donates cells for her diabetic daughter, alleviating diabetic
symptoms (22).
* Scientists find that patients can serve as their own donors, converting their liver cells to
insulin-secreting cells (22).

2006 : *Transplant of nasal stem cells into 7 patients with spinal cord injury; patients
regain some motor function and sensation, 2 patients showed bladder control
improvement (22).
*Scientists show they can grow pancreatic cells for long periods and turn them into
insulin-secreting cells (22).
*Team demonstrates human umbilical cord blood stem cells form insulin-secreting cells
(22).

*Young stroke victim treated with umbilical cord stem cells (22).
June 2007 - Research reported by three different groups shows that normal skin cells can
be reprogrammed to an embryonic state in mice(23). In the same month, scientist
Shoukhrat Mitalipov reports the first successful creation of a primate stem cell line
through somatic cell nuclear transfer (24).
October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel
Prize for Physiology or Medicine for their work on embryonic stem cells from mice using
gene targeting strategies producing genetically engineered mice (known as knockout
mice) for gene research (25).
November 2007 - Human induced pluripotent stem cells: Two similar papers released by
their respective journals prior to formal publication: in Cell by Kazutoshi Takahashi and
Shinya Yamanaka, "Induction of pluripotent stem cells from adult human fibroblasts by
defined factors"(26), and in Science by Junying Yu, et al., from the research group of James
Thomson, "Induced pluripotent stem cell lines derived from human somatic cells"(27) :
pluripotent stem cells generated from mature human fibroblasts. It is possible now to
produce a stem cell from almost any other human cell instead of using embryos as needed
previously, albeit the risk of tumorigenesis due to c-myc and retroviral gene transfer
remains to be determined.

January 2008 - Robert Lanza and colleagues at Advanced Cell Technology and UCSF
create the first human embryonic stem cells without destruction of the embryo (28) .
January 2008 - Development of human cloned blastocysts following somatic cell nuclear
transfer with adult fibroblasts (29) .
February 2008 - Generation of pluripotent stem cells from adult mouse liver and stomach:
these iPS cells seem to be more similar to embryonic stem cells than the previously
developed iPS cells and not tumorigenic, moreover genes that are required for iPS cells
do not need to be inserted into specific sites, which encourages the development of non-
viral reprogramming techniques (30) .
March 2008-The first published study of successful cartilage regeneration in the human
knee using autologous adult mesenchymal stem cells is published by clinicians from
Regenerative Sciences (31)
October 2008 - Sabine Conrad and colleagues at Tübingen, Germany generate pluripotent
stem cells from spermatogonial cells of adult human testis by culturing the cells in vitro
under leukemia inhibitory factor (LIF) supplementation (32) .
30 October 2008 - Embryonic-like stem cells from a single human hair (33) .
1 March 2009 - Andras Nagy, Keisuke Kaji, et al. discover a way to produce embryonic-
like stem cells from normal adult cells by using a novel "wrapping" procedure to deliver
specific genes to adult cells to reprogram them into stem cells without the risks of using a
virus to make the change(34-36). The use of electroporation is said to allow for the
temporary insertion of genes into the cell (37-40).
28 May 2009 Kim et al. announced that they had devised a way to manipulate skin cells
to create patient specific "induced pluripotent stem cells" (iPS), claiming it to be the
'ultimate stem cell solution (41) .
 11 October 2010 First trial of embryonic stem cells in human(42).
25 October 2010 - Ishikawa et al. write in the Journal of Experimental Medicine that
research shows that transplanted cells which contain their new host's nuclear DNA could
still be rejected by the invidual's immune system due to foreign mitochondrial DNA.
Tissues made from a person's stem cells could therefore be rejected, because
mitochondrial genomes tend to accumulate mutations (43) .

Sources of Stem Cells:

1-Adult (somatic) stem cells (ASC):

ASCs are found in many tissues throughout the body. These cells divide and
differentiate to replenish the supply of differentiated cells that die as part of the natural
“life cycle” of the tissue or to repair damaged tissue. Tissues like the blood, skin, liver,
gut and bone are replenished and repaired almost constantly and while the ability to
maintain and repair our tissues diminishes as we get older, the fact that we can live for
several decades demonstrates clearly the capacity of ASCs for self-renewal. In the body,
ASCs usually differentiate to a particular cell type or range of cell types and these are
usually associated with the tissue in which they are located. In this regard ASCs are
considered to be multipotent cells.

However, evidence is accumulating to suggest that ASCs might in fact have

pluripotency and this is supported by several lines of experimental investigation:

(i) Stem cells from the bone marrow have been differentiated into neural Cells (44)and
likewise stem cells from the brain have been differentiated into blood cells (45,46) .
(ii) Studies on bone marrow transplantation in rodents and humans have demonstrated
that small numbers of marrow-derived stem cells can migrate to various tissues and
organs around the body and at least colonize, but possibly also differentiate into or
stimulate cells in those tissues (47,48) .
(iii) Bone marrow stem cells have been used in clinical trials to help promote repair of
heart tissue damaged after a heart attack.(49,50)
(iv) The expression of genes and proteins associated with pluripotency (e.g. those
expressed by ES cells) have been detected in some ASCs (51,52) .

2-The adult stem cell niche:

The capacity of ASC to self-renew and to differentiate into mature cell types is a
tightly regulated process which is controlled by a number of tissue-specific environmental
factors. In order to maintain tissue homeostasis, these cells often reside in what is referred
to as a “stem cell niche”. (figure3)This “niche” is essentially a microenvironment that
limits the exposure of stem cells to differentiation, apoptotic and other signalling events
that would otherwise deplete stem cell reserves. In addition, the “niche” tightly controls
stem cell division to avoid overpopulation of a tissue with these cell types, which would
give rise to cancer. When required, stem cells can be activated to produce transit
amplifying or progenitor cells that are at that point committed to producing the mature
cell types of that tissue. Hence, the dynamic interplay between stem cells and their niche
is essential in the maintenance of healthy tissues and so it is important to understand these
relationships in order to utilise these cell types effectively for therapeutic applications
(53,54)
.

Components of the stem cell niche.

Edited from http://rstb.royalsocietypublishing.org/content/363/1489/123.full

Figure 3 Components of the stem cell niche. (a) Cell–cell and cell–extracellular
environment interactions. Membrane-associated receptors and ligands (red and yellow)
mediate cell–cell contacts and cell fate decisions, including self-renewal and
differentiation. In addition, gap junctions (purple), intercellular channels that allow the
passage of ions and metabolites, both coordinate behaviour between coupled cells and can
tether cells together. Hemi-channels allow communication between the cell and the
environment. (b) Diffusible factors. Diffusible factors (purple spheres) can direct stem
cells to either self-renew or generate differentiated progeny. The availability of diffusible
factors that bind to receptors (green) in turn can be regulated by ligand inhibitors (blue
half-moons), which can sequester these factors and prevent signalling. (c) Basal lamina
and blood vessels. An extracellular matrix ECM-rich basal lamina (black), which can be
associated with blood vessels (red) or cells, has several functions in stem cell niches,
including anchoring cells to the niche (grey spheres), sequestering and presenting
diffusible signals (purple and brown spheres), and linking cells and the ECM. In addition,
proteolytic fragments of ECM components (blue squiggles) may regulate stem cells.
Endothelial cells and the vasculature are emerging as integral components of stem cell
niches, where they can regulate stem cell fate decisions through either diffusible signals
and/or direct cell–cell contact. (d) Oriented cell division. Both cell–cell contact (via
adherens junctions, red) and tethering of cells to the basal lamina (black) can influence
cell fate by orienting the plane of cell division, such that key intracellular determinants
are symmetrically or asymmetrically distributed. Oriented segregation of these factors
may influence cell fate. Short-range diffusible factors (purple and brown spheres)
secreted by stromal cells (green) that promote self-renewal only influence immediately
adjacent cells, allowing differentiating daughter cells to escape the niche.
3- Bone marrow stem cells:

The bone marrow is one of the most abundant sites for ASCs and these are also
probably the most studied and best understood of all ASC types. Part of the reason why
marrow stem cells have been studied so extensively, compared to other ASCs is
accessibility — samples of bone marrow can be collected relatively easily by introducing
a needle directly into the bone marrow, usually at the iliac crest and aspirating marrow
tissue. The marrow tissue can be then cultured for further investigation. Stem cells from
the bone marrow often enter the circulation and can be also found in peripheral blood
samples (55,56) .

There are in fact two distinct types of ASC within the bone marrow — the
haematopoietic stem cell (HSC) that classically gives rise to the entire blood cell
lineage(57) and the bone marrow stromal stem cell (BMSC), also called the mesenchymal
stem cell (MSC), that classically gives rise to various connective tissues notably bone,
cartilage and adipose tissue(58). Other cell types are also found in bone marrow aspirates,
notably red blood cells, endothelial cells, fibroblasts,adipocytes and osteoblasts. There is
also evidence for distinct subpopulations of stem cells with the marrow environment,
notably multipotent adult progenitor cells (MAPCs), which appear to share similar
properties to ES cells (59,60) .With such a heterogeneous mix of cells it is necessary to apply
specific methods to isolate and characterise the stem cells, such as density centrifugation,
cell sorting techniques like fluorescence-activated cell sorting( FACS) or
immunomagnetic cell sorting( MACS) and differential adhesion (61,62) .

In terms of characterisation of a stem cell population such as from the bone marrow,
the classic experiment is the colony forming unit (CFU) assay,(63,64) where the cell fraction
is diluted to specific densities or number of cells, including down to a single cell
(determined from a total mononuclear cell count). The cells are cultured for several days
and then observations made for growth of distinctive colonies of differentiating cells
which will be the progeny of any stem cells present the culture. The colonies can be
characterised by morphology and also by more detailed molecular and biochemical
assays.(65) The colonies can be also counted and collectively with data from the other
assays this can be used to assess the relative abundance or frequency of a particular stem
cell type within the bone marrow. It is believed that the stem cell population within the
marrow is somewhere between one cell in 10,000 down to 100,000 cells (66-68) .

There are some more rudimentary differences between the HSCs and BMSCs, which
are that HSCs tend not to adhere directly to cell culture plates (are either co-cultured on a
feeder layer of growth-arrested stromal cells or fibroblasts or on a semi-solid support
matrix such as methylcellulose, which is a gel-like material), whereas BMSCs will, in
general adhere to cell culture plates. Thus, a simple adhesion assay performed over an
hour or so can be used to initially differentiate (termed a differential adhesion assay)
between the HSCs and BMSCs fractions.(67,68)

4. Haematopoietic stem cells (HSCs):

Of all the different types of stem cell, HSCs are the best studied and are also an
example of a very successful stem cell therapy, forming the basis of bone marrow
transplantation to treat various types of leukaemia. Within the body, HSCs are found
mainly in the bone marrow (may also circulate in the blood and occupy other tissues like
the spleen) and they function to generate the entire blood cell lineage (e.g. red blood cells,
leucocytes and lymphocytes),(69). Although the marrow is a diffuse tissue, the proliferation
and differentiation of HSCs is tightly controlled within microenvironments* or niches.
(70,71).
The exact nature of the HSCs niche is not known, but physical contact with stromal
cells (including BMSCs) and osteoblasts is important, as is the presence of various
growth factors, in particular stem cell factor (SCF) and also a number of specific
cytokines (colony stimulating factors),(72,73).There are a number of “markers” expressed on
the surface of HSC such as CD34 (and many others that are used in varying
combinations) which can be used sort fractions of HSC (74,75).
5. Bone marrow stromal stem cells (BMSCs)/mesenchymalstem cells (MSCs):

This stem cell type “shares” the marrow environment with HSCs (and several other
cell types) and as indicated above there is a certain amount of interaction between the
different cell populations. Again the exact nature of the microenvironment or niche in
which BMSCs reside is not known, but the marrow and the bone tissue that confines it are
dynamic environments and the key function of BMSCs is to help maintain these
environments, differentiating into cells such as osteoblasts, adipocytes and stromal cells /
fibroblasts.(76) For example, throughout our life, bone is constantly being resorbed by
osteoclasts (derived from HSCs) and replaced by osteoblasts in response to normal
physiological demand, e.g. mechanical loading associated with walking, running, etc.
and metabolic turnover, with bone tissue serving as a reservoir of essential mineral ions
and cytokines. Our ability to repair fractured or broken bones also serves as powerful
illustration of the function of BMSCs.

Selection of BMSCs from marrow aspirates is probably less efficient than that for
HSCs because fewer specific “markers” exist for BMSCs.(77). Initial isolation can be
achieved by differential adhesion assays, which will remove the HSC fraction but other
cells present in the marrow like adipocytes, and stromal cells/fibroblasts will also adhere
to cell culture plates. There are some “markers” that are useful for selection of BMSCs
including Stro-1, an as yet uncharacterised cell surface antigen, CD44, a glycoprotein that
binds hyaluronan, a component of the extracellular matrix and CD105, also called
endoglin, and a receptor for the cytokine transforming growth factor beta. Both CD44 and
CD105 can be also used for selecting HSC, but some distinction can be achieved by
subsequent adhesion of the selected cells.(78-81)

The BMSC can, in general, be maintained and expanded in culture quite easily,
without the need to add specific growth factors, such as required for HSC. The
differentiation potentials of BSMCs can be assessed by CFU assays and also by adding
specific growth factors to stimulate differentiation into a particular cell type. Such
experiments reveal that BMSCs can be readily differentiated in vitro to osteoblasts,
chondrocytes, adipocytes, stromal cells, tendon cells and muscle cells and possibly several
other cell types.(82) Based on the potential to differentiate into these various connective
tissue cell types, BMSCs have been used in a number of tissue engineering studies, for
example to repair damaged bones and joints in both animal models and also humans.

There are also a few clinical examples where BMSCs have been used to help treat
genetic diseases of the bone, such as osteogenesis imperfecta, where the osteoblasts have
an impaired capacity to synthesise bone matrix protein (collagen type 1), resulting in
brittle bones. Implantation of donor BMSCs can help restore differentiation of normal
osteoblasts, leading to synthesis of normal bone tissue (83,84).

6. Multipotent adult stem cells (MAPCs):

MAPCs were isolated as a subset of BMSCs/MSCs and so are extremely rare cells.
They express cell surface “markers” that are distinct from BMSCs and also express
“markers” associated with ES cells and pluripotency. Relatively few experiments have
been performed to date to explore and confirm the biology of these cell (85,8)

7. ASCs from other tissues:

Many tissues have now been shown to contain a population of stem cells (or cells with
stem cell-like characteristics) that serve to replace or repair cells that are lost due to
normal wear and tear or injury. Notable examples of such tissue include skin,gut and
liver, which as part of their normal function have high cell attrition rates. However, many
other tissues, which appear to a have a much more limited capacity for normal repair,
such as cartilage,heart,teeth and brain have also been shown to contain stem cells or
putative stem cells. However, it is not known why these tissues are unable to effect self
repair as efficiently as tissues like the skin,(87-91). Clearly, the isolation and therapeutic
application of ASC from some tissues is going to be challenging, however, their study
using animal models or human surgical and cadaveric samples is still important for
understanding their biology and potentially designing methods, such as scaffold delivery
systems to stimulate these cells in the body (92-95).

8. Cord blood stem cells and foetal stem cells:

Blood collected from the umbilical cords of newborn babies has been shown to contain
populations of stem cells with characteristics similar to both HSCs and BMSCs and could
potentially be useful in allogenic transplants,(92,93). Stem cells have also been isolated from
the cord blood during early pregnancy,using a needle guided by ultrasound. Again these
foetal cord blood stem cells have characteristics similar to both HSCs and BMSCs, but
unlike cord blood collected from term pregnancies, these foetal stem cells may be less
developed immunologically and potentially would cause fewer problems with immune
rejection. More controversially foetal stem cells can be potentially isolated from elective
abortions (94,95).

9. Embryonic stem cells :

Embryonic stem cells are derived from the inner cell mass of the pre-implantation
blastocyst and have been obtained from a number of different species including mice,
non-human primates and humans .
Manipulation of the human embryo is restricted to within the first 14 days of its
creation.This time point marks a specific stage in embryonic development with the onset
of organogenesis and in particular the formation of the primitive streak, which is the
rudimentary central nervous system. ES cells are usually isolated before day 5, when the
entire embryo is comprised of few hundred cells (96-98)

10. Epiblast stem cells:

 The epiblast is a tissue that forms at a later stage than ES cells, after the developing
embryo implants into the uterus. Cells derived from the epiblast of the mouse and rat and
have been shown to behave as pluripotent stem cells. Perhaps more interesting is the fact
that rodent epiblast stem cells have characteristics that are more similar to human ES cells
(99)

Embryonic Stem Cells vs. Adult Stem Cells

Pluripotent stem cells are suggested to be more versatile in therapeutic potential and
facing formidable technical challenges. Before practicing in human disease treatment,
considerable control over the stem cell development and acceptance of patient's immune
system has to be assured. Embryonic stem (ES) cells are relatively better candidate than
adult stem cells due to the reason that it is capable to give rise to cells found in all tissues
of the embryo instead of being merely multipotent, as adult stem cells are thought to be.
However, little evidence points out that multipotent adult stem cells may be able to
change course and provide the flexibility just as good as ES cells. In addition, searching
effort of the pluripotent potential of adult stem cells is limited by the low multiplication
capacity of adult stem cells, which is a disadvantage in relative of ES, making it difficult
to isolate and purify. Finally, by comparing the ‘experiences’, adult stem cells are
exposed to DNA abnormalities at a higher rate. Using of own adult stem cells would not
be rejected by the immune system because the patient's own cells could be expanded in
culture and then reintroduced into the patient. ES cells transferred from a donor into a
patient could cause transplant rejection. However, the recipient would reject donor ES
cells has not been determined in human experiments (100) .

Similarities and differences between embryonic and adult stem cells

Human embryonic and adult stem cells each have advantages and disadvantages
regarding potential use for cell-based regenerative therapies. Of course, adult and
embryonic stem cells differ in the number and type of differentiated cells types they can
become. Embryonic stem cells can become all cell types of the body because they are
pluripotent. Adult stem cells are generally limited to differentiating 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(101).

Large numbers of embryonic stem cells can be relatively easily grown in culture, while
adult stem cells are rare in mature tissues and methods for expanding their numbers in cell
culture have not yet been worked out. This is an important distinction, as large numbers
of cells are needed for stem cell replacement therapies(101).

A potential advantage of using stem cells from an adult is that the patient's own cells
could be expanded in culture and then reintroduced into the patient. The use of the
patient's own adult stem cells would mean that the cells would not be rejected by the
immune system. This represents a significant advantage as immune rejection is a difficult
problem that can only be circumvented with immunosuppressive drugs (102)
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. (102)

The unique potential contribution of human embryonic stem cells to therapies is a

product of both their longevity and their capacity to produce a wide range of specialised
cells in the laboratory. By contrast, adult stem cells that are grown in the laboratory
appear to have much shorter lifespans than embryonic stem cells. This reduces their
capacity to form new cell types. Stem cells can also be obtained from aborted fetuses and
umbilical cord blood, but it is not clear whether the full range of cell types that are
required for treatments could eventually be generated from these sources alone (103) .

Stem Cells in Culture

All stem cells, whether isolated from adults or embryos, have the ability to proliferate
in culture and can differentiate into many different kinds of cells. Differentiation of these
cells occurs spontaneously or through directed differentiation *. Researchers choose the
initial culturing conditions that prevent spontaneous differentiation. This involves
growing the cells on a layer of feeder cells that secrete substances into the culture media
that help nourish the ES cells (figure 10). For the most part, the identity of these
substances is unknown. The feeder cells in most in vitro experiments are mouse
embryonic fibroblasts that have been exposed to γ-radiation, a form of radioactivity that
destroys the cell’s ability to replicate(103)

Feeder cells help maintain the stem cells in an undifferentiated state; they also provide
a favorable substrate for the ES cells to grow on.

When the stem cells are needed for a directed differentiation experiment, they are
transferred to fresh culture plates that lack a feeder cell layer, then given culture media
containing one or more growth factors. In some cases, directed differentiation simply
involves transferring the cells to fresh plates, lacking a feeder layer, so the cells can form
embryoid bodies (104) .

Directed differentiation of adult stem cells has confirmed previous in vivo studies,
which concluded that adult stem cells have less plasticity than ES cells.When stem cells
from an adult mouse brain are placed in culture, they differentiate into different kinds of
neural tissue but not the variety of cell types that are produced by ES cells. Likewise,
adult stem cells from the bone marrow differentiate, in vitro, into blood cells, neurons,
and fat cells, but not glandular tissue .Moreover, scientists have less control over the
differentiation process of adult stem cells than that of ES cells. Adult bone marrow stem
cells differentiate spontaneously when placed in culture, and there is no way to stop them.
Consequently, it is very difficult to maintain a continuous culture of these cells. So far,
only mouse ES cells are able to proliferate in vitro for an indefinite period of time while
retaining an embryonic phenotype (104) .

Figure 4. Scheme for the generation of new cells and tissues from embryonic stem cells.
ES cells are often cultured upon a lawn of animal feeder cells that provide growth factor
support for the expanding ES cells. Appropriate manipulation of the culture results in the
formation of embryoid bodies, and a defined chemical cocktail can orchestrate
differentiation towards a specific cell fate.

Edited from http://www.kidney.org.uk/Medical-Info/transplant/stem-cells.html

The gradual loss of plasticity in some stem cells, mouse or human, may not be a serious
problem, because some ES cells have been maintained for more than two years in an
undifferentiated state. Moreover, even after months of culturing, stem cells will still
respond to directed differentiation. This responsiveness is extremely important if these
cells are going to be used to treat a disease. For example, to repair a damaged heart,
cultured ES cells, kept in stock for years, would be stimulated to differentiate into
myocytes, after which they would be injected into the circulatory system of the patient.
The trick here is to ensure that the cells are only partially differentiated (that is, they are
myocyte precursors); otherwise they would lose the power to proliferate and be unable to
effect repairs (109) .Simply injecting freshly isolated stem cells into the patient would lead
to the formation of teratomas, as occurs when stem cells are injected into mice. Injected
myocyte precursors, on the other hand, will home to the parent organ, the heart, and, it is
hoped, initiate repairs. Of course, this scenario is very hypothetical and may contain a
large dose of wishful thinking. For the present, we simply do not know what a myocyte
precursor will do when it reaches the heart, and, perhaps more important,we do not know
how the heart itself will respond (104) .

Directed differentiation of cultured mouse or human stem cells has led to the
production of many cell types, but in most cases scientists do not understand the details of
the process.Manipulating the culture conditions by adding a growth factor or a molecule
that influences gene expression may cause the stem cells to differentiate into nervous
tissueor bone, but the intermediate steps that lead to the transformation are still obscure
(104)
.

Consequently, directed differentiation requires a good deal of guesswork. Various

compounds known to influence gene expression can be added to the culture media to see
if they will stimulate differentiation and, if they do, which kind of cell is produced. In this
way, scientists are in the process of constructing a catalog of culture additives that will
lead to the production of specific cell types from cultured embryonic and adult stem cells.
The study of directed differentiation may also help scientists understand the molecular
nature of plasticity and the factors that distinguish embryonic and adult stem cells. This
information could pave the way to effective therapies based solely on the use of adult
stem cells (104) .

Xeno-free derivation and culture of human embryonic stem cells: current status,
problems and challenges

Since the first publication in 1998 , human embryonic stem cells (hESC) have attracted
significant attention due to their dual ability to self-renew and to differentiate into all cell
types of the body. This makes them excellent candidates for cell- and tissue-replacement
therapies, as well as for the basic scientific research on human embryogenesis and
diseases . However, most hESC lines available to date have been directly or indirectly
exposed to animal material during their derivation and/or propagation in vitro .(106) .
Although there are efforts to use the currently available hESC lines for clinical trials, such
xeno-contaminated cells are generally judged unsuitable for transplantation because of the
risk of zoonosis transmitted by animal pathogens and the potential activation of animal
retroviruses, not to mention the possibility of immune rejection . For therapeutic
purposes, all steps for the production of hESC must avoid the use of animal components,
i.e., culture systems using animal-free derivation methods, animal-free culture media, and
animal-free substrates should be established to create and propagate clinical-grade hESC
lines. .(106)
Derivation of hESC
hESC lines are conventionally derived from the inner cell mass (ICM) of pre-
implantation stage blastocysts, of both good and poor quality , which have been donated
for research and would otherwise be discarded. Morula-stage embryos or late-stage
blastocysts (7-8 days) may also be used to create hESC lines. Although all the hESC
lines derived worldwide share the expression of characteristic pluripotency markers ,
many differences are emerging between lines that may be more associated with the wide
range of culture conditions in current use than with the inherent genetic variations of the
embryos from which hESC were derived . The reported success rate for hESC derivation
is highly variable, possibly depending on the embryo quality and the culture protocol
used. Very recently, Klimanskaya et al. published a new method for the derivation of
hESC lines from single blastomeres obtained by a procedure similar to the one used for
preimplantation genetic diagnosis, which could, theoretically, avoid embryo destruction
in the future . Alternatively, hESC lines were also successfully obtained from arrested
embryos which stopped cleavage and failed to develop to morula and blastocysts in
vitro ..(106)
Isolation of ICM: immunosurgical versus mechanical method

Isolation of the ICM from blastocysts is a very important step in the derivation of
hESC lines. Although several reports demonstrated the derivation of hESC lines from
intact blastocysts after removal of zona pellucida , the outgrowth of trophectoderm at
an early stage might inhibit the expansion of ICM and further reduce the success rate of
hESC derivation . Nearly all reported hESC lines to date have been efficiently obtained
from ICM isolated by immunosurgery , a procedure that removes the outer trophectoderm
epithelial cell layer from the blastocyst using anti-human whole-serum antibodies and
guinea pig complement. However, the possible xeno-contamination in antiserum may
limit its clinical use. Mechanical isolation of ICM serves therefore as a better alternative
for the derivation of clinical-grade hESC lines, avoiding the exposure of blastocysts to
animal antibodies. Human ESC lines have been successfully derived from manually
isolated ICM . In our laboratory, laser-hatching technology is employed to mechanically
isolate the ICM. Efficiently used in assisted reproductive medicine, laser beams can be
precisely delivered to drill the trophectoderm and favor ICM expulsion. Compared with
the manual microdissection, laser-hatching technology requires minimal
micromanipulation skills and maximally avoids xenogenic contamination by reagents. .(106)
Feeder-dependent derivation of hESC

To obtain ESC, the isolated ICM need to be placed on specific substrates and cultured
in appropriate media (Figure 11). In most cases, ICM are cultured on mitotically
inactivated feeder cells. Although irradiated or treated with mitomycin, feeder cells are
still able to stimulate ESC growth and inhibit their differentiation through the secretion of
specific growth factors and cytokines . Like mouse ESC, hESC lines were firstly
established on mouse embryonic fibroblasts (MEF) , in a medium containing fetal bovine
serum (FBS). Such conditions are unsuitable for human cell transplantation since the
exposure to animal components presents a serious risk of transmitting unidentified
retroviruses and other pathogens to the patients. Although there is a report showing no
evidence for hESC infection by animal feeder-derived viruses , concerns still remain over
the clinical use of these cells.
Furthermore, hESC cultured under xeno-contaminated conditions can present a non-
human sialic acid, which is immunogenic to humans . For therapeutic purposes and to
eliminate, or at least to reduce, the xenogenic contamination from animal feeder cells and
sera, human feeder cells and serum replacement (SR) have been recently employed for
both culture and derivation of hESC lines . One report also shows hESC lines established
on feeder cells derived from hESC themselves . However, none of these culture systems
can be considered entirely animal-free since FBS was normally used to culture feeder
cells, and SR is a reconstituted formulation still containing large amounts of bovine serum
albumin as well as other proteins. In order to completely remove animal substances from
the culture system, several teams have employed human serum in culture media , hESC
lines derived under such conditions are expected to meet the criteria for clinical
applications. .(106)
Figure 5 Derivation and culture methods for human embryonic stem cells. Here it is
illustrated all embryo stages showed in the literature to give rise to human embryonic
stem cell lines. IVF: In-vitro fertilization; ICSI: Intra-cytoplasmic sperm injection;
PN:Pronucleus.

Edited from http://www.nature.com/cr/journal/v17/n8/fig_tab/cr200761f1.html#figure-title

Feeder-free derivation of hESC

Like serum, feeder cells are a source of variability in experimental conditions and are
also a concern for future transplantation of hESC derivatives to patients. A recent study
successfully established a hESC line on the extracellular matrix (ECM) prepared from
MEF . Despite the immunosurgical ICM isolation and the culture in SR-supplemented
medium, the use of such ECM represents a significant advance as, if proven efficient,
ECM of human origin would avoid the above-mentioned pathogenic risks, as well as the
possible interference of living feeder cells in future transplantation therapies. In addition,
feeder cell-derived ECM can be easily sterilized and stored for clinical applications.
Another recent report described the derivation of two hESC lines in a defined culture
medium containing components solely derived from recombinant sources or purified from
human material, on substrate formed by human ECM components. .(106)

Although these culture conditions are laborious and costly, and may lead to abnormal
karyotype during long-term culture, hESC lines derived and propagated in this xeno-free
and feeder-independent system would be more directly applicable to clinical use. Such
culture conditions also provide a basis for further simplifying culture requirements in
studies investigating the molecular mechanisms of hESC self-renewal and differentiation.
We are presently testing the capability of human feeder cell-derived ECM to support
prolonged undifferentiated growth of hESC, aiming at the establishment of clinical-grade
lines in suitably defined xeno-free culture conditions. .(106)
Clonal derivation of hESC

hESC derived from ICM of blastocysts should be considered as heterogenous .

Isolation of a homogenous pool of differentiated hESC is necessary for basic studies like
drug development and toxicity testing. Moreover, for therapeutic applications, the
removal of undifferentiated, potentially tumorigenic cells will also be a requirement, or at
least malfunctioning cells should be removed using cell ablation strategies, before and/or
after engraftment. .(106)
New hESC lines have been clonally derived from the existing ones . However, unlike
mouse ESC, the clonal efficiency of hESC is extremely low, as hESC are sensitive to
single-cell disaggregation and recover poorly when plated at clonal density. Indeed, cell-
cell interactions seem critical for efficient hESC propagation, since the loss of gap
junctions between hESC can increase cell apoptosis and inhibit hESC colony growth . For
these reasons, hESC are routinely passaged in cell clumps, produced by mechanical
cutting and/or mild enzymatic treatment , which is an obstacle to large-scale propagation
of hESC for clinical use. Nevertheless, clonal survival of hESC can be enhanced by
culturing in physiologic oxygen (2%) , or in the presence of neurotrophins . Therefore, the
efficiency of hESC derivation might be improved at low oxygen concentration and/or
with neurotrophins in culture medium. .(106) of p
Passaging of hESC: mechanical versus enzymatic methods
When the feeder cells become old or the colonies are too dense or too large, hESC
need to be split for continuing culture. Passaging of hESC falls into two categories –
mechanical and enzymatic. Clearly, the passaging technique is critical for maintaining
undifferentiated and karyotypically stable hESC lines. Mechanical passaging is performed
by dissociating the colonies into small clumps through manually scraping and cutting and
transferring them to new culture plates covered with feeder cells or appropriate substrates.
This is labor-intensive and time-consuming, and it normally generates variable cluster
size and results in inconsistent cell distribution. On the other hand, enzymatic passaging
dissociates the hESC colonies using enzymes such as animal-derived collagenase,
dispase, and trypsin , xeno-free recombinant enzyme or enzyme-free cell dissociation
buffer , allowing a relatively consistent and standardized passaging of hESC. .(106)
In view of clinical applications, enzymatic passaging is advantageous since it enables
large-scale expansion of hESC. However, emerging evidence suggests that excessive
and/or frequent dissociation to single cells may lead to karyotype abnormalities . In
contrast, mechanical methods of passaging allow selective transfer of exclusively
undifferentiated colonies and seem to better maintain genetic stability. Since the process
of manual colony dissection limits the practical use of mechanical passaging in bulk
hESC culture, many researchers combine both enzymatic and mechanical passaging
methods for long-term hESC maintenance . Indeed, hESC lines initially derived and
passaged by mechanical methods have been subsequently adapted to enzymatic passaging
for practical reasons . Automated mechanical passaging of hESC, which incorporates the
advantages of mechanical dissection without sacrificing the practical benefits of
enzymatic passaging, was also developed . To overcome the low resistance of hESC to
single-cell dissociation during passaging, recent studies have achieved the culture of
newly derived hESC lines which keep stable karyotype in long-term culture by enzymatic
bulk passage . These single-cell dissociation-resistant hESC lines would be valuable for
clinical applications. Nevertheless, hESC in long-term culture need to be routinely
checked for their chromosomal stability. .(106)
Propagation of hESC
Considerable progress has been made towards the definition of xeno-free culture
conditions allowing the propagation of undifferentiated hESC, thus significantly
improving the clinical potential of these cells. .(106)
Feeder-dependent propagation of hESC

To eliminate the xeno-contamination of mouse feeder cells, human feeder cells have
been derived and shown to support the undifferentiated growth of hESC . The existing
hESC lines can maintain their pluripotency on several types of human feeder cells , or on
the feeder cells derived from hESC themselves . However, most commonly used human
feeders have been exposed to animal components during their isolation and culture , and
the animal-derived SR is still needed to maintain hESC growth under these conditions,
potentially making hESC xeno-contaminated and unsuitable for cell replacement
therapies. In addition, hESC cultured in SR medium present immunogenic sialic acids on
their surface; although the contamination with non-human sialic acids can be diluted (and
probably lost) in hESC after long-term culture under xeno-free conditions, they may still
represent a risk to transplantation . In view of clinical applications, it is mandatory to
establish clinical-grade feeder cell lines for hESC derivation and propagation, while
animal serum and SR should be eliminated from culture media for both feeder cells and
hESC. Most recently, Ellerstrom et al. established xeno-free human foreskin fibroblast
feeders for hESC derivation, in a medium supplemented with human serum and devoid of
any animal material. The hESC lines derived and propagated in such systems are
therefore advantageous for possible therapeutic purposes. .(106)
Feeder-free propagation of hESC

The necessity of feeder cells limits the capacity to meet the large-scale culture demands
for clinical applications, as well as for genetic manipulation of hESC in basic research.
Furthermore, establishment of clinical-grade feeder cell lines for hESC culture is costly.
Xu et al. first demonstrated a successful feeder-free hESC culture system in which
undifferentiated cells can be maintained in the long term on a Matrigel layer in MEF-
conditioned medium. Alternatively, hESC-derived fibroblasts can also be used to produce
conditioned medium capable of supporting undifferentiated growth of hESC on Matrigel .
.(106)

However, Matrigel is a soluble basement membrane extract from mouse tumors

containing several types of ECM and other unknown factors, and MEF-conditioned
medium also contains undefined animal components, which limits its use for clinical-
grade hESC culture. As a step forward, a single ECM component such as laminin or
fibronectin, of both animal and human origin, has also been successfully used to support
undifferentiated growth of hESC in either MEF-conditioned medium or medium
supplemented with SR and various growth factors . In these conditions the possible xeno-
contamination in substrates is reduced, but in culture media it remains and a risk to future
clinical applications may still persist. .(106)
In order to optimize the culture conditions, great efforts have been made in identifying
conditioned media components essential for hESC self-renewal, as a crucial prerequisite
for the eventual applications of hESC in the treatment of human diseases. High
throughput screening methods have been employed to investigate the protein composition
of media conditioned by feeder cells of either animal or human origin, providing
preliminary insight into the possible feeder cell-secreted factors that support the growth of
hESC. Basic fibroblast growth factor (bFGF) appears to play a key role in sustaining
hESC self-renewal, and is included in nearly all the reported medium formulations for
hESC derivation and propagation. Indeed, certain existing hESC lines have been
successfully maintained over a long term in unconditioned medium supplemented with a
high concentration of bFGF .Suppression of bone morphogenetic proteins (BMP) also
seems to be important for hESC propagation. Noggin, a BMP antagonist, synergizes with
bFGF in sustaining the proliferation of undifferentiated hESC in the absence of feeder
cells and conditioned medium . (106)
Although the possible xeno-contamination of MEF-conditioned medium is avoided in
these culture systems, exogenous factors in unconditioned medium (e.g. SR) and in
culture substrates (e.g. Matrigel) still pose a risk to therapeutic applications. To
circumvent this issue, Stojkovic et al. reported the growth of undifferentiated hESC on
human serum-coated plates in SR-containing medium conditioned by human embryonic
fibroblasts derived from hESC, which reduces the exposure of hESC to animal
ingredients. Recently, several chemical-defined culture media, in which the SR is
replaced with a cocktail of recombinant growth factors and cytokines, have been
developed to sustain undifferentiated propagation of existing hESC lines on Matrigel or
on human-derived ECM in short-term studies . (106)
Such animal-free media should significantly facilitate the use of hESC in therapeutic
applications. However, as discussed above, most available hESC cell lines are xeno-
contaminated and their propagation in animal-free medium cannot completely eliminate
the exogenous and immunogenic factors in culture . For clinical purposes, it would
require starting with newly derived hESC that have never been exposed to animal
products. As a step forward, Ludwig et al. successfully derived and propagated two new
hESC lines in defined medium on human-derived substrates, although immunosurgery is
still employed in their work to isolate the ICM. Most recently, Fletcher et al. reported the
first hESC line derived without direct exposure to any undefined animal products. We
expect such approaches to facilitate the clinical applications of hESC and to provide a
platform for further optimizing culture requirements for undifferentiated growth of hESC.
(106)

It should be noted that, compared with the culture on feeder cells, the feeder-free
system is less optimal for sustaining the undifferentiated growth of hESC, as
differentiation on the edges of the colonies can frequently be seen in these conditions.
Furthermore, feeder-free cultures of hESC might result in chromosomal changes, such as
gain of chromosomes 17q and 12 , which should be a concern for the future applications
of these cells in transplantation therapies. (106)
 Stem Cell Markers

Identifying a stem cell is not always easy. Adult stem cells account for only one out of
100,000 cells of the total cell population, so the odds of finding one, at the best of times,
are small indeed. Stem cells have a simple morphology, and one might think this would
set them apart from other cells in the body, but there are many differentiated cells that
have a similar size and shape. Consequently, it is almost impossible to separate stem cells
from differentiated cells by simple visual inspection. The situation with ES cells is a little
better, since their source and identity are known without question. However, all stem cells
change somewhat when placed in culture, making it necessary to monitor their behavior
and to track any changes in their state of differentiation (106) .
To this end, scientists have developed a set of markers that work for both mice and
humans, which simplify the identification of stem cells and the evaluation of their
phenotype. There are many different stemcell markers, but they all fall within one of three
groups: glycoprotein receptors that are embedded in the cell membrane; cell-specific gene
expression; and cell-specific molecules such as hormones, enzymes, or structural proteins
(106)
.

GLYCOPROTEIN RECEPTORS

White blood cells carry cell-surface receptors called cluster of differentiation (CD4)
and (CD8), which are specific for mature T lymphocytes. The protein that binds to these
receptors is called a ligand and can be detected with a procedure called
immunofluorescence. Briefly, this involves placing the cells on a microscope slide and
covering them with a solution containing the ligand. During incubation, the ligand binds
to the receptors (if present) after which the sample is covered with a solution containing a
fluorescent-labeled antibody that will attach specifically to the ligand. After an
appropriate incubation period, the slide is examined under a fluorescent microscope. Cells
carrying CD4 or CD8 receptors will be colored blue or green, whereas all the other cells
will be colorless. A negative reaction—where all cells are colorless—indicates that the
stem cells have not differentiated, or that they have not differentiated into T-
lymphocytes(107) .
Figure 6 showing the immunofluorescence procedure .

Edited from http://schools-wikipedia.org/wp/a/Antibody.htm

 Fluorescent markers are often used in conjunction with a machine called a
fluorescence activated cell sorter (FACS). A suspension of thousands of cells is treated
with the immunofluorescence procedure to tag any stem cells that may be present with a
fluorescent dye, after which the sample is injected into the FACS machine. The cell
suspension passes through a very thin tube, which forces the cells to move past a laser
beam in single file. The laser beam gives each cell an electric charge: Those that carry the
fluorescent marker receive a negative charge; the rest receive a positive charge. An
electromagnetic field directs the negatively charged cells into one tube, while the
positively charged cells are directed to a separate tube. A FACS machine can isolate a
single stem cell from a population of more than 100,000 cells in less than an hour (107) .

CELL-SPECIFIC GENE EXPRESSION

The expression of certain genes in specific cell types is another kind of stem cell
marker. Some neurons are known to express a gene called noggin, which is not expressed
in nonneural tissue. Using a procedure called fluorescent in-situ hybridization (FISH), it
is possible to detect the cells that express the noggin gene. Like the immunofluorescence
procedure just described, the final product of the FISH reaction is a field of cells on a
microscope slide that are either colored or not. Cells expressing noggin will be blue; those
that do not express this gene will be colorless. Stem cells that are differentiating into
neural tissue will be colored. Stem cells that are not differentiating, or are differentiating
into nonneural tissue, will be colorless (107) .

CELL-SPECIFIC MOLECULES

Some cells produce special hormones, macromolecules, and enzymes that may be used
as markers because they are not found in all cells. The β cells in the pancreas are the only
cells in the body that produce insulin, so this hormone makes an excellent marker for the
differentiation of a stem cell into a β cell. All cells make a protein called tubulin, which is
an important structural protein, but neurons make their own special neurotubulin, which
can serve as a marker of neural differentiation. Embryonic stem cells produce a protein
called genesis, which plays an important role in gene transcription. ES cells also have
special glycoproteins in their membranes, such as stem cell antigen-number 1 (Sca-1) and
embryonic antigen-3 (Ea-3). Thus, genesis, Sca-1, and Ea-3 are markers for the
undifferentiated state of a stem cell. The disappearance of these molecules is an early
indication the cell is beginning to differentiate, even before an outward change in
appearance is evident. Detection of cells carrying these markers is possible using
immunofluorescence or FISH (107) .

A special DNA sequence called a telomere may also be used in conjunction with the
standard markers to help identify a potential stem cell. A telomere is not a gene but a
simple repetitive sequence located at the tip of each chromosome that is needed for the
proper duplication of each chromosome during the cell cycle.With each round of cell
division, the telomere shrinks in length, but an enzyme called telomerase later restores it.
Actively dividing cells have high levels of telomerase, whereas postmitotic cells have
none. Consequently, the presence of telomerase is an indication that the cells are actively
dividing, as stem cells usually are (107) .

Another approach to monitoring a stem cell’s differentiation status employs the

technique of gene therapy to introduce a reporter gene, called GFP, into the genome of a
stem cell. The GFP gene codes for a fluorescent protein that emits a green light and can
be engineered so it is active only when the cell is in an undifferentiated state and is turned
off when the cell begins to differentiate. Consequently, differentiation of the cell is
(107)
associated with a loss of the green color.

An alternative use of the GFP gene is to use it to give cells a permanent green color.
Dr. Fred Gage and his colleagues at the Salk Institute recently obtained stunning results
on stem cell differentiation by using the GFP gene. Their intention was to study the
ability of astrocytes (a kind of neuron) to stimulate differentiation of adult stem cells into
neurons. The in vitro experiments were conducted using cells isolated from the rat. After
transfecting stem cells with a GFP reporter gene, Gage’s team added them to a culture
plate containing astrocytes (a reporter gene is always turned on, so the GFP reporter gene
gave the cells a permanent green color). Each time the stem cells divided, they produced
green daughter cells, so their fate was easily monitored. This procedure gave clear
evidence that the astrocytes could induce stem cells to differentiate into neurons, since
green neurons appeared that could only have originated from the green stem cells (107) .

Stem cells are generally characterized through the examination of more than one
marker and are referred to accordingly. This produces a very specific, though somewhat
awkward, naming convention.A neural stem cell (NSC), isolated from the brain, and
evaluated for the expression of CD8, genesis and noggin would be referred to as NSC
(CD8–/low, genesis–, noggin+), meaning the expression of CD8 is absent to low, genesis
is absent, and noggin is present. The use of these markers was established in studies
involving research animals, such as the mouse, but they are now an indispensable part of
the more recent research effort to identify and characterize human stem cells (107) .

Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize

Differentiated Cell Types
Marker Name Cell Type Significance
Blood Vessel
Fetal liver kinase-1 Cell-surface receptor protein that identifies endothelial
Endothelial
(Flk1) cell progenitor; marker of cell-cell contacts
Smooth muscle cell- Smooth muscle Identifies smooth muscle cells in the wall of blood
specific myosin vessels
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
heavy chain
Vascular endothelial Identifies smooth muscle cells in the wall of blood
Smooth muscle
cell cadherin vessels
Bone
Bone-specific
Enzyme expressed in osteoblast; activity indicates bone
alkaline phosphatase Osteoblast
formation
(BAP)
Minerlized bone matrix that provides structural integrity;
Hydroxyapatite Osteoblast
marker of bone formation
Mineral-binding protein uniquely synthesized by
Osteocalcin (OC) Osteoblast
osteoblast; marker of bone formation
Bone Marrow and Blood
Bone Important for the differentiation of committed
morphogenetic Mesenchymal stem and mesenchymal cell types from mesenchymal stem and
protein receptor progenitor cells progenitor cells; BMPR identifies early mesenchymal
(BMPR) lineages (stem and progenitor cells)
White blood cell Cell-surface protein markers specific for mature T
CD4 and CD8
(WBC) lymphocyte (WBC subtype)
Hematopoietic stem Cell-surface protein on bone marrow cell, indicative of a
CD34 cell (HSC), satellite, HSC and endothelial progenitor; CD34 also identifies
endothelial progenitor muscle satellite, a muscle stem cell
CD34+Sca1+ Lin- Mesencyhmal stem cell Identifies MSCs, which can differentiate into adipocyte,
profile (MSC) osteocyte, chondrocyte, and myocyte
Absent on HSC
Cell-surface molecule that identifies WBC lineages.
CD38 Present on WBC Selection of CD34+/CD38- cells allows for purification of
lineages HSC populations

A type of cell-adhesion molecule used to identify specific

CD44 Mesenchymal
types of mesenchymal cells
Cell-surface receptor on BM cell types that identifies
HSC and MSC; binding by fetal calf serum (FCS)
c-Kit HSC, MSC
enhances proliferation of ES cells, HSCs, MSCs, and
hematopoietic progenitor cells
CFU assay detects the ability of a single stem cell or
Colony-forming unit progenitor cell to give rise to one or more cell lineages,
HSC, MSC progenitor
(CFU) such as red blood cell (RBC) and/or white blood cell
(WBC) lineages
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
An individual bone marrow cell that has given rise to a
Fibroblast colony-
Bone marrow colony of multipotent fibroblastic cells; such identified
forming unit (CFU-
fibroblast cells are precursors of differentiated mesenchymal
F)
lineages
Fluorescent dye that binds DNA; HSC extrudes the dye
Hoechst dye Absent on HSC
and stains lightly compared with other cell types
Leukocyte common
WBC Cell-surface protein on WBC progenitor
antigen (CD45)
HSC, MSC Thirteen to 14 different cell-surface proteins that are
Lineage surface Differentiated RBC markers of mature blood cell lineages; detection of Lin-
antigen (Lin) and WBC lineages negative cells assists in the purification of HSC and
hematopoietic progenitor populations
Cell-surface protein specific for mature granulocyte and
Mac-1 WBC
macrophage (WBC subtypes)
Cell-surface protein (immunoglobulin superfamily) found
Bone marrow on bone marrow fibroblasts, which may be important in
Muc-18 (CD146)
fibroblasts, endothelial hematopoiesis; a subpopulation of Muc-18+ cells are
mesenchymal precursors
Cell-surface protein on bone marrow (BM) cell,
Stem cell antigen
HSC, MSC indicative of HSC and MSC Bone Marrow and Blood
(Sca-1)
cont.
Cell-surface glycoprotein on subsets of bone marrow
Stromal stromal (mesenchymal) cells; selection of Stro-1+ cells
(mesenchymal) assists in isolating mesenchymal precursor cells, which
Stro-1 antigen
precursor cells, are multipotent cells that give rise to adipocytes,
hematopoietic cells osteocytes, smooth myocytes, fibroblasts, chondrocytes,
and blood cells
Cell-surface protein; negative or low detection is
Thy-1 HSC, MSC
suggestive of HSC
Cartilage
Collagen types II
Chondrocyte Structural proteins produced specifically by chondrocyte
and IV
Principal protein of skin; identifies differentiated
Keratin Keratinocyte
keratinocyte
Sulfated Molecule found in connective tissues; synthesized by
Chondrocyte
proteoglycan chondrocyte
Fat
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
Adipocyte lipid-
binding protein Adipocyte Lipid-binding protein located specifically in adipocyte
(ALBP)
Fatty acid
Adipocyte Transport molecule located specifically in adipocyte
transporter (FAT)
Adipocyte lipid-
binding protein Adipocyte Lipid-binding protein located specifically in adipocyte
(ALBP)
General
Male-specific chromosome used in labeling and detecting
Y chromosome Male cells
donor cells in female transplant recipients
Karyotype Most cell types Analysis of chromosome structure and number in a cell
Liver
Principal protein produced by the liver; indicates
Albumin Hepatocyte functioning of maturing and fully differentiated
hepatocytes
Cell-adhesion molecule important in cell-cell
B-1 integrin Hepatocyte interactions; marker expressed during development of
liver
Nervous System
Cell-surface protein that identifies neural stem cells,
CD133 Neural stem cell, HSC
which give rise to neurons and glial cells
Glial fibrillary
acidic protein Astrocyte Protein specifically produced by astrocyte
(GFAP)
Microtubule-
Dendrite-specific MAP; protein found specifically in
associated protein-2 Neuron
dendritic branching of neuron
(MAP-2)
Myelin basic protein Protein produced by mature oligodendrocytes; located in
Oligodendrocyte
(MPB) the myelin sheath surrounding neuronal structures
Intermediate filament structural protein expressed in
Nestin Neural progenitor
primitive neural tissue
Important structural protein for neuron; identifies
Neural tubulin Neuron
differentiated neuron
Important structural protein for neuron; identifies
Neurofilament (NF) Neuron
differentiated neuron
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
Cluster of primitive neural cells in culture of
Embryoid body (EB),
Neurosphere differentiating ES cells; indicates presence of early
ES
neurons and glia
A neuron-specific gene expressed during the
Noggin Neuron
development of neurons
Cell-surface marker on immature, developing
O4 Oligodendrocyte
oligodendrocyte
Cell-surface marker that characterizes mature
O1 Oligodendrocyte
oligodendrocyte
Neuronal protein located in synapses; indicates
Synaptophysin Neuron
connections between neurons
Tau Neuron Type of MAP; helps maintain structure of the axon
Pancreas
Cytokeratin 19 CK19 identifies specific pancreatic epithelial cells that
Pancreatic epithelium
(CK19) are progenitors for islet cells and ductal cells
Glucagon Pancreatic islet Expressed by alpha-islet cell of pancreas
Insulin Pancreatic islet Expressed by beta-islet cell of pancreas Pancreas
Insulin-promoting Transcription factor expressed by beta-islet cell of
Pancreatic islet
factor-1 (PDX-1) pancreas
Structural filament protein indicative of progenitor cell
Nestin Pancreatic progenitor
lines including pancreatic
Pancreatic
Pancreatic islet Expressed by gamma-islet cell of pancreas
polypeptide
Somatostatin Pancreatic islet Expressed by delta-islet cell of pancreas
Pluripotent Stem Cells
Embryonic stem (ES),
Alkaline Elevated expression of this enzyme is associated with
embryonal carcinoma
phosphatase undifferentiated pluripotent stem cell (PSC)
(EC)
Protein expressed during development of primitive
Alpha-fetoprotein
Endoderm endoderm; reflects endodermal differentiation Pluripotent
(AFP)
Stem Cells
Bone
Growth and differentiation factor expressed during early
morphogenetic Mesoderm
mesoderm formation and differentiation
protein-4
Brachyury Mesoderm Transcription factor important in the earliest phases of
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
mesoderm formation and differentiation; used as the
earliest indicator of mesoderm formation
Cluster designation
ES, EC Surface receptor molecule found specifically on PSC
30 (CD30)
Gene for growth factor expressed by ES cells, primitive
Cripto (TDGF-1) ES, cardiomyocyte
ectoderm, and developing cardiomyocyte
GATA-4 gene Endoderm Expression increases as ES differentiates into endoderm
Antibody to a specific extracellular-matrix molecule that
GCTM-2 ES, EC
is synthesized by undifferentiated PSCs
Transcription factor uniquely expressed by ES cells
Genesis ES, EC
either in or during the undifferentiated state of PSCs
Germ cell nuclear
ES, EC Transcription factor expressed by PSCs
factor
Hepatocyte nuclear Transcription factor expressed early in endoderm
Endoderm
factor-4 (HNF-4) formation
Ectoderm, neural and Intermediate filaments within cells; characteristic of
Nestin
pancreatic progenitor primitive neuroectoderm formation
Neuronal cell-
Cell-surface molecule that promotes cell-cell interaction;
adhesion molecule Ectoderm
indicates primitive neuroectoderm formation
(N-CAM)
Transcription factor unique to PSCs; essential for
OCT4/POU5F1 ES, EC
establishment and maintenance of undifferentiated PSCs
Transcription factor expressed as ES cell differentiates
Pax6 Ectoderm
into neuroepithelium
Stage-specific
Glycoprotein specifically expressed in early embryonic
embryonic antigen-3 ES, EC
development and by undifferentiated PSCs
(SSEA-3)
Stage-specific
Glycoprotein specifically expressed in early embryonic
embryonic antigen-4 ES, EC
development and by undifferentiated PSCs
(SSEA-4)
Stem cell factor Membrane protein that enhances proliferation of ES and
(SCF or c-Kit ES, EC, HSC, MSC EC cells, hematopoietic stem cell (HSCs), and
ligand) mesenchymal stem cells (MSCs); binds the receptor c-Kit
An enzyme uniquely associated with immortal cell lines;
Telomerase ES, EC
useful for identifying undifferentiated PSCs
Antibody to a specific extracellular matrix molecule is
TRA-1-60 ES, EC
synthesized by undifferentiated PSCs
 Table 1 : Markers Commonly Used to Identify Stem Cells and to Characterize
Differentiated Cell Types
Marker Name Cell Type Significance
Antibody to a specific extracellular matrix molecule
TRA-1-81 ES, EC
normally synthesized by undifferentiated PSCs
Ectoderm, neural and Intermediate filaments within cells; characteristic of
Vimentin
pancreatic progenitor primitive neuroectoderm formation
Skeletal Muscle/Cardiac/Smooth Muscle
Transcription factors that direct differentiation of
MyoD and Pax7 Myoblast, myocyte
myoblasts into mature myocytes
Secondary transcription factors required for
Myogenin and MR4 Skeletal myocyte
differentiation of myoblasts from muscle stem cells
A component of structural and contractile protein found
Myosin heavy chain Cardiomyocyte
in cardiomyocyte
A component of structural and contractile protein found
Myosin light chain Skeletal myocyte
in skeletal myocyte

Table 1 Edited from http://stemcells.nih.gov/info/scireport/appendixe.as

Stem cells applications

Uses in Research

Much is left to be discovered and understood in all aspects of human biology. What
has been frequently lacking are the tools necessary to make the initial discoveries, or to
apply the knowledge of discoveries to the understanding of complex systems. These are
some of the larger problems in basic and clinical biology where the use of stem cells
might be the key to understanding (108).

Models of human disease that are constrained by current animal and cell culture models.

Investigation of a number of human diseases is severely constrained by a lack of in

vitro models. A number of pathogenic viruses including human immunodeficiency virus
and hepatitis C virus grow only in human or chimpanzee cells. ES cells might provide cell
and tissue types that will greatly accelerate investigation into these and other viral
diseases. Current animal models of neurodegenerative diseases such as Alzheimer’s
disease give only a very partial representation of the disease’s process (108).

Transplantation.

Pluripotent stem cells could be used to create an unlimited supply of cells, tissues, or
even organs that could be used to restore function without the requirement for toxic
immunosuppression and without regard to tissue matching compatibility. Such cells,
when used in transplantation therapies, would in effect be suitable for “universal”
donation. Bone marrow transplantation, a difficult and expensive procedure associated
with significant hazards, could become safe, cost effective, and be available for treating a
wide range of clinical disorders, including aplastic anemia and certain inherited blood
disorders. This would be especially important in persons who lost marrow function from
toxic exposure, for example to radiation or toxic agents. Growth and transplant of other
tissues lost to disease or accident, for example, skin, heart, nervous system components,
and other major organs, are foreseeable (109) .

Gene Therapy.

In gene therapy, genetic material that provides a missing or necessary protein, or

causes a clinically-relevant biochemical process, is introduced into an organ for a
therapeutic effect. For gene-based therapies (specifically, those using DNA sequences), it
is critical that the desired gene be introduced into organ stem cells in order to achieve
long-term expression and therapeutic effect. Although techniques for delivering the
therapeutic DNA have been greatly improved since the first gene therapy protocol almost
10 years ago, there are as yet no bona fide successes. Besides delivery problems, loss of
expression or insufficient expression is an important limiting factor in successful
application of gene therapy and could be overcome by transferring genes into stem cells
(which presumably will then differentiate and target correctly) ,(110).

Somatic Nuclear Transfer ("Therapeutic Cloning")

When cells are transferred from one person into another person’s body, the foreign
cells maybe recognized by the immune system and rejected. An approach to solving this
problem is to remove the fertilized egg’s DNA, replace it with the complete nucleus of a
cell from the patient, and grow it to the blastocyst stage (five to six days) to produce ES
cells. Those stem cells would be near-identical in their DNA makeup to the original
patient.

Figure 7 showing Somatic Cell Nuclear Transfer (SCNT) .

 Edited from http://stemcells.nih.gov/info/2006report/2006Chapter8.htm

Reprogramming through Somatic Cell Nuclear Transfer (SCNT) : In SCNT (see

Figure7) human oocytes (eggs) are collected from a volunteer donor who has taken drugs
that stimulate the production of more than one oocyte during the menstrual cycle.
Scientists then remove the nucleus from the donated oocyte and replace it with the
nucleus from a somatic cell, a differentiated adult cell from elsewhere in the body. The
oocyte with the newly transferred nucleus is then stimulated to develop. The oocyte may
develop only if the transplanted nucleus is returned to the pluripotent state by factors
present in the oocyte cytoplasm. This alteration in the state of the mature nucleus is called
nuclear reprogramming. When development progresses to the blastocyst stage, the ICM is
removed and placed into culture in an attempt to establish a pluripotent stem cell line. To
date, the technique has been successfully demonstrated in two primates: macaque
monkeys(119) and humans (111) .

However, successful SCNT creates an embryo-like entity, thereby raising the ethical
issues that confront the use of spare IVF embryos. However, pluripotent cell lines created
by embryos generated by SCNT offer several advantages over ES cells. First, the nuclear
genes of such a pluripotent cell line will be identical to the genes in the donor nucleus. If
the nucleus comes from a cell that carries a mutation underlying a human genetic disease
such as Huntington’s disease, then all cells derived from the pluripotent cell line will
carry this mutation. In this case, the SCNT procedure would enable the development of
cellular models of human genetic disease that can inform our understanding of the
biology of disease and facilitate development of drugs to slow or halt disease progression.
(111)

Alternatively, if the cell providing the donor nucleus comes from a specific patient, all
cells derived from the resulting pluripotent cell line will be genetically matched to the
patient with respect to the nuclear genome. If these cells were used in transplantation
therapy, the likelihood that the patient’s immune system would recognize the transplanted
cells as foreign and initiate tissue rejection would be reduced. However, because
mitochondria also contain DNA, the donor oocyte will be the source of the mitochondrial
genome, which is likely to carry mitochondrial gene differences from the patient which
may still lead to tissue rejection (111) .

Some Examples of Treatments for Major Diseases

Table 2:CURRENT HUMAN CLINICAL APPLICATIONS USING ADULT

STEM CELLS
ANEMIAS & OTHER BLOOD AUTO-IMMUNE DISEASES
CONDITIONS
Sickle cell anemia Systemic lupus (auto-immune condition that
can affect skin, heart, lungs, kidneys, joints,
 and nervous system)
Sideroblastic anemia Sjogren’s syndrome (autoimmune disease
w/ symptoms similar to arthritis)
Aplastic anemia Myasthenia (An autoimmune
neuromuscular disorder)
Red cell aplasia (failure of red blood cell Autoimmune cytopenia
development)
Amegakaryocytic thrombocytopenia Scleromyxedema (skin condition)
Thalassemia (genetic [inherited] disorders Scleroderma (skin disorder)
all of which involve underproduction of
hemoglobin)
Primary amyloidosis (A disorder of plasma Crohn’s disease (chronic inflammatory
cells) disease of the intestines)
Diamond blackfan anemia Behcet’s disease
Fanconi’s anemia Rheumatoid arthritis
Chronic Epstein-Barr infection (similar to Juvenile arthritis
Mono)
BLADDER DISEASE Multiple sclerosis
End-stage bladder disease Polychondritis (chronic disorder of the
cartilage)
OCULAR Systemic vasculitis (inflammation of the
blood vessels)
Corneal regeneration Alopecia universalis
CANCERS Buerger’s disease (limb vessel constriction,
inflammation)
Brain tumors—medulloblastoma and Juvenile (Type-I) Diabetes
glioma
Retinoblastoma (cancer) CARDIOVASCULAR
Ovarian cancer Acute Heart damage
Skin cancer: Merkel cell carcinoma Chronic coronary artery disease
Testicular cancer IMMUNODEFICIENCIES
Lymphoma Severe combined immunodeficiency
syndrome
Non-Hodgkin’s lymphoma X-linked lymphoproliferative syndrome
Hodgkin’s lymphoma X-linked hyper immunoglobulin M
syndrome
Acute lymphoblastic leukemia LIVER DISEASE
Acute myelogenous leukemia Chronic liver failure
Chronic myelogenous leukemia Liver cirrhosis
Chronic myelomonocytic leukemia NEURAL DEGENERATIVE DISEASES
& INJURIES
Juvenile myelomonocytic leukemia Parkinson’s disease
Cancer of the lymph nodes: Spinal cord injury
Angioimmunoblastic lymphadenopathy
Multiple myeloma (cancer affecting white Stroke damage
blood cells of the immune system)
Myelodysplasia (bone marrow disorder) WOUNDS & INJURIES
Breast cancer Limb gangrene
Neuroblastoma (childhood cancer of the Surface wound healing
nervous system)
Renal cell carcinoma (cancer of the kidney) Jawbone replacement
Soft tissue sarcoma (malignant tumor that Skull bone repair
begins in the muscle, fat, fibrous tissue,
blood vessels)
Ewing’s sarcoma OTHER METABOLIC DISORDERS
Various solid tumors Hurler’s syndrome (hereditary genetic
disorder)
Waldenstrom’s macroglobulinemia (type of Osteogenesis imperfecta (bone/cartilage
lymphoma) disorder)
Hemophagocytic lymphohistiocytosis Krabbe Leukodystrophy (hereditary genetic
disorder)
POEMS syndrome (osteosclerotic Osteopetrosis (genetic bone disorder)
myeloma)
Myelofibrosis Cerebral X-linked adrenoleukodystroph

* There are no current clinical trials in humans with embryonic stem cells:

“It is nearly certain that the [human] clinical benefits of the [embryonic stem cell]
research are years or decades away. This is a message that desperate families and
patients will not want to hear.”
Table 2 Edited from http://stemcellsafaen.blogspot.com/2011/03/stem-cell-enhancer-support-
natural.html

Stem Cell and Type 1 Diabetes

Basically, insulin is synthesized by pancreas for converting the glucose to glycogen for
storage. However, for type I diabetic patients, the pancreatic cells that contribute to
insulin production are destroyed by the immune system. It is possible to use the stem cell
research for inducing the differentiation of human ES cells in cell culture to form
insulinproducing cells. This cell is transferred to the patient by transplantation for
diabetic’s therapy (112) .
 Firstly, mouse ES cells were derived from the blastocyst and cultured under specific
conditions. The ES cells were expanded and differentiated. The cells were selected for
further differentiation and characterization. The growing cells in culture can form three-
dimensional clusters which have identical structure to the normal pancreatic islets. The
cells finally can secrete insulin and the amount of insulin production will increase with
the increasing concentration of glucose level in theculture media (112) .

When these pancreatic islet-like cells are implanted into diabetic mice, the
vascularization of the cells observed. The synthesis of the insulin can be detected and the
cell shows the pancreatic physical characteristics like the normal cells. The pancreatic-
like cells that can secrete insulin can be easily produced from mouse ES cells by using
this research method (112) .

Figur8 Treatment of Type1 diabetes by using stem cells .

by Kirschstein.R, Skirboll.L.R. Stem cells and diabetes. Journal of Stem cells:

scientific progress and future research directions. National Institutes of Health. p.67.
 Nervous System Diseases.

Many nervous system diseases result from loss of nerve cells. Mature nerve cells
cannot divide to replace those that are lost. Thus, without a “new” source of functioning
nerve tissue, no therapeutic possibilities exist. In Parkinson’s disease, nerve cells that
make the chemical dopamine die. In Alzheimer’s disease, cells that are responsible for the
production of certain neurotransmitters die. In amyotrophic lateral sclerosis, the motor
nerve cells that activate muscles die. In spinal cord injury, brain trauma, and even stroke,
many different types of cells are lost or die. In multiple sclerosis, glia, the cells that
protect nerve fibers are lost. Perhaps the only hope for treating such individuals comes
from the potential to create new nerve tissue restoring function from pluripotent stem
cells (113).
Remarkably, human clinical experiments have demonstrated the potential
effectiveness of this approach to treatment. Parkinson’s patients have been treated by
surgical implantation of fetal cells into their brain with some benefit. Although not
completely effective, perhaps owing to lack of sufficient numbers of dopamine secreting
cells, similar experiments using appropriately differentiated stem cells should overcome
those obstacles. More complex experiments have already been successfully conducted in
rodent models of Parkinson’s. Similar approaches could be developed to replace the dead
or dysfunctional cells in cortical and hippocampal brain regions that are affected in
patients with Alzheimer’s (113) .

Primary Immunodeficiency Diseases.

Pluripotent stem cells could be used in treatment of virtually all primary

immunodeficiency diseases. Presently, there are more than 70 different forms of
congenital and inherited deficiencies of the immune system that have been recognized.
These are among the most complicated diseases to treat with the worst prognoses.
Included here are diseases such as severe combined immunodeficiency disease (the
“bubble boy” disease), Wiskott-Aldrich Syndrome, and the autoimmune disease lupus.
The immune deficiencies suffered as a result of acquired immune deficiency syndrome
(AIDS) following infection with the human immunodeficiency virus are also relevant
here. These diseases are characterized by an unusual susceptibility to infection and often
associated with anemia, arthritis, diarrhea, and selected malignancies. However, the
transplantation of stem cells reconstituted with the normal gene could result in restoration
of immune function and effective normalization of life span and quality of life for these
people (114)

Diseases of Bone and Cartilage.

Stem cells, once appropriately differentiated, could correct many diseases and
degenerative conditions in which bone or cartilage cells are deficient in numbers or
defective in function. This holds promise for treatment of genetic disorders such as
osteogenesis imperfecta and chondrodysplasias. Similarly, cells could be cultivated and
introduced into damaged areas of joint cartilage in cases of osteoarthritis or into large
gaps in bone from fractures or surgery (115) .

Cancer.

At the present time, bone marrow stem cells, representing a more committed stem
cell, are used to rescue patients following high dose chemotherapy. Unfortunately, these
recovered cells are limited in their capacity to restore immune function completely in this
setting. It is hoped that injections of properly-differentiated stem cells would return the
complete repertoire of immune response to patients undergoing bone marrow
transplantation. Complete and functional restoration will be required if, for example,
immune/vaccine anticancer therapy is to work. More importantly, success would permit
use of very toxic (and effective) chemotherapeutic regimens that could not currently be
utilized for lack of an ability to restore marrow and immune function (116) .

stem cells & cancer

Two defining features of stem cells are their ability to self-renew and their ability to
give rise to differentiated cell types of one or more cell lineages. Upon cell division, one
daughter cell maintains the characteristics of a stem cell including the ability to self-
renew and the other daughter cell shows characteristics of commitment towards
differentiation (Figure 9). The feature of self-renewal is shared with tumor cells.This
common feature has led to two proposals for the relevance of stem cell biology to
carcinogenesis. The first proposal is that self-renewal provides increased opportunities
for carcinogenic changes to occur. The second proposal suggests that altered regulation
of self-renewal directly underlies carcinogenesis. We will explore each of these below
where we will find that the concepts from both proposals will intertwine. Note that
research into this topic is relatively in its infancy and these proposals require further
investigation and substantiation.(117)

Figure 9 Features Of Stem Cells .

 Edited from l.pecorino, molecular biology of cancer ,2005

Self-renewal provides an extended window of time for mutation

Stem cells are long-lived targets for chance mutations compared with many
differentiated cells that die within days or months. The accumulation of mutations
necessary for carcinogenesis is more likely to occur in stem cells that self-renew over the
life-time of an individual rather than in mature cells that exit the cell cycle and/or
undergo apoprosis after a brief period. This concept supports the proposal the tumors are
likely to arise from stem cells. Because restricted progenitors cells self-renew for shorter
periods of time they are less likely than stem cells to become oncogenic. (117)
Let us look at the process of skin carcinogenesis. Skin is a tissue with a clearly
defined hierarchical organization of differentiation. The differentiation pathway begins in
the basal layers and leads to the formation of the dead, cornified outer layer of the skin.
Epithelial cells of the skin have a turnover rare of 60 days in humans. However,
malignant transformation involving the accumulation of specific mutations takes 18
months or more, so the 60-day life span of a differentiating celt is not long enough to
allow a sufficient number of mutations to accumulate. Self-renewal is a quality of stem
cells that allows for the accumulation of transforming mutations since an individual
mutation may be passed on to daughter cells which are themselves susceptible to
additional mutations and can pass on accumulated mutations to future progeny cells. This
rationale suggests that the accumulation of mutations required for the initiation of cancer
is likely to occur in the normal stem cell compartment. (117)
Deregulation of self-renewal
Stem cells must maintain a balance between self-renewal and differentiation. One
proposal for the relevance of stem cell biology to carcinogenesis is that the loss of this
balance by stem cells can lead to unregulated self-renewal, a hallmark of cancer.
Therefore, tumor cells may arise from stem cells. Alternatively, differentiated cells may
acquire a mutation that reactivates a self-renewal program. Perhaps in a manner similar
to that in which viruses 'hijack' the cell's machinery for replication, tumor cells "hijack
"the cell’s machinery of self-renewal for oncogenesis. Both of these proposals, that
cancer can initiate either in a stem cell that has lost regulation of self-renewal or in a
differentiated cell that has obtained the ability to self-renew, are supported by the
identification of cancer stem cells. (117) These are rare cells within a tumor that have the
ability to self-renew and to give rise to phenotypically diverse cancer cells. Some
evidence suggests that they drive tumongcnesis.
It has been shown in several types of cancer that tumors are maintained in a growing
cancerous state by only a small fraction of particular tumor cells. These cells have
surface proteins called markers, which are characteristic of the stem cell normally
present in the tissue. For example, it has been established that only about one in a million
acute myeloid leukemia cells can develop into new leukemias when transferred in vivo
and that these cells expressed the same markers (CD34+; CD38—) as normal
hematopoietic stem cells. Also, brain cancer stem cells display normal neural stem cells
markers. Interestingly, the proportion of brain cancer Stem cells identified in a variety of
brain cancers correlates with the course of the disease, or prognosis. Fast-growing tumors
such as glioblastomas had more brain cancer stem cells than slow-growing tumors like
astrocytomas. Evidence for the existence of breast cancer cells was obtained by testing
whether human breast cancer cells could give rise to new tumors when grown in
immunocompromised mice. A minority subpopulation of breast cancer stem cells was
isolated based on the expression of cell surface markers (CD44+ CD24-/lOW) and these
cells showed a 10- to 50-fold increase in ability to form tumors in animals, compared
with the bulk of breast tumor cells.
Furthermore, these cells were not only able to demonstrate the ability to self-renew but
were also able to give rise to cells with different characteristics or phenotypes that made
up the bulk of the tumor. These observations support the concept these cells are breast
cancer stem cells. (117)
Mammary stem cells react to physiological cues, such as hormones, to provide a
source of proliferation and differentiation during pregnancy (or the creation of a milk-
generating breast. A major factor that protects women from breast cancer is an early,
first, full-term pregnancy .It is suggested that the depletion of stem cells as a result of the
burst of differentiation that occurs during pregnancy is the reason why pregnancy is
protective against breast cancer- There may be fewer breast stem cells that have the
potential of becoming breast cancer stem cells over time in women who have had
children in early adulthood. (117)
In summary, a small minority of cancer stem cells may drive tumorigenesis in some
cancers, similar to the small number of adult stem cells that drive the growth of normal
tissues.
Molecular mechanisms of self-renewal
Let us examine the molecular mechanisms of self-renewal. The molecular mechanisms
that regulate self-renewal of stem cells are just beginning to . be understood. There is
some evidence that the Wnt signaling pathway, which is important for regulating pattern
formation during development, may be involved in the self-renewal process of stem cells
during development, in the adult, and also in cancer. When the Wnt regulated
transcription factor, Tef (see below) is deleted by a gene knockout in mice, animals born
lack stem cells of the intestines. In addition, hematopoieric stem cells respond to Wnr
signaling in viuo and require Wnr signaling for self-renewal . Data from DNA arrays
show that the gene expression pattern in response to Wnt signaling is similar between
colon stem cells and colon cancer cells, but differs in differentiated colon cells,
suggesting that Wnt signaling plays a role in stem (cancer) cell self-renewal. The
Hedgehog signaling pathway too, which is also important for regulating pattern
formation in the embryo, has been implicated in the process of stem cell self-renewal as
well. Both the Wnt and Hedgehog signaling pathways will be discussed below. (117)
The Wnt signaling pathway
Wnt proteins (>19 members) are secreted intercellular signaling molecules that act as a
ligand to trigger a specific signal transduction pathway (Figure 10).

Figure 10The Wnt signaling pathway .

Edited from l.pecorino, molecular biology of cancer ,2005

It is easiest to examine the cell in the absence of Writ ligand first (Figure 10a). In this
state, several proteins associate together in the cytoplasm to form a degradation complex.
The degradation complex consists ofglycogen synthase kinase (GSK3β), axin, and
adenomatous polyposis coli (APC) protein. Axin and APC form a structural scaffold for
GSK3β, which is a serine/threonine kinase. This complex modifies an important
transcriptional coactivator called p-catenin via phosphorylation and ubiquitinarion. These
modifications act as molecular flags that target β-catenin for degradation by the
proteosome. Since β-catenin is not available in unsrimulated cells, target genes under the
regulation of β-catenin are repressed. (117)
Upon binding of Wnt ligand to its seven-pass transmembrane receptor, Frizzled, and co-
receptor LRP ( low-density liprotein receptor related protein), axin is recruited to
coeceptor LRP and this disrupts the assembly of the degradation complex (Figure 10 b).
Li addition, an inhibitor of GSK3p, dishevelled protein, is activated via phosphorylation
(not shown). These events allow B-catenin to escape degradation and move into the
nucleus where it can act as a co-activator of the Tcf/LEF (T-cell factor/lymphoid
enhancing factor) family of transcription factors to regulate specific target genes (e.g. c-
myc, cyclin D, and adhesion molecules from the EPH-family). (117)
Wnt signaling and cancer
Wnt1 was one of the first proto-oncogenes discovered. Viral integrationinduced
oncogene activation and subsequent cancer of the mammary gland. Mutations that
constitutively activate he Wnt signaling pathway have been identified in several
particular cancers. For example, such mutations are responsible for 90% of colorectal
cancer. This translates in human terms to 50,000 lives in the USA alone per year. Most
of the mutations inactivate the function of APC or activate ti-cacenin, but rarely alter the
ligand Wnt- Colorectal cancer can be classified incu two forms; familial forms and
sporadic forms. Patients with the inherited cancer predisposition syndrome, familial
adenomatous polyposis coli (FAP), carry a germline mutacion in the APC gene and
develop high numbers of polyps in the colon (polyposis) in early adulthood. As a result
of having many polyps, these patients have an increased risk of colorectal cancer. (117)
The APC gene acts as a true tumor suppressor gene in that both copies of the A PC
gene are inactivated in colorecral tumors. Most mutations occur in the coding sequences
for the central region of the APC protein (codons 1250-1500) referred to as the mutation
cluster region, in both germline and somatic cases (Figure 7.11). (117)
The intestines is a highly regenerative tissue whereby stem cells and epithelial
progenitors that reside in the crypts give rise to more differentiated cells that migrate
along the villi (Figure 12a). Upon reaching the top of the villi, fully differentiated cells
undergo apoptosis. Normally, Wilt signaling is required for maintaining the Stein cell
characteristics of the crypt cells. It has been proposed that acquiring constitutive
activation of Wnt leads to colon cancer stem cells. (117)
Alternatively, one study reported that APC mutations associated with colorectal
cancers were only present in dysplastic cells found at the top of the crypts in
precancerous lesions (adenomas), and not in the stem cells at the base of the crypt.
Microscopic examination of precancerous lesions revealed an abrupt transition between
the dysplastic compartment at the top and the normal epithelium at the bottom of the
crypts. The researchers propose that the morphogenesis of a colorectal tumor occurs in a
top-down direction as illustrated in Figure 12b. (117)
Mutations in the Wnt signaling cascade also promote other types of cancers. Activating
mutations of β-catenin that affect the regulatory sequences essential for its targeted
degradation can lead to skin tumors. Mutations in the axin gene are found in
hepatocellular carcinoma. Many of the axin gene mutations lead to protein truncations
that delete the axin- β-catenin binding sites. Therefore, these observations suggest that
some transforming mutations may function to reactivate the self-renewal pathway. The
cells carrying these mutations can be thought of as de novo stem cells, that is cells that
have acquired stem cell characteristics as a result from mutation, and were not produced
from self-renewal of other stem cells. (117)

Figure 11 The mutation cluster region of APC .

Edited from l.pecorino, molecular biology of cancer ,2005

Figure 12 morphogenesis of a colorectal tumor (a) Stem cells reside in thr crypts of
intestinal villi ( b) colorectal tumors may initiate the top of the crypts .
Edited from l.pecorino, molecular biology of cancer ,2005

The Hedgehog signaling pathway

The Hedgehog (Hh) signaling pathway also plays important roles in embryonic
development, tissue self-renewal, and carcinogcnesis. It is essential for pattern formation
in many tissues including the neural tube , skin, and-gut. Similar to Wnt proteins, Hh
proteins (three members: Sonic, Desert, and Indian) are secreted intercellular signaling
molecules that act as a ljgand to trigger a .specific signal transduction pathway (Figure
13).
Two transmembrance proteins, Patched and Smoothened, are responsible for signal
transduction by Hh. (117)
In the absence of Hh (Figure 13a), Patch inhibits Smoothened and thus suppresses the
pathway. upon binding of Hh to Patched (Figure 13b), inhibition of Smootheried is
relieved. The signal is transduced into the cell and causes a large microtuhulc_complex
to dissociate and release the Gli zinc finger transcnptionai acrivators so that they can he
translocated to the nucleus to regulate the expression of target genes. (117)

Figure13 The Hedgehog signaling pathway .

Edited from l.pecorino, molecular biology of cancer ,2005

Hedgehog signaling and cancer

Patched is defined as a rumor suppressor gene; patients with Gorlin's syndrome carry a
germline mutation in one copy of Parched and have a predisposition ro develop skin,
cerebellar, and muscle tumors (basal cell carcinoma (BCC), meduHoblasromas, and
rhabdornyosarcomas respectively). Inactivating mutations in Patched and activating
mutations of Smoorhened were identified in sporadic (opposed ro familial) human 'BCC
tumors and G1M expression was found in nearly all BCCs. In fact, all sporadic BCCs
possess an activated Hh signaling pathway. (117)
It has been suggested that since BCC are tumors that include hairfollicle
differentiation, they may originate from a source of stem cells that reside in a structure of
the hair follicle, called the hair follicle bulge, which has acquired an aberrant Hh
signaling pathway. These cancer stem cells could self-renew and give rise to the
differentiated hair follicle cells observed in BCC tumors. Similarly, it has been proposed
that medulfoblastoma, the most common childhood malignant brain tumor, arises from
interneuron precursors that possess an inappropriately activated Hh pathway. Activation
of this pathway by mutation is observed in 30% of sporadic medulloblastomas.
Molecular evidence of an activated Hh pathway was also reported for gliomas. Gli was
originally identified as an amplified gene in cultured glioma cells. Unlike Wnt, where
mutations involved in carcinogenesis rarely affect this ligand, Hh is overexpressed in
upper gastrointestinal tumors. Taken together, it is apparent that the Hh signal pathway is
relevant for a several types of human cancer. (117)
Additional properties of stem cells and tumor cells
Cancer stem cells, in addition ro being able to self-renew, also have the ability to give
rise to more differentiated cell types with limited proliferative capacity. Teratocarcinoma
is an obvious example whereby the tumor contains undiffereitiated stem cells and
n^nprol iterative differentiated cells such as bone and cartilage. Although rhe degree of
differentiation for other cancers is less obvious, cancer stem cells from other cancers
have been shown to generate more cells with different phenotypes. (117)
The cell heterogeneity present in a tumor may be derived from cancer stem cells that
not only can self-renew bur can simultaneously give rise to more differentiated cells. A
striking demonstration has suggested that tumor cells can be reprogrammed to become
normal and rotipotent during experimental manipulation (like fully differentiated cells
during cloning experiments). When placed in early mouse embryos, teratocarcinoma
cells can mimic stem cells and can contribute to normal development. (117)
In addition, the degree of differentiation of a cell may also affect the outcome of
oncogene activation. The use of selective gene promoters to drive the expression of a Ras
oncogene in different cell populations with different degrees of differentiation (e.g. stem
cells or committed progeny cells) resulted in rumors with different malignant potential
(i.e. malignant carcinomas vs. benign papillomas). Although additional studies are
needed, this suggests that the activation of an oncogene may be carcinogenic in some
states of differentiation and not in others within a particular cell lineage. (117)
Another feature of stem cells that is shared with tumor cells is the ability to migrate to
other tissues of the body. In contrast, most differentiated cells remain localized to a
specific tissue. Transplantation of stem cells has demonstrated the migratory nature of
stem cells. For example, bone marrow cells migrate to several nonhematopoietic tissues
such as the brain, liver and lung- Metastasis of rumor cells from a primary site to
secondary sites is a characteristic that makes cancer lethal. It has been proposed that the
origin of the transformed cell determines the po'.ential for metastasis: tumors arising
from a stem cell are more likely to metastasize compared with tumors arising from more
differentiated cells which are less likely to spread. The inherent ability of a srem cell to
migrate may cause these cells to be aggressively metasratic, if transformed. The myeloid
leukemias support this view: transformed stem cells are likely to be malignant while
transformed committed progenitor cells are likely to be benign. (117)
Moreover, telomerase activity, which is necessary for tumor proliferation and
progression and is present in 90% of human cancers, is present in normal stem cells and
proliferative cells.This supports the hypothesis that cancer cells are derived from normal
stem cells .
Role of lineage-specific transcription factors in blocked differentiation
Acute myeloid leukemia serves as an important paradigm for examining how
disruption of transcription-factor function can interfere with differentiation, and lead to
cancer. Acute myeloid leukemia (AML) is a disease characterized by a block in the
differentiation of the granulocyre or monocyte lineage (Figure 14). (117)
There are several subtypes of acute myeloid leukemia. The classification system of
this disease is still evolving but should eventually reflect moleclar features at the point of
the differentiation block. The lineage is organized as a hierarchy that begins with
pluripotent hematopoietic stem cells (HSCs). These cells self-renew and form progenitor
cells. The progenitor cells differentiate into several types of precursor cells including
myeloid precursor cells. (117)

Figure14 Haematopoietic lineages : disruption in the granulocyte or monocyte lineage

leads to AML .
Edited from l.pecorino, molecular biology of cancer ,2005

Myeloid precursor cells are common to both the monocytes and granulocyte lineages.
Several transcription factors have been identified to be important in hematopoietic
lineage development. One factor, AML1, is involved in almost all lineages Others are
lineage-specific factors (differentiation factors), such as PU.l and CCAAT/enhancer
binding protein u (C/EBPα). Lineage specific transcription factors activate a particular
set of lineage-specific genes and/or inhibit the cell cycle for terminally differentiated
celts. PU.l is involved in the differentiation of the common myeloid progenitor cell
(CMP) and, later on, in the differentiation of monocyres/macrophages. Most myeloid
specific genes have PU.l sites in their promoters. C/EBPα, a zinc finger transcription
factor, functions in the differentiation of granulocytes. (117)
Many mutations that are typically found in acute myeloid leukemia affect specific
transcription factors; both chromosomal translocations [e.g. t(8;21)] and coding region
mutations are common. The gene for the AML1 transcription factor is disrupted in the
t(8;21) translocation and this translocation leads to acute myeloid teukemia. The
chromosomal translocation, t(8;21) is identified in both hematopoietic stem cells and
more differentiated cells in patients, thus providing additional evidence that the
transforming mutations of acute myeloid leukemia occur in hematopoietic stem cells. (117)
Mutations in lineage-specific transcription factors are found in patients with acute
myeloid leukemia subtypes that are consistent with their role in normal hematopoiesis.
PU.l mutations are found in the earliest stage (MO: very immature leukemia) arid in
monocyric leukemias reflecting PU-l's early role in myeloid precursor cells and in the
development of monocytes/macrophages. Approximately 10% of acute myeloid
leukemia patients carry a mutation in c/EBPα and most of these cases are associated with
the granulocyric subtype reflecting c/EBPα's role in granulocyte differentiation. Thus,
mutation of transcription factors involved in differentiation is an important mechanism
behind oncogenesis. (117)
In acute myeloid leukemia, most leukemic cells have a limited capacity for
proliferation but are replenished by rare leukemic stem cells. Therefore, the self-renewal
ability of stem cells is important in the maintenance of this disease. A transcriptional
represser, Bmi-1, has been demonstrated to be essential for the control of self-renewal in
hematopoieric stem cells and in leukemic stem cells .In vitro, leukemic stem cells that
lack Bmi-1 show growth arrest in the Gl phase of the cell cycle and begin to
differentiate. In uiro, mice with a Bmi-1 gene knockout show a progressive depletion of
all blood cells indicating Bmi-Ts essential role m hematopoietic stem cells. In addition,
in mouse models using leukfmic stem cells lacking Bmi-1, lower numbers of leukemic
cells are detected in the peripheral blood compared with controls, indicating that these
cancer Stem cells have proliferative defects. This is an example of how a common gere
csn regulate self-renewal in both normal and cancer stem cells. Bmi-1 normally exerts its
effects partially by repressing [he expression of two cyclin-dcpendent kinase inhibitors
pl6 and pl4 via chromatin remodeling. Bmi-l's role as a human oncogene is supported by
the identification of Bmi-1 gene amplification in some mantle cell lymphomas. (117)
Acute promyelocytic leukemia, a subtype of acute myeloid leukemia, is most often
characterized by [he chromosomal trans location, t(15;17), ithar results in the fusion of
the PML gene with the retinoic acid receptor alpha (RARa) gene to create a hybrid
protein with altered functions. It has been proved that RARs (α, β, and γ) are members
of the steroid hormone receptor superfamily and act as ligand dependent transcription
factors that are important effectors of retinoic acid's essemial role in cell differentiation.
The wild-type receptors bind ro the retinoic acid response element in target genes as
RAR-RXR heterodimers. In the absence of retinoic acid (RA), the receptors associate
with HDAC-corepressor complexes that silence target genes by histone deacerylarion
and subsequent chromatin compaction (Figure 15a). Upon binding of RA, the receptor
undergoes a change in shape that causes the receptor to dissociate from the HDAC-co-
repressor complex and allows the receptor to interact with co-activators in order to
transcriptionally induce its target genes (Figure 15b). (117)

Figure15 The activity of RAR and the PML-RAR fusion protein vs. retinoic acid
concentration .
Edited from l.pecorino, molecular biology of cancer ,2005

Co-activators recruit HATs and also mediate interactions with the basal
transcriptional machinery. The oncogenic fusion protein maintains both the DNA
binding domain and the ligand binding domain of the RARa receptor. It has a higher
affinity for HDAC and does not dissociate in the presence of physiological
concentrations of RA (Figure 15c). In addition, it is likely that the fusion protein forms
homodimers that may act in a dominant negative manner by blocking wild-type RAR-
RXR heterodimers or by recruiting novel co-repressors. The normal role of the PML
protein may also be disrupted in the fusion protein. PML protein is normally found in
nuclear organelles called nuclear bodies and, as a co-activator of p53, acts as a pro-
apoptotic protein. Thus, although altered gene expression of retinoic acid target genes is
most likely the predominant mechanism of PML-RAR's oncogenic ability, additional
possibilities such as affecting PML Function, exist. (117)
Therapeutic strategies
The concept of cancer stem cells has important implications for the design and
testing of new cancer drugs. First, since cancer stem cells support the growth and
migration of the tumor, drugs need to target this small subset ''if cells within the rumor.
Many existing drugs give hopeful initial responses that are followed by disappointing
latter reoccurrences. Drugs targeted ar cancer stem cells may prevent reoccurrence and
actually cure metastatic cancer. In addition, the efficacy of such new drugs should be
determined by its effect on the cancer stem cell population and not on overall tumor
regression. Drugs may be successful at killing all of the cells of a tumor except cancer
stem cells, so that measuring tumor regression would not reflect the fact that the most
dangerous tumor cell types remained unaffected. (117)
Differences in drug resistance between cancer stem cells versus other tumor cells is a
possible explanation for such a scenario- Below is a sample of drug strategies that target
self-renewal or difierentiation pathways. (117)
Inhibitors of the Wnt pathway

The importance of the Wnt pathway in several cancers, particularly colorectal cancer,
suggests that the molecular components of this parhway are good rargers for new
therapeutics. Disruption of the protein-protein interaction between p-catenin and the Tcf
transcription factors (Figure 16) is one strategy that has been investigated . This
interaction occurs down stream of the APC degradation complex and is the end-point
effect pf Wnt signaling. Drugs acting at this stage would counter both in activating
mutations in APC, Axin GSK3β, and activating mutations in p-catenin that cause
inappropriate formation of β-catenin/Tcf complexes. The observation that TCf4
inhibition induces the differentiation of colorectal cancer cells into epithelial villi is
evidence that supports this approach. identified three natural compounds from a high-
throughput screen that acted as inhibitors of the β-catenin-Tcf interaction. Also, these
compounds that share a core chemical structure inhibited the expression of two Tcf-
target genes and inhibited colorectal cancer cell proliferation. Although in its early
stages, this strategy holds promise for the development of new cancer therapeutics. Since
these drugs target a molecular pathway that is important in selfrenewai, they have a
greater chance of tumor eradication rather than only tumor regression. (117)
Figure 16 Drug strategy to inhibit the β -catenin−Tef interaction .
Edited from l.pecorino, molecular biology of cancer ,2005

Inhibitors of the Hh pathway

Inhibitors of the Hh signaling pathway are being investigated as cancer therapeutics
(Figure 17). One example is cyclopamine, a sceroidal alkaloid that is found in high
level's in wild corn lilies, [ its name comes from the cyclonic effects 'formation of a
single eye) observed by its action as terarogen. Pregnant sheep that ingested high
quantities of wild corn lilies gave birth to cyclopic lambs. There are obvious implications
for treating woman oi' child-bearing Jge with this terarogen. Cyclopamine suppresses the
Hh pathway by inhibiting the activity of the transmembrane protein, Smoothened. In
animal studies, cyclopamine treatment blocked growth of medulloblastomas celts and
affected the regulation of genes involved in differentiation . Expression of a neuronal
stem cell marker, neurofilament nestin, was decreased while expression of a marker of
neurona! differentiation, Neuro D, was increased. Genetech is involved in the
development of small molecule antagonists and Hedgehog blocking antibodies as means
of inhibiting the Hh pathway for new cancer treatments. (117)

Figure17 inhibition of the Hedgehog pathway by cyclopamine .

Edited from l.pecorino, molecular biology of cancer ,2005
 Ethical problems on stem cell research

Moral Status on Human Stem Cells

Human embryonic germ (EG) cells* are derived from the tissue of aborted fetus within
five to eight weeks after pregnancy. The procedure is similar to the harvesting the organs
from a dead bodies. The ethical issue is concerned about the participation of stem cell
research on abortion which is regarded as against the legality. There are arguments related
to the ethical status of human ES cells on whether it should be embryos or specialized
tissue. (118)

Adult stem cells (obtained from adult tissues, cord blood, etc.) do not pose any ethical
controversies, since they do not destroy complete human beings. However, embryonic
stem cells are isolated by destroying human embryos - the form in which all of us began
our lives. Some say these embryos are not persons. (118)

They believe that embryo should morally be protected because it has potential to
develop into an individual human being with cognitive abilities. Besides, the possession
of unique human genome of an individual will cause impact on the next generations.
There were debates about the “natural potentiality” on ES cells. To clone the ES cells,
mammalian cloning technology has to be carried out with facilities and genetic
engineering process. Those who are handling the cloning process are disturbing the
natural potentiality of the stem cells which are carrying a life. In addition, it will be
cloning with the other cells that are cloned. The full moral protection of every human cell
is a necessity since the scientific modification of the stem cell leads to some ethical
problems in natural cells.
It can be concluded that multiple moral values should be considered in stem cell
research even at the life’s beginnings. However, some peoples believe that stem cells are
not equivalent to the same moral status that the embryos have. They think that stem cells
may be regarded as any other form of human tissue. It is quite complicating and
confusing while defining the potentiality. Basically, biology explains that the cell
development happens continuously during human growth and maturation. The stem cell
research can improve future human health and the respect for the democratic society is to
be discussed. (118)

Controversy On The Source Of The Stem Cell

There are three possible sources of stem cells: adult stem cells extracted from children
or adult donors, embryo germ cell stem cells (EG cells) derived from aborted fetuses and
ES cells taken from disaggregated preimplantation embryos. Useful EG cells from
spontaneous aborted tissue can be extracted and cultured by using the advanced
technology. Unfortunately, the amount of the product is limited even it is placed under the
optimum conditions. Research shows that specific fetal anomalies and specific
chromosomal abnormalities are found in most of the derived aborted tissue in culture. It is
difficult to produce sufficient amount of the “normal” tissue naturally. The process is time
consuming since the EG cells can only be taken during a narrow developmental phase
that is within the first eight weeks after conception. (118)

Besides, the deliberate creation of embryos allows the use of “spare embryos” to be
used in the infertility procedures. For the points stated above is relevance to early
embryonic life damage which are illegally wrong. The person who is dealing with the
stem cell research may have to be blamed as a crime for this morally unacceptable killing
of a fetus and embryo. This raises the question of “complicity” or “cooperation” with evil
among the community. (118)

List of Figures
Figure 1: A general overview of the concept of stem cell self-renewal and
differentiation.
Figure 2: Embryonic and Adult Stem Cells.
Figure 3: Components of the stem cell niche.
Figure 4: Scheme for the generation of new cells and tissues from embryonic stem cells
Figure 5: Derivation and culture methods for human embryonic stem cells.
Figure 6: Showing the immunofluorescence procedure .
Figure 7: Showing Somatic Cell Nuclear Transfer (SCNT) .
Figure 8: Treatment of Type1 diabetes by using stem cells .
Figure 9: Features of Stem Cells .
Figure 10: The Wnt signaling pathway .
Figure 11: The mutation cluster region of APC.
Figure 12: Morphogenesis of a colorectal tumor .
Figure 13: The Hedgehog signaling pathway .
Figure 14: Haematopoietic lineages : disruption in the granulocyte or monocyte lineage
leads to AML .
Figure 15: The activity of RAR and the PML-RAR fusion protein vs. retinoic acid
concentration .
Figure 16: Drug strategy to inhibit the β -catenin−Tef interaction .
Figure 17: Inhibition of the Hedgehog pathway by cyclopamine .

List Of Tables

Table 1 : Markers Commonly Used To Identify Stem Cells And To Characterize

Differentiated Cell Types .

Table 2: Current Human Clinical Applications Using Adult Stem Cells

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