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Welcome

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Stem Cell
Research
Prof. D. Amal Mangoud

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Topics:
• Stem cell Glossary
• What are Stem Cells?
• Why is stem cell research important?
• Types of Stem Cells
• Adult stem cells VS. embryonic
stem cells.
• Potential Application of Stem Cell
Research
• Use of stem cells in cancer
therapy.
• Pros and Cons of Using Embryonic Stem Cell
• Pros and Cons of Using Adult Stem Cell 3
•Blastocoel.
• Germ cell layers •Blastocyst.
• Hematopoietic stem cell •Inner cell mass .
• Human embryonic stem cell •Long-term self-renewal .
• In vitro •Cell-based therapies.
• In vivo •Cell culture .
• Inner cell mass •Clone
• Long-term self-renewal •Culture medium
• Mesenchymal stem cells •Differentiation.
• Mesoderm •Directed differentiation.
• Neural stem cell •DNA .
• Neurons •Ectoderm
• Nuclear transfer •Endoderm
• Ontogeny •Feeder layer
• Passage •Fertilization
• Plasticity •Fetus
• Pluripotent •Gene
• Progenitor cells •Microenvironment
• Proliferation •Subculturing
• Regenerative or reparative medicine •Surface markers
• Signals •Transdifferentiation.
• Somatic stem cells •Undifferentiated
• Stromal cells
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Stem Cells - Definition
• Are undifferentiated
Stem cell
“master” cell that do
not yet have a specific
function

Di
l
• Can change to one or

wa

ffe
ne

re
re

nt
several different cell

lf-

iat
Se

e
types (differentiate)
under proper
conditions
Stem cell Specialized cell
• Can undergo unlimited (e.g., white blood cell)
cell division (self-
renewal)
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Why is stem cell research important?
1) To create cells and
tissue for transplants
and therapies..

2) To improve our
understanding of
human development
and the causes of
birth defects, cancer
and other
degenerative
diseases.

3) To teach us more
about how drugs
work in the human
body and how to
make them safer.

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Types of Stem
Type
Cells
Source Differentiated
Embryonic stem Harvested from 5-6 days old cell type
Any of the 200 cell types present
cells embryo (blastocyst) in the human body (e.g., skin
cells, liver cells, heart cell, etc.)

Fetal stem cells Taken from germline tissues All fetal tissues in the fetus
that will make up the ovaries before birth
or testes of aborted fetuses

Umbilical cord Taken from umbilical cord All blood cell types (red blood
stem cells blood, which contains stem cells, white blood cells, and
cells similar to those found in platelets)
bone marrow of newborns
Adult stem cells Resided in developed tissues, Develop into the same cell types.
like bone marrow, liver, skin, E.g., blood stem cells can
and the lining of develop into several blood cell
gastrointestinal tract types, but cannot develop into
brain, kidney, or liver cells.
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Embryonic stem cells
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Directed
differentiation of
mouse
embryonic stem
cells.

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Where do Embryonic Stem
Cells Come From?

• Human beings start their lives from a single fertilized egg.


• The fertilized egg divides and forms two cells; each of those cells
divides again, and so on.
• About five days after conception, there is hollow ball of about 150
cells called the blastocyst.
• The blastocyst contains two types of cells, the trophoblast and the
inner cell mass.
• Embryonic stem cells are the cells that make up the inner cell
mass. 16
Two ways to make
embryos Fertilization (sexual reproduction) –
a child gets half its genes from its mother (in her egg) and half from its father
(in his sperm).

Cloning
is an asexual form of reproduction. All the child's genes would come from a body
cell of a single individual.

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Embryonic
stem cell
Success

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ADULT STEM CELL RESEARCH: THE CLEAR
WINNER
• For EVERY treatment success claimed by embryo stem
cell proponents, there is an

• Ethical therapy either available or in the pipeline that is


much more promising.

• Adult stem cells have treated over 58


diseases in human patients in published

• Clinical studies. Embryonic stem cells


have not treated even ONE patient.

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Adult stem cells
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Hematopoietic and stromal stem cell differentiation. 24
Plasticity of adult stem
cells.
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Heart muscle repair with adult stem cells. 26
Schematic illustration of the experiments carried out to
A single colony of hybrid cells (in investigate the switching of adult cells into embryonic stem
blue) resulting from fusion between  cells. The Edinburgh scientists fused mouse embryonic
stem cells with mouse brain stem cells, to produce single
mouse embryonic stem cells and hybrid cells. In some cases the cells were made to produce
brain stem cells, in a dish. extra Nanog. If reprogramming is successful, colonies of
hybrid cells that behave like embryonic stem cells form;
failure to reprogramme results in death of the hybrid cells
and no colony remains.
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Adult Stem Cells

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Adult Stem Cells

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a- Neural stem cells, labelled with
-galactosidase, were cultured with myoblast
cells or embryoid bodies, producing
-galactosidase-labelled muscle cells. This
was interpreted as evidence that the stem
cells received signals that caused them to
transdifferentiate into muscle cells. But there
are other possibilities. The stem cells might
have been contaminated with muscle
precursor cells; a few stem cells might have
mutated; or the stem cells might have fused
with myoblasts or embryoid-body cells.

b, Ying et al.2 cultured puromycin-resistant


neural stem cells that expressed green
fluorescent protein (GFP) with hygromycin-
resistant embryonic stem (ES) cells. After
selection, the surviving cells expressed GFP,
were resistant to puromycin and hygromycin,
and had double the normal amount of DNA
(4n versus 2n). This suggests that the cells
arose by fusion.

c, Terada et al.3 cultured GFP-labelled,


puromycin-resistant bone marrow cells with
ES cells. The resulting cells expressed GFP,
were resistant to puromycin and had ES-cell
properties. They also had double the usual
DNA content. 30
Embryonic
Vs.
Adult stem cells

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How Many Human
Embryonic Stem Cell
Lines are There?
• The actual number of human
embryonic stem cell lines is a matter
of some debate.
• To date, more than 100 human
embryonic stem cell lines have been
derived worldwide.
• However, most of those lines are not
adequately characterized yet.
• Only 22 cell lines are eligible for
federal funding in the USA. 32
Potential Application of
Stem Cell Technology

• Replace damaged or defected


tissues/organs – cell therapy
• Drug testing

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How Does Cell
Therapy Work?
• Stem cells can be used to generate healthy
and functioning specialized cells, which can
then replace diseased or dysfunctional cells.
• It is similar to the process of organ
transplantation only the treatment consists of
transplanting cells instead of organs.

“Special sauce” Bone


(largely unknown) tissues

Day 5-6
Blastocyst Nerve Heart
tissues tissues
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How Does Cell
Therapy Work?
• Bone marrow transplants are an example of
cell therapy in which the stem cells in a
donor's marrow are used to replace the blood
cells of the victims of leukemia.
• Cell therapy is also being used in experiments
to graft new skin cells to treat serious burn
victims, and to grow new corneas for the
sight-impaired.
• In all of these uses, the goal is for the
healthy cells to become integrated into the
body and begin to function like the
patient's own cells. 36
What Diseases Can be
Cured by Stem Cell
Therapies?
• Any disease in which there is tissue
degeneration can be a potential candidate for
stem cell therapies

Alzheimer’s disease
Parkinson’s disease Spinal cord injury
Heart disease
Severe burns
Diabetes
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Drug Testing
• Stem cells could allow scientists
to test new drugs using human
cell line which could speed up
new drug development.
• Only drugs that were safe and
had beneficial effects in cell line
testing would graduate to whole
animal or human testing.
• It would allow quicker and safer
development of new drugs.
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Cancer stem cell theory

New cancer model:


1) Tumors arise from cells termed cancer stem cells that
have properties of normal stem cells, particularly self-
renewal and multipotentiality (a minority) of tumor
cells.

2) Unregulated cell growth is due to a disruption in the


regulatory mechanism in stem cell renewal.

3) Cancer is a stem cell disorder and not a simple


mechanism whereby cell proliferation is disrupted.
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Cancer stem cell theory

• These CSCs cells persist in tumors as a


distinct population that likely causes
relapses and metastasis.

• This theory explains why are many


cancers so difficult to treat.
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Cancer stem cell theory

Why stem cells?

• Only stem cells have the ability to self renew


and neoplasia is essentially dysregulated self
renewal
• Stem cells are long-lived cells which can
acquire the necessary number of sequential
mutations to convert a normal cell into a
malignant one.
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Are we targeting the right cells?

• Conventional chemotherapies kill differentiated or


differentiating cells, which form the bulk of the tumor but
are unable to generate a new one.

• A population of CSCs, which gave rise to it, remains


untouched and may cause a relapse of the disease.

• Development of specific therapies targeted at CSCs holds


hope for improvement of survival and quality of life of
cancer patients, especially for sufferers of metastatic
disease, where little progress has been made in recent
years.
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WRONG TARGET. Traditional cancer therapies (top) kill rapidly dividing tumor
cells (blue) but may spare stem cells (yellow) that can give rise to a new tumor. In
theory, killing cancer stem cells (bottom) should halt a tumor's growth lead to its
disappearance.

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What are Cancer Stem Cells?
Cells that have properties of normal stem cells:
1) The abilities to self-renew.
2) Tha ability to differentiate into multiple cell
types.
3) They form a distinct population in tumors that
likely causes disease relapse and metastasis.

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Self-renewal of stem cells

The concept of self-


renewal is crucial to
understand CSC, and
also to get insight on the
mechanism by which
current therapies might
evade the available
treatments.

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Self-renewal of stem cells

 Provides the cell with the ability to undergo infinite


cellular divisions with only few of the stem cells
dividing at a particular time.

2) The doubling time of most stem cells is relatively long, as


compared to their immediate progenitors, which
replicate with shorter doubling times (Repair of DNA
damage).

3) In some stem cells at division the `mother’ cell retain the


original chromosome while providing the daughter
with the newly formed chromosome (Chromosomal
preservation) → minimizes mutation in the mother
cell. 46
CSC Development
• The molecular pathways for stem cell
differentiation are complex indicating
that dysregulation could occur at
multiple sites to turn off the
homeostatic balance and create
abnormal cells, or cancer cells, also
referred as malignant cells or
transformed cells.

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Normal Stem Cells vs. Cancer Stem
Cells

• The stem cells in tumors (CSCs) are not the


same type of stem cells being explored as
potential therapies to treat degenerative
diseases.
• But they develop because of mutations that
accumulate over years and often decades. The
mutations are thought to promote the tumor
stem cells' ability to proliferate, eventually
leading to cancer 48
CSC- PATHWAYS

• A normal stem cell may be transformed into a


cancer stem cell through disregulation of the
proliferation and differentiation pathways
controlling it.
• The first findings in this area were made using
haematopoietic stem cells (HSCs) and their
transformed counterparts in leukemia.
• However, these pathways appear to be shared
by stem cells of all organs.
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CSC- PATHWAYS
Bmi-1
This group of transcriptional repressor
was discovered as a common oncogene
activated in lymphoma and later shown
to specifically regulate HSCs and neural
stem cells. This pathway appears to be
active in CSC of pediatric brain tumors
and CRC

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CSC- PATHWAYS
Bmi-1
** In normal cells BMI-1 inhibits the transcription of
CDNK2A which encodes two cyclin dependent kinase
inhibitors, INK4A and ARF.

** Cell cycle progression is promoted in the absence of


INK4A and pro-apoptotic genes are inhibited in the
absence of ARF. Hence, BMI-1 promotes proliferation
and inhibits apoptosis.

**In the case of cancer, BMI-1 is circumvented and


CDNK2A is no longer inhibited, thereby resulting in
unregulated proliferation and self-renewal.

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CSC- PATHWAYS
Notch
• The Notch pathway has been known to developmental
biologists for decades.

• Its role in control of stem cell proliferation has now


been demonstrated for several cell types including
haematopoietic, neural and mammary stem cells.

• Components of the Notch pathway have been proposed


to act as oncogenes in mammary and other tumors.

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CSC- PATHWAYS
Wnt/β-catenine
• This pathway is strongly implicated as stem cell
regulators.

• It is commonly hyperactivated in tumors and is


required to sustain tumor growth.

• Their role has been illustrated especially in


gliomas (the Gli transcription factors), CRC
and mammary tumors.

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The Wnt-B-catenine pathway
• In the normal Wnt pathway the levels of the
transcription factor ß-catenin mediates self-
renewal.

• ß-catenin could be turned off by a destruction


complex as a feedback mechanism.

• However, in cancer the control process is


circumvented and ß-catenin levels constantly
thrive, hence causing continual proliferation
and self- renewal.

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CSCs in different solid tumors
Stem cells may cause some forms of bone cancer, University of
Florida

• Osteosarcoma occurs right next to the most active centers of growth,


the growth plates in long bones.

• These areas of the skeleton contain many stem cells undergoing rapid
growth and developing into bone during the adolescent growth spurt.

• A stimulated, abnormal stem cell might therefore be the cell of origin of


osteosarcoma.

• stem-like cells were isolated from tumors. About one in 1,000 cells in
the samples had features of embryonic stem cells. The researchers also
found abundant levels of the two key factors that help maintain
embryonic stem cells in a very primitive state.

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The Real Problem in Breast
Tumors: Cancer Stem Cells
• At the University of Michigan
researchers have identified a
small population of cells in
breast tumors that can seed the
growth of new cancers. These
cancer-causing cells, which
make up a tiny fraction of cells
within tumors, have properties
similar to those of stem cells.

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CSCs in Colorectal carcinoma

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CSCs in Colorectal carcinoma

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CSCs in Hepatocellular
carcinoma

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CSC hypothesis & Drug Resistance
• The CSC hypothesis states: “the cancer-initiating cell
is a transformed tissue stem cell, which retains the
essential property of self-protection through the
activity of multiple drug resistance transporters.

• This resting constitutively drug-resistant cell remains


at low frequency among a heterogeneous tumor mass.

• The mutation allows for unbridled cell growth and


resistance to chemotherapeutic efforts since CSCs
express genes for drug resistance and anti-apoptotic
mechanism, .

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• Stem cells are immature cells that can replicate, or
renew themselves, and are able to differentiate into all
cells types.

• mutations and rearrangements of the genomes of stem


cells give rise to CSCs. These changes could underlie the
development of cancers in many tissues.

• Stem cells are more difficult to kill. Because they are so


important throughout a person's lifetime, they have
developed mechanisms that protect themselves.
Therefore, tumor stem cells are able to resist toxic
substances, such as cancer drugs.

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• The next step is to figure out what makes the CSC
different from the other cells in the tumor.

• DNA microarrays could be used to identify genes that are


active in the cancer-causing cells (CSCs) compared to
other tumor cells. Some of these genes might control the
cell's ability to replicate and metastasize.

• Identifying these genes may suggest new drug targets that


could selectively kill the cancer cells

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Obstacles of Stem Cell
Research
• How to find the right type of stem cells?
• How to put the stem cells into the right
place?
• Will the stem cells perform the desired
function in the body?
• Differentiation protocols for many cell
types have not been developed.

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Summary of Policies Defined
Around the World Human Embryo Cloning Use of Stem Cell Use of Superfluous
Countries
(=creating embryo) Lines Embryos

France, Spain Prohibited Authorized Authorized

Italy, Austria, Ireland Prohibited Prohibited Prohibited

U.K. Denmark Authorized Authorized Authorized


Israel, Sweden, Prohibited Authorized Authorized
Belgium, India
Germany Prohibited Authorized Authorized
(imported)
U.S.A. Prohibited (public) Authorized under Authorized
Free (private) restricted condition (in most states)
(public)
Free (private)
Canada Prohibited Under consideration Under consideration

Japan, Netherlands, Authorized Authorized Authorized


Korea
Australia Currently prohibited Authorized Authorized
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but under
consideration
Divergent Religious View
on Research and Clinical
Use of ESC
Religions Embryonic Stem Cell (ESC) Research
Catholic Prohibited (life begins at conception)
Muslim Acceptable (fetus has moral existence
only at the end of the 4th month)
Jewish Acceptable (embryo has no moral
status until 40 days)
Buddhist Prohibited (life begins at conception)

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Ethical Considerations
of Stem Cell Research
• When does human life/personhood
begin?

• Adult stem cells or embryonic


stem cells?

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Religious Debate over
Harvesting
Embryonic Stem Cells
The pro-life group generally
believes that:
• Personhood happens at, or
shortly after, conception.
• Thus, they consider the
removal of stem cells from an
embryo -- a procedure which
kills the stem cells -- to be a
form of murder of a human
being.
• They argue that no potential
health benefits to even Day 5-6
hundreds of millions of people
can justify the murder of other Blastocyst
humans. 67
Religious Debate over
Harvesting
Embryonic Stem Cells
The pro-choice group
generally believes that:
• Personhood is attained much later
in pregnancy, perhaps when the
fetal brain develops
consciousness during the third
trimester.
• Thus, extracting stem cells from
an five or ten-day old pre-embryo
is not murder.
• Killing a pre-embryo, which is only
a potential human being, is
justified if it has the potential to
cure diseases and extend the lives Day 5-6
of people.
Blastocyst
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Pros and Cons of Using Adult
Stem Cell
Pros Cons
• Difficult to obtain, grow
• Do not require the slower, and are less robust
killing of an when compared to cells
extracted from embryos.
embryo. • Stem cells for all cell and
• Adult stem cell tissue types have not yet
been found in the adult
research is more human.
advanced because • Stem cells in adults are
often present in only
of its four decade minute quantities, are
head start over difficult to isolate and
purify, and their numbers
embryo stem cell may decrease with age.
studies.
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Pros and Cons of Using Embryonic
Stem Cell
Pros Cons
• Stem cells can • Require the killing of
an embryo.
potentially
• Difficult to develop
develop into all and maintain.
cell types. • Unstable and mutate
in culture.
• Pure embryonic stem
cells cultures are
difficult to obtain.

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Embryonic Stem Cells
are Unstable and Mutate
in Culture
• Like ordinary cells, stem cells
accumulate significant numbers of
mutations over time, including several
that could cause them to become
tumors.

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Pure Embryonic Stem
Cells Cultures are
Difficult to Obtain
• Some embryonic stem cell lines
approved for research are no longer
"pure" human lines since being
exposed to mouse "feeder" cells to
help keep them viable.

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