KOLEKSI DAN APLIKASI
STEM SEL
EDITED BY ;
PROF.DR.PRATIWI TS,MS
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Stem Cell – Definition
 A cell that has the ability to continuously divide
and differentiate (develop) into various other
kind(s) of cells/tissues
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Introduction
 Can we vs. should we
 Dramatic advances of modern molecular
genetics
 Should we ask the morality questions before
attempting the “can we” questions?
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Stem Cell/Cloning Topics
 What are stem cells?
 History of stem cell/cloning research
 Possible uses of the technology
 Current status/knowledge
 Questions and known problems
 Legal considerations
 Politics
 Moral considerations
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Kinds of Stem Cells
Stem cell
type
Totipotent
Totipotent
Pluripotent
Pluripotent
Multipotent
Multipotent
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Description
Examples
Cells from early
Each cell can develop
(1-3 days)
into a new individual
embryos
Cells can form any
(over 200) cell types
Some cells of
blastocyst (5 to
14 days)
Cells differentiated,
Fetal tissue, cord
but can form a number blood, and adult
of other tissues
stem cells
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Stem Cell/Cloning Topics
 What are stem cells?
 History of stem cell/cloning research
 Possible uses of the technology
 Current status/knowledge
 Questions and known problems
 Legal considerations
 Politics
 Moral considerations
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Kinds of Stem Cells
Stem cell
type
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Description
Examples
Totipotent
Totipotent
Each cell can develop
into a new individual
Cells from early
(1-3 days)
embryos
Pluripotent
Pluripotent
Cells can form any
(over 200) cell types
Some cells of
blastocyst (5 to
14 days)
Multipotent
Multipotent
Cells differentiated, but Fetal tissue, cord
can form a number of
blood, and adult
other tissues
stem cells
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Stages of Embryogenesis
Day 2
2-cell embryo
Day 1
Fertilized egg
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Day 11-14
Tissue
Differentiation
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Day 3-4
Multi-cell embryo
Day 5-6
Blastocyst
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Derivation and Use of Embryonic Stem
Cell Lines
Isolate inner cell mass
(destroys embryo)
Outer cells
(forms placenta)
Inner cells
(forms fetus)
Day 5-6
Blastocyst
Culture cells
“Special sauce”
(largely unknown)
Liver
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Kidney
Heart
repaired
Heart muscle
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Bone Marrow Stem Cells
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Possible Uses of Stem Cell
Technology
 Replaceable tissues/organs
 Repair of defective cell types
 Delivery of genetic therapies
 Delivery chemotherapeutic agents
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Early Successes – Adult Stem Cells
 Human mesenchymal stem cells turned on genes
found in bone, cartilage, adipose, muscle,
hematopoiesis-supporting stromal, endothelial,
and neuronal cells.
 Multipotent adult progenitor cells have been
shown to differentiate into functional,
hepatocyte-like cells.
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Early Successes – Adult Stem Cells
 Human neural stem cells can migrate extensively
in the brain after injection.
 Adult stem cells have been isolated from amniotic
fluid, peripheral blood, umbilical cord blood,
umbilical cord, brain tissue, muscle, liver,
pancreas, cornea, salivary gland, skin, tendon,
heart, cartilage, thymus, dental pulp, and adipose
tissue.
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Early Successes – Human Cloning
 2001 – First cloned human embryos (only to six cell stage)
created by Advanced Cell Technology (USA)
 2004* – Claim of first human cloned blastocyst created and a cell
line established (Korea) – later proved to be fraudulent
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*Hwang, W.S., et al. 2004. Evidence of a Pluripotent Human
Embryonic Stem Cell Line Derived from a Cloned Blastocyst. Science
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303: 1669-1674.
Cloned Embryonic Stem Cells –
Advantages/Problems
 Advantages
 No rejection
 “Prefect match”
 Problems
 Only 10% of cloned oocytes became embryos
 0% (0 out of 2061) survived to become a cell line
 Genetic donor was same as egg donor (i.e., won’t
work for males!)
 Cost is high (health insurance probably won't pay)
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Challenges to Stem Cell/Cloning
Research
 Stem cells need to be differentiated to the appropriate
cell type(s) before they can be used clinically.
 Recently, abnormalities in chromosome number and
structure were found in three human ESC lines.
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Challenges to Stem Cell/Cloning
Research
 Stem cell development or proliferation must
be controlled once placed into patients.
 Possibility of rejection of stem cell transplants
as foreign tissues is very high.
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Challenges to Stem Cell/Cloning
Research
 Contamination by viruses, bacteria, fungi,
and Mycoplasma possible.
 The use of mouse “feeder” cells to grow
ESC could result in problems due to
xenotransplantation (complicating FDA
requirements for clinical use).
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At Conception, It Is Only a Single Cell
Claim:
 Fertilized eggs are single cells, like blood cells or other parts
of the body
Rebuttal:
 This single cell is unique from both the father’s and mother’s
cells and is the beginning of every new human being
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Only a Small Percentage of Embryos
Implant
Claim:
 Embryos are only potential life. Most do not result in births
Rebuttal:
 25-33% of women become pregnant in the first month
 33% of implanted embryos die before birth
 There are countries in which over 25% of children die
before age 5. Should we allow killing of children?
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Unexpected Phenotypes
 Phenotype more severe than expected:
- Early lethal
- Lack of inductive signals
 Phenotype less severe than expected:
- Incomplete gene disruption
- Genetic redundancy
- Functional redundancy (compensation)
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Applied research - DNA
Applied research arising out
of the discovery of DNA
includes disease diagnosis,
drug development, gene
therapy and, more recently,
genetically-modified
organisms
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Transgenic Animals and Products
 Mice- transgenetic mice have been used in
several ways.
 One of the best known is to produce human
antibodies.
 Cattle- are used to control disease such as
mastitis in dairy cows.
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Methods of creating transgenetic
animals
 Step One- collect embryos
 With proper stimulation far more
embryos can be obtained than would be
the natural result of the reproductive
process.
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Methods of creating transgenetic
animals
 Step Two- Inject embyros.
 A pro nucleus is the haploid nucleus of
the sperm or ovum that have united in
fertilization to form a zygote.
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Embryo Transfer
Embryo transfer is the harvesting of
fertilized ova from a donor and
implanting them into a recipient.
The harvested embyros are
transferred to a recipient.
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Clone Birth Defects
• Cloned offspring often suffer from large offspring
syndrome, where the clone and the placenta that nourished
it are unusually large.
• Cloned offspring often have serious inexplicable respiratory
or circulatory problems, which causes them to die soon after
birth.
• Clones tend to have weakened immune systems and
sometimes suffer from total immune system failure.
• Very few clones actually survive to adulthood.
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 Clones appear to age faster than
normal.
 Clones experience problems associated
with old age, such as arthritis, while they
are still young.
 This may be due to the fact that clones
have shorter telomeres
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Transgenic Animals:
Animal biotechnology is the field to engineer
transgenic animals, i.e., animals that carry genes
from other species.
 The technology has already produced transgenic
animals such as mice, rats, rabbits, pigs, sheep,
and cows.
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Transgenic Animals
 Definition:
An organism (typically a mouse) that is
engineered to carry a foreign gene, or
transgene of choicem as part of its own
genetic material.
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Transgenic Animals
 Purpose:
These animals are very useful for
delineating the function of newly
discovered genes as well as for
producing useful proteins in large
animals.
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Transgenic Animals
 In some of the eggs, the genetic material
integrates at a random site on a
chromosome and so becomes part of the
mouse cell's genetic material the animal
resulting from that egg will therefore
carry that gene and so is referred to as a
"transgenic animal".
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What is a transgenic animal?
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A transgenic animal is one whose genome has
been changed to carry genes from other
species.
For example, an embryo can have an extra, functioning
gene from another source artificially introduced into
it, or a gene introduced which can knock out the
functioning of another particular gene in the embryo.
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Transgenic Animals
 Animals that have their DNA
manipulated in this way are known as
transgenic animals.
 Transgenic animals are useful as disease
models and producers of substances for
human welfare.
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Why are these animals being
produced?
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Some transgenic animals are produced for
specific economic traits.
E.g., transgenic cattle were created to produce
milk containing particular human proteins,
which may help in the treatment of human
emphysema.
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How are transgenic animals produced?
DNA microinjection
Introducing the transgene DNA directly into the zygote at
an early stage of development.
 No vector required.
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Retrovirus-mediated gene transfer:
Infecting mouse embryo with a retrovirus which carry the
new gene.
 Using virus as a vector .
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Embryonic stem cell-mediated gene transfer
The blastocyst (inner layer of a fertilized egg) is
harvested and mixed with recombinant DNA and
inserted back in the blastocyst.
Sperm-mediated transfer
Use of “Linker protein" to attach DNA to sperm which
transfer the new DNA during fertilization.
Gene gun
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Embryonic stem cell-mediated gene transfer
This method involves:
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Isolation of totipotent stem cells (stem cells that can
develop into any type of specialized cell) from embryos.
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The desired gene is inserted into these cells.
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Cells containing the desired DNA are incorporated into
the host's embryo.
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Procedure for Producing Transgenic
Mice
 First Breeding Pair:
 Fertile male + superovulated female
 Fertile male
 Superovulated female = immature female induced to superovulate
Pregnant mare’s serum (=FSH) on day 1
 Human Chorionic Gonadotropin (=LH) on day 3
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 Mated on day 3
 Fertilized oocytes microinjected on day 4 with foreign DNA
construct.
 Microinjected oocytes are transferred to the oviducts of surrogate
mothers at end of day 4.
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Procedure for Producing Transgenic
Mice
 Second breeding pair:
 Sterile male + surrogate mother
 Sterile male produced through vasectomy
 Surrogate mother must mate to be suitable recipient
of injected eggs
 Mated on day 3
 Microinjected oocytes from first breeding pair are
transferred to oviducts on day 4
 Embryos implant in uterine wall and are born 19 days
later.
 Southern blotting techniques confirm presence and
copy number of transgenes.
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Procedure for Producing Transgenic
Mice
 Third breeding pair:
 Foster parents
 Fertile male + female mated to give birth on same day surrogate mother
 Serves as foster parent if caesarian section is required for surrogate
mother
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Totipotent and
pluripotent cells
isolated directly
from the inner cell mass
of embryos
at the blastocyst stage.
Totipotent =
meaning that
its potential is total.
(IVF-IT surplus embryos
in case of humans)
pluripotent =
they can give rise
to many types of cells
but not all types of cells
(no fetus developed).
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More about stem cells
Embryonic stem cells
Truly pluripotential
several countries
have sanctioned deriving
human ES-cell lines
from ‘surplus’ embryos
created through
in vitro fertilization
although several human
ES-cell lines have been made,
they will not be immunologically compatible
with-PROF
most
patients
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who require cell transplants.
Adult stem cells
More restricted
pattern of differentiation
medical gain without ethical pain
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Transgenic mice
The growth hormone gene has been engineered to be expressed
at high levels in animals.
The result: BIG ANIMALS
metallothionein promoter
regulated as heavy metals
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Mice fed heavy metals are 2-3 times larger
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Studies Utilizing Transgenic Mice
 “Pharm” animals
(transgenic livestock)
 Bioreactors whose cells have been engineered to synthesize
marketable proteins
 DNA constructs contain desired gene and appropriate
regulatory sequences (tissue-specific promoters)
 More economical than producing desired proteins in cell
culture
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Antifreeze gene promoter
with GH transgene in atlantic salmon
GH gene comes from
larger salmon
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Wild and domestic trout respond differently
to overproduction of growth hormone.
So in some cases, GH not effective.
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Improving Agricultural Products with
Transgenics
Transgenic technology holds great potential in agriculture,
medicine, and industry
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The benefits of these animals to human welfare can be
grouped into areas:
 Agriculture
 Medicine
 Industry
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1. Agricultural Applications
A) Breeding
 Traditional cross breeding have been used for
ages to create chickens, cows, pigs etc.
 Farmers have always used selective breeding to
produce animals that exhibit desired traits
(e.g., increased milk production, high growth
rate).
 Traditional breeding is a time-consuming,
difficult task.
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Researchers have now used gene transfer to
improve the productivity of livestock.
Now it is possible to develop traits in animals in a
shorter time and with more precision.
It also offers farmers an easy way to increase
yields.
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Scientists can improve the size of livestock
genetically.
Transgenic cows exist that produce more milk or milk
with less lactose or cholesterol.
Transgenic cows have been used to produce milk
which are richer in proteins and lower in fat.
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B) Quality
Herman, a transgenic bull carries a human gene
for Lactoferrin (gene responsible for higher iron
content)
Pigs and cattle that have more meat on them.
Sheep that grow more wool.
Eggs can be made healthier with high quality
protein.
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C) Disease resistance
Disease-resistant livestock is not a reality just yet.
But there has been improvement in disease
reduction in animals.
The Foot- and- Mouth disease in England in 2000
led to destruction of herds of cattle, sheep and
goat.
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 Scientists are attempting to produce
disease-resistant animals, such as influenzaresistant pigs, but a very limited number of
genes are currently known to be responsible
for resistance to diseases in farm animals.
 Transgenic disease protection promises a
long term cost effective method of battling
animal diseases.
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2. Medical Applications
A) Xenotransplantation
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Transplant organs may soon come from transgenic
animals.
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B) Nutritional supplements and pharmaceuticals
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Products such as insulin, growth hormone, and
blood anti-clotting factors may soon be or have
already been obtained from the milk of transgenic
cows, sheep, or goats.
The first transgenic cow (Rosie ) produced human
protein-enriched milk at 2.4 grams per liter.
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This transgenic milk is a more nutritionally
balanced product than natural milk and could be
given to babies or the elderly with special
nutritional or digestive needs.
A transgenic cow exists that produces a substance
to help human red cells grow.
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C) Human gene therapy
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Human gene therapy involves adding a normal
copy of a gene (transgene) to the genome of a
person carrying defective copies of the gene.
Finland produced a calf with a gene that makes
the substance that promotes the growth of red
cells in humans.
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3. Industrial Applications
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By extracting polymer strands from the milk and weaving
them into thread, the scientists can create a light, tough,
flexible material that could be used in such applications as
military uniforms, medical microsutures, and tennis racket
strings.
Biosteel is an extraordinary new product that may be soon
used in bullet proof vests and in suture silk for stitching
wounds.
Animals have been used as “Bioreactors” to produce
proteins. Genes for desired proteins are introduced via
transgenics to the target cells .
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The target cells are cloned and several such cells are raised
into adults.
These adults may produce milk or eggs (due to the
presence of introduced gene rich in desired protein).
Toxicity-sensitive transgenic animals have been produced
for chemical safety testing.
Microorganisms have been engineered to produce a wide
variety of proteins, which in turn can produce enzymes
that can speed up industrial chemical reactions.
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Transgenic animals have been used to produce
pharmaceutical protein: example a human gene called AT
III has been transferred to goats.
Goats milk contain this protein that prevents blood
clotting (goats multiply faster than cows)
“Hen bioreactor” eggs are used to enrich protein by
recombinant DNA technology.
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Transgenic Goats That Produce Valuable Proteins in
Their Milk – “Biorectors”
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A Summary of Animal Cloning
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Although there has been limited success in
cloning some animals, it's still seen as a viable
technology.
Ever since the announcement of the birth of Dolly,
additional sheep, cows, goats, pigs, and mice have
been cloned.
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There are still obvious problems as evidenced from
the numerous deaths of cloned animals that occur just
before or after birth.
Cloning is a big first step. Genetic manipulation of
cloned animals is the future direction of the cloning
frontier.
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