10/27/2014

Stem cell research &

Cloning


DR. jacinta sayao
Stem cell therapies are not new.
Doctors have been performing bone
marrow stem cell transplants for
decades. But when scientists learned
how to remove stem cells from human
embryos in 1998, both excitement and controversy ensued.

The excitement was due to the huge potential these cells have
in curing human disease. The controversy centered on the
moral implications of destroying human embryos. Political
leaders began to debate over how to regulate and fund
research involving human embryonic stem (hES) cells.
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Human suffering
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What are stem cells?
They have the remarkable potential to develop
into many different cell types in the body during
early life and growth

Totipotent
Pluripotent
Multipotent
Unipotent/
Oligopotent

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Properties of stem cells
Self renewal
dividing essentially without
limit to replenish other cells
as long as the person or
animal is still alive
in undifferentiated state

Potency
to develop into different cells
(differentiation)
Regenerative medicine
Given their unique regenerative abilities, stem
cells offer new potentials for treating diseases
such as diabetes and heart disease. (Cell-based
therapies , also referred to as regenerative or
reparative medicine)

Stem cells are used to screen new drugs and to
develop model systems to study normal growth
and identify the causes of birth defects


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Regenerative medicine
Study of early human development
Therapeutic tool for various diseases
Develop and test new therapies

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Types of stem cells

Adult or Somatic Embryonic

Adult - Bone marrow, cord blood, fat, brain tissue,
and muscles. Fat yields the largest numbers
of mesenchymal stem cells, while bone
marrow or umbilical blood yield more stem
cells that will become blood cells

Embryonic - donated unused IVF embryos
Pregnancy terminations
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Embryonic stem cells (ESC)
"are cells that are derived from
the inner cell mass of blastocyst
stage (3-5 day old) human
embryos, are capable of
dividing without differentiating
for a prolonged period in
culture, and are known to
develop into cells
and tissues of the three primary
germ layers.
National Institute of Health
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Embryonic stem cells (ESC)
Embryonic cells
are pluripotent
and virtually
immortal.


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hESC (human embryonic stem cells)
To study drug toxicity-

liver cells & cardiomyocytes derived from
SC- useful to study drug toxicity


Davila ,Toxico Sci 2004;79:214-23
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Fetuses from pregnancy terminations
Their ability to renew themselves is limited.

Animal studies have shown that it is more
difficult to produce normal tissues from
these cells.

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Adult stem cells
A relatively rare undifferentiated cell found
in many organs and differentiated tissues
with a limited capacity for both self renewal
(in the laboratory) and differentiation.

Vary in their differentiation capacity, but it
is usually limited to cell types in the organ
of origin.
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Adult stem cells
A persons own stem cells should be
the best source of cells for
transplantation.

Adult stem cells will eventually
substitute for embryonic stem cells.


Umbilical cord stem cells
Stem cells collected from
the umbilical cord at birth
that can produce all of the
blood cells in the body
(hematopoietic). Cord blood
is currently used to treat
patients who have
undergone chemotherapy to
destroy their bone marrow
due to cancer or other
blood-related disorders
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Adult Stem Cells and Postnatal
Stem Cells
Advantages
No ethical controversy.
None to minimal risk of immune rejection.
Reduced cost and time.
Genetic stability.

Potential disadvantage
?Limited plasticity
?Limited lifespan in culture, may carry defective
gene


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Stem Cell Cultivation
University of Wisconsin-Madison
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FDA TRIALS USING ADULT OR CORD
BLOOD STEM CELLS
>70 current human clinical applications using adult
stem cells

there are no current human clinical trials involving
human 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.

Science, J une 17, 2005
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Debate over hESC
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Conservative vs. Liberal
Conservative = Literal Translation of the Bible
Liberal = Compromising Translation of Bible
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Objections and concerns..
An embryo should be accorded full
human status from the moment of its
creation
A first step on a slippery slope towards
human reproductive cloning.
The Ethical Question
Stem cell research thus raised difficult questions:

Does life begin at fertilization, in the womb, or at birth?
Is a human embryo equivalent to a human child?
Does a human embryo have any rights?
Might the destruction of a single embryo be justified if it
provides a cure for a countless number of patients?
Since ES cells can grow indefinitely in a dish and can, in
theory, still grow into a human being, is the embryo really
destroyed?

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Question to consider:

What is the Value of the Embryo?

Value of the Embryo
The leading views speculate that embryos have the
status of:
persons, or
potential persons, or
divine creations, or
subjects of moral 'harm', or
the beginnings of human life, with intrinsic value, or
organic material with no more moral standing than
other body parts.

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Embryos have Status as Human
Beings or Persons
there is no non-arbitrary point in the physical growth
continuum between embryo and developed human that
counts as a morally significant dividing line.
need to indicate the developmental point at which
personhood, or status as a human being
All individuals, by virtue of being persons, have
fundamental rights not to have their basic human interests
interfered with in certain ways, and most importantly, their
interest in the maintenance of their life and bodily integrity
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Others argue that embryos do not have the psychological,
physiological, emotional, intellectual properties that we
tend to centrally associate with personhood.

Embryos, particularly the very early pre-implantation
blastocysts involved in stem cell research, do not, for
instance, have consciousness, individuality, the ability to
reason, or the ability to form courses of action in life and to
choose between them
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Embryos Status as Potential Persons
It is to concede that embryos do not currently exhibit
the properties of personhood, but they will, if allowed to
develop and fulfill their potential therefore they ought to
still be accorded the moral respect and dignity that
personhood warrants.

We still treat humans as persons when they are
temporarily unconscious or asleep. While in these
incapacitated conditions, individuals are not conscious, can
not reason, and can not form and choose courses of
actionthe characteristics we associate with personhood.
But we still see it as morally wrong to harm them or violate
their basic rights.
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This same reasoning is also applied to embryos and the
fact they will exercise these capacities when they
eventually become fully developed humans.

Three types of concern have been expressed about this
argument:
the probability of IVF embryos developing into full-
term successful births is low.
The probability of an IVF embryo becoming a
successful birth depends heavily on human action and
intervention , as well as other biological conditions
Some would argue, that it is not clear why something
that could become a person should be morally regarded
as if it actually were a person.



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Embryos have Status as Divine
Creations
For theistic ones, embryos warrant special
moral importance because they are divine
creations in being the beginnings of human
life.
In other words, embryos are not ours to
destroy (nor create).
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Embryos are Harmed by their
Destruction
loss of life ... is a harm that can be
inflicted on any organism; plants and non-
human animals. Human organisms of every
stage of development including the
embryonic can all suffer loss of life.
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Embryos have Status as Human
Life with Intrinsic Value
A single human life has value no matter in what form
or shape
because of the complex creative investment it represents
because of our wonder at the divine or evolutionary
processes that produce new lives from old ones,
through which a human being will continue hundreds of
generations of cultures and forms of life and value
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Embryos have Status as Human
Life with Intrinsic Value
Finally, when mental life has begun and flourishes, at the
process of internal personal creation and judgment by
which a person will make and remake himself, it will be
the most powerful and inevitable source of empathy and
communion we have with every other creature.
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Embryos have the Status
of Mere Body Parts
Some might hold that embryos are merely parts of other
people's bodies until they reach a certain autonomous or
independent developmental stage.

Accordingly, embryos have no independent moral status at
all, and are merely the property of the people from whose
body they came. The only respect due to embryos is the
respect that should be accorded other people's property.
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Question to consider:
Is it ethical to delay research

using embryonic stem cells until adult stem
cells are fully capable of treating any disease?
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Novel methods
Extraction of single blastomere without damaging
embryo and developed into independent hESC
lines

Chung et al ,Nature 2006; 439:216-19

Altered Nuclear Technique (ANT)
genetically modifying the somatic nucleus so that
induced pluripotent stem cells are produced

meissener & Jaenisch Nature 2006;439:212-15
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First clinical trial using hESC
GERON Co, 2005-2006


Aldhous, Nature 2005;434:94-6
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Existing lines of hESC
414 lines are available in 20 countries

Characterization of these lines is limited
- only 49% of lines are published in peer
reviewed journals

Guhr et al Stem cells 2006;24:2187-91
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Maintenance of hESC
Immortal cells in undifferentiated state

Optimal growth conditions for future therapeutic
applications

Majority of hESC have been isolated and
maintained in fetal calf serum and mouse
embryonic fibroblasts as feeder cell layers
(Xenoproteins and xenosupports)
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Problems
Transmission of interspecies virus transfer

Incorporation of foreign sugar molecule to
hESC leading to immune response

It may also lead to impairment of cell
function & tissue development

Varki Am J Phys Anthropol Suppl 2001
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Research focus is on.
Use of human components instead of animal
sources to avoid zoonosis

Fetal, adult muscle tissue, skin, Fallopian tube,
endometrium, foreskin cells

Successful undifferentiated growth of hESC using
xenobiotic free feeder system

Richards ,Stem cells 2003;21:546-56
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Differentiation
Direct hESC to differentiate into specific cell lines

- drug development
- cell replacement therapeutics

Specific germ cell layers can be directed by
adding specific growth factors




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Scientific Stem Cell Challenges
Stem cells represents a very small fraction of cells in
tissue.
Isolate a small number of stem cells (finding a needle in a
haystack).
Expand the number of stem cells for research and clinical
applications.
Maintain genetic stability in culture and in recipient.
Culture media has to be free of animal protein.
Deliver cells to tissue of interest.
Stem cells have to be functional.
Avoid or restrict tissue rejection.

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Challenges to Stem Cell/Cloning
Research
differentiation to the
appropriate cell
type(s) before using
clinically.

Recently, chromosome
abnormalities 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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Beware
Private companies promising wonders with stem
cells - mostly in countries with unregulated laws

Ilic D, Regenerative med 2006;1:1-4

UKRAINE capital of ESC ?
but the references they quote do not have
international authenticity in peer reviewed
journals
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Stem Cell Research Legislation
Governments around the globe have passed legislation to
regulate stem cell research. In the United States, laws
prohibit the creation of embryos for research purposes.
Scientists instead receive "leftover" embryos from fertility
clinics with consent from donors. Most people agree that
these guidelines are appropriate.

Disagreements surface, however, when political parties
debate about how to fund stem cell research. The federal
government allocates billions of dollars each year to
biomedical research. Legislators have had the unique
challenge of encouraging advances in science and
medicine while preserving a respect for life.




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Stem Cell Research Legislation
U.S. President Bush, for example, limited federal funding
to a study of 70 or so hES cell lines back in 2001. While
this did slow the destruction of human embryos, many
believe the restrictions set back the progress of stem cell
research.

President Obama overturned Bush's stem cell policy in
2009 to expand the number of stem cell lines available to
researchers.





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2009 GUIDELINES ON HUMAN STEM CELL RESEARCH
National Institutes of Health Guidelines on
Human Stem Cell Research

On March 9, 2009, President Barack H. Obama issued
Executive Order 13505: Removing Barriers to Responsible
Scientific Research Involving Human Stem Cells. The
Executive Order states that the Secretary of Health and
Human Services, through the Director of NIH, may
support and conduct responsible, scientifically worthy
human stem cell research, including human embryonic
stem cell (hESC) research, to the extent permitted by law.

This helps ensure that NIH-funded research in this area is
ethically responsible, scientifically worthy, and conducted
in accordance with applicable law.
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2009 GUIDELINES ON HUMAN STEM CELL
RESEARCH
National Institutes of Health Guidelines on
Human Stem Cell Research

These guidelines are based on the following
principles:
Responsible research with hESCs has the potential to
improve our understanding of human health and illness
and discover new ways to prevent and/or treat illness.
Individuals donating embryos for research purposes
should do so freely, with voluntary and informed
consent.

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The Stem Cell Debate: Is it over?
New breakthroughs may soon
bring this debate to an end.
Scientists have larned how to
stimulate a patient's own cells to
behave like embryonic stem cells.
These so-called induced
pluripotent stem (iPS) cells are
reducing the need for human
embryos in research and opening
up exciting new possibilities for
stem cell therapies.

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THE STORY OF IPS CELLS

In 2007, scientists were able to turn a
differentiated cell back into a stem cell
with the potential to become any type of
cell in the body.

The difference between a stem cell and a
differentiated cell is reflected in the cells'
DNA. In a stem cell, the DNA is arranged
loosely, with its genes ready to spring
into action. As signals enter the cell and
differentiation begins, genes that will not
be needed are shut down, and genes that
will be required for a specialized function
remain open and active.





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By introducing a cocktail of 24 different genes, the
scientists were able to convert differentiated cells into stem
cells. They gradually eliminated genes from the mixture,
and in the end they were able to turn differentiated cells
into stem cells by activating just 4 genes. These genes
appear to be remodeling the cells' DNA, unlocking the
genes that were shut down during differentiation.

Armed with the ability to reverse the differentiation
process, scientist are exploring new ways to use stem cells
in research and medicine.

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Induced Pluripotent Stem Cells
Induced pluripotent stem cells
(iPSCs) are adult cells that have
been genetically reprogrammed
to an embryonic stem celllike
state by being forced to express
genes and factors important for
maintaining the defining
properties of embryonic stem
cells
Human iPSCs also express stem
cell markers and are capable of
generating cells characteristic of
all three germ layers.
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With iPS cells now available
as an alternative to hES cells,
the debate over stem cell
research is becoming
increasingly irrelevant. But
ethical questions regarding
hES cells may not entirely go
away.
Inevitably, some human
embryos will still be needed
for research. iPS cells are not
exactly the same as hES cells,
and hES cells still provide












important controls: they
are a gold standard against
which the "stemness" of
iPS cells is measured.


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Some experts believe it's
wise to continue the study
of all stem cell types,
since we're not sure yet
which one will be the
most useful for cell
replacement therapies.
An additional ethical
consideration is that iPS
cells have the potential to
develop into a human
embryo, in effect
producing a clone of the
donor. Many nations are
already prepared for this,
having legislation in place
that bans human cloning
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GENE CLONING
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Cloning
Different processes for duplicating biological
material.
Diff types = technologies can be used for other
purposes besides producing the genetic twin of
another organism.
1. Embryo cloning,
2. Reproductive cloning
3. Therapeutic cloning.
Recombinant DNA Technology or DNA
Cloning
1970s = common practice in MB labs

Recombinant DNA technology," "DNA cloning,"
"molecular cloning or "gene cloning

Transfer of a DNA fragment of interest from one
organism to a self-replicating genetic element
eg. bacterial plasmid.

The DNA of interest can then be propagated in
a foreign host cell.

Scientists studying a particular gene often use
bacterial plasmids to generate multiple copies of
the same gene.
How?
DNA fragment containing
the gene of interest is cut
from chromosomal DNA
using RE.
United with a plasmid that
has been cut with the same
RE.
Fragment of chromosomal
DNA + cloning vector =
"recombinant DNA
molecule."
The recombinant DNA can
then be reproduced along
with the host cell DNA.
Reproductive Cloning
Adult DNA cloning
This technique which is intended to produce a duplicate of
an existing animal.
It has been used to clone a sheep and other mammals.
The DNA from an ovum is removed and replaced with the
DNA from a cell removed from an adult animal. Then, the
fertilized ovum, now called a pre-embryo, is implanted in a
womb and allowed to develop into a new animal.
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Reproductive Cloning
As of 2002-Jan, it had not been tried on humans. It is
specifically forbidden by law in many countries.

It has the potential of producing a twin of an existing
person.

Based on previous animal studies, it also has the potential
of producing severe genetic defects.

Many medical ethicists consider it to be a profoundly
immoral procedure when done on humans.
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Reproductive cloning
Technology used to generate an animal that has the same
nuclear DNA as another currently or previously existing
animal.

Somatic cell nuclear transfer (SCNT), = transfer of GM
from the nucleus of a donor adult cell to an egg whose
nucleus, and GM has been removed.

The reconstructed egg containing the DNA from a donor
cell must be treated with chemicals/ electric current to
stimulate cell division.
Once the cloned embryo reaches a suitable stage, it is
transferred to the uterus of a female host where it continues
to develop until birth.
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Somatic Cell Nuclear Transfer (SCNT)
technique used in CLONING
May be ethically acceptable as you are not using embryos by conventional
methods

Dolly the sheep
The first mammal to be cloned
was put down by lethal injection
Feb. 14, 2003.
Prior to her death, Dolly had
been suffering from lung cancer
and arthritis.
Studies showed that telomeres
are shortened, a phenomenon
that is associated with cellular
aging
Reproductive cloning
The possibility of human cloning, raised when
Scottish scientists at Roslin Institute created Dolly
(Nature 385, 810-13, 1997),
Since Dolly, sheep, goats, cows, mice, pigs, cats,
and rabbits all using nuclear transfer technology.
Attempts at cloning certain species such as
monkeys, chickens, horses, and dogs, have been
unsuccessful as some species
some may be more resistant to somatic cell
nuclear transfer than others.
Process can be traumatic
How to utilize repro cloning
Can be used to develop efficient ways to
reproduce animals with special qualities. Mass
production of , drug-producing animals or
animals that have been genetically altered to
serve as models for studying human disease.

Could be used to repopulate endangered
animals or animals that are difficult to breed. Eg.
In 2001, the first clone of an endangered wild
animal was born, a wild ox called a gaur. The
young gaur died from an infection about 48
hours after its birth.

In 2001, scientists in Italy reported the successful
cloning of a healthy baby endangered wild
sheep, now living in Sardinia.

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Problems of repro cloning
Reproductive cloning is expensive and highly inefficient.
> 90% of cloning attempts fail to produce viable offspring.
> 100 nuclear transfer procedures could be required to
produce one viable clone.
Cloned animals tend to have compromised immune
function and higher rates of infection, tumor growth, and
other disorders.
Japanese studies have shown that cloned mice live in
poor health and die early.
1/3 calves born alive have died young, and many of them
were abnormally large.
Dont live long enough to generate good data.
In 2002, researchers at Cambridge reported that the genomes
of cloned mice are compromised = certain of genes function
abnormally.
Therapeutic Cloning
Biomedical cloning/ Research Cloning

It involves the process of somatic cell nuclear transfer in
which the nucleus of a cell from a human patient's body is
injected into a human ovum which has had its nucleus
removed. The pre-embryo dies in the process

The goal of therapeutic cloning is to develop organs for
transplant that have an identical DNA structure to the
organ recipient. It does not involve the attempt to create a
newborn.

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Human cloning:
the most controversial debate of the decade.

Aroused worldwide interest and concern
because of its scientific and ethical implications

Is it morally acceptable?
Questions to be raised
Is self-engineering acceptable?
Will failures, eg. deformed offspring, be
acceptable?
Will cloning lead to designer babies ?
Who is socially responsible for cloned humans?
Rights and legal protection?

Opinions
95% public say no to cloning,
but 95% of scientists say yes
Reasons for cloning

Provide valuable research
Answer to infertility?
Scenario 1
Scenario 1: A husband and wife who
wish to have children but both are
carriers of a lethal recessive gene.

Rather than risk the one in four
chance of conceiving a child who
will suffer a short and painful
existence
Scenario 2
Scenario 2 : Parents of a terminally ill
child are told that only a BMT can
save the child's life:

With no donor available, parents
attempt to clone a human being
from cells of the dying child. If
successful, the new child could be a
match for BMT
Concerns of human cloning
Act of human arrogance = Playing God
Safety concerns = Due to the inefficiency
of animal cloning (1-2 viable offspring for
every 100 experiments) and the lack of
understanding about reproductive
cloning = unethical to attempt to clone
humans.
Technology only in animals, in men =
possibility of mutation/ bio damage?
Individuality and Uniqueness = Fear
that a clone would not be an
individual but merely a carbon
copy.
Would it have a soul?
Clone would be constantly
compared = burdened with
oppressive expectations.


Quotations on reproductive cloning
(creating duplicate humans):

Conservative position: "...scientists who envision medical
breakthroughs using stem cells from human embryos are
now moving on to human cloning -- breeding people for
the purpose of harvesting their tissues and organs from
their bodies, then disposing of them."


Liberal position: "Human cloning allows man to fashion
his own essential nature and turn chance into choice. For
cloning's advocates, this is an opportunity to remake
mankind in an image of health, prosperity, and nobility; it
is the ultimate expression of man's unlimited potential."
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Quotations on therapeutic cloning (creating
human organs for transplanting):

Conservative position: "Cloning, even so-called
therapeutic or experimental cloning, creates a new life
without a father, and reduces a mother to the provider of
an almost emptied egg. Nonetheless, it is a new human life
and the determination to destroy it and limit its use to
scientific research for therapeutic ends compound further
the moral issues rather than protect mankind. As such,
cloning embryonic human life under any circumstance
crosses an ethical line, takes an irrevocable step, from
which science can never turn back."
3


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Quotations on therapeutic cloning (creating
human organs for transplanting):

Liberal position: "Therapeutic cloning will in time allow
scientists to create organs that are a perfect match for
those in need of a transplant. The cloned organ would be
based on the recipients genetic material and would not
require the use of debilitating immunosuppressive
therapies. There would also be no chance of rejection,
which is fatal. Therapeutic cloning represents the ideal in
organ transplantation, as it would provide an unlimited
source of organs to anyone who needs them. The need for
these organs is dire."
4


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Religious Objections
The Roman Catholic Church, under the papacy of Benedict
XVI, has condemned the practice of human cloning, in the
magisterial instruction Dignitas Personae, stating that it
represents a "grave offense to the dignity of that person as
well as to the fundamental equality of all people".

Sunni Muslims consider human cloning to be forbidden by
Islam.The Islamic Fiqh Academy, in its Tenth Conference
proceedings, which was convened in Jeddah, Saudi Arabia
in the period from June 28, 1997 to July 3, 1997, issued a
Fatw stating that human cloning is haraam (prohibited by
the faith).

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Thank you for listening!
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