Stem Cells

1
Stem Cells
There is a growing gulf between what medicine can
do and what the health service can
afford. Richard Gardner, chair of the Royal
Societys working group on stem cells and
therapeutical cloning

• 2014
• Marek Vácha

2
Stem Cell

• 1. It is not itsef terminally differentiated
  (that is, it is not at the end of a pathway of
  differentiation)
• 2. It can divide without limit (or at least for
  the lifetime of an animal)
• 3. When it divides, each daughter has a choice
  it can either remain a stem cell, or it can
  embark a course that commits it to terminal
  differentiation.
• (Alberts, B., Jonnson, A., Lewis, J., et al.
  (2002) Molecular Biology of The Cell. 4th ed.
  Garland Science. New York.)

3
The image created by Conrad Waddington to
represent the epigenetic landscape. The position
of the ball represents different cell fates. C.H.
Waddington Organisers genes. Cambridge
University Press, 1940
4
Embryonic Stem Cells

• were firstly isolated from mice in1981.
• Human Embryonic Stem Cells were firstly isolated
  in 1998 by
• James Thomson (Wisconsin university funded
  by Geron corporation).
• Dr. John Gearhart of Johns Hopkins University

James A. Thomson says he thought long and hard
before doing stem cell research.
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Embryonic Stem Cells
ES cells that have proliferated in cell culture
for 6 or more months without differentiating, and
which have pluripotent and appear genetically
normal, are referred to as an embryonic stem cell
line.
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• Thomson and Gearhart, using different approaches,
  had isolated these very early precursor cells and
  spread them out on a feeder layer of mouse cells
  to produce an immortalized pluripotent human stem
  cell culture.

James Thomson (left) and John Gearhart (right)
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James A. Thomson (2007)

• His laboratory was one of two that reported a new
  way to turn ordinary human skin cells into what
  appear to be embryonic stem cells without ever
  using a human embryo.
• The fact is, Dr. Thomson said in an interview, he
  had ethical concerns about embryonic research
  from the outset, even though he knew that such
  research offered insights into human development
  and the potential for powerful new treatments for
  disease.
• If human embryonic stem cell research does not
  make you at least a little bit uncomfortable, you
  have not thought about it enough, he said. I
  thought long and hard about whether I would do
  it.
• (New York Times, 22. 11. 2007)

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iPS - Induced pluripotent stem cells
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iPS

• induced pluripotent stem cells
• by addition of four human human genes to human
  skin cells they can be reprogrammed back to the
  state of stem cells from an early embryo, with
  the ability to develop into every cell type in
  the body

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iPS

• iPS thus promise to provide a source of
  immunologically compatible tissues for treating
  patients currently suffering from incurable
  genetic diseases such as Alzheimers disease,
  without encountering the ethical objections
  raised by using ESC from human embryos or cybrids

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• In the long-term the scientific view is that it
  will be possible to re-programme adult stem cells
  with the full potential of embryonic cells but
  without the morally contestable need to create an
  embryo.
• Liam Donaldson, the Chief Medical Officer in the
  UK

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James A. Thomson (2007)

• Now with the new technique, which involves adding
  just four genes to ordinary adult skin cells, it
  will not be long, he says, before the stem cell
  wars are a distant memory. A decade from now,
  this will be just a funny historical footnote,
  Dr. Thomson said in the interview.
• http//www.nytimes.com/2007/11/22/science/22stem.h
  tml?_r1refscienceorefslogin

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2012 Shinya Yamanaka wins Nobel Prize
Shinya Yamanaka and John Gurdon
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Shinya Yamanaka

• originally trained as a clinician and became an
  orthopaedic surgeon
• a goal to find a way of creating pluripotent
  cells from differentiated cells in a lab.
• He started this stage of his work with a list of
  24 genes which were vitally important in ES cells

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• Yamanaka decided to test if combinations of these
  genes would drive differentiated cell backward to
  a more primitive developmental stage
• they worked with mice
• using neoR gene which got switched on only if the
  cell it was in had become pluripotentn
• Oct4, Sox2,Klf4,c-Myc
• Its incredible to think that mammalian cells
  carry about 20 000 genes, and yet it only takes
  four to turn a fully differentiated cell into
  something that is pluripotent.

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• Shinya Yamanaka with his postdoctoral research
  associate Kazutoshi Takahashi

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2012 Shinya Yamanaka wins Nobel Prize

• "He deserves not only a Nobel Prize for Medicine,
  but a Nobel Prize for Ethics."

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2012 Shinya Yamanaka wins Nobel Prize

• "He deserves not only a Nobel Prize for Medicine,
  but a Nobel Prize for Ethics.
• (Julian Savulescu, Uehiro Chair in Practical
  Ethics Director, Oxford Uehiro Centre for
  Practical Ethics)
• This is a rare example of a scientific discovery
  that may solve more ethical problems than it
  creates. Many ethical objections to stem cell
  research have focused on the need to destroy
  human embryos. iPS cell technology may ultimately
  enable scientists to evade these objections by
  deriving pluripotent stem cells from adult
  tissue. For the moment, though, iPS cell research
  will need to run parallel to research with
  embryonic stem cells.
• (Dr Tom Douglas, Wellcome Trust Research
  FellowOxford Uehiro Centre for Practical
  EthicsUniversity of Oxford)

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• In the long-term the scientific view is that it
  will be possible to re-programme adult stem cells
  with the full potential of embryonic cells but
  without the morally contestable need to create an
  embryo.
• Liam Donaldson, the Chief Medical Officer in the
  UK

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• More recently, however, several research groups
  have uncovered differences between iPS and ES
  cells in gene expression and other cellular
  functions, such as cell divisions.
• At least until these differences are fully
  understood, the stuidy of ES cells will continue
  to make important contributions to the
  development of stem cell therapies
• in fact, ES cells will likely always be a focus
  of basic research as well
• (Reece, J.B., Urry, L.A., (2011) Campbell
  Biology. 9th. ed. Pearson Publication, Inc. , New
  York. p. 462)

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iPS cells potential uses

• cells from patients suffering from diseases can
  be reprogrammed to become iPS cells, which can
  act as model cells for studying the disease and
  potential treatments. Human iPS cell lines have
  already ben developed frma indeviduals with type
  1 diabetes, Parkinsons disease, and at least a
  dozen other diseases
• a patients own cells could be reprogrammed into
  iPS cells and then used to replace nonfunctional
  tissues

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This technique has already been used succesfully
to treat sickle-cell disease in a mouse that had
been genetically engineered to have the disease
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Classification

• (ESC)Human embryonic stem cells,
• Embryonic Stem Cell (ESC) Therapy - theoretically
  involves the use of cells extracted from a five
  day old in vitro fertilized embryo (in this
  embryo there is approximately 100 cells, ICM is
  30 of them). ESCs have been scientifically
  substantiated to be pluripotent for all cell
  types.
• (FSC)Human embryonic germ cells,
• Fetal Stem Cell (FSC) therapy involves the use of
  human fetuses aborted between 1 and 3 months.
  Tissue with imbedded stem cells is scraped from
  the liver, neural or gonadal ridges. Commercial
  providers of these potentially pluripotent cell
  types do not scientifically classify the cell
  populations.
• (ASC)Human Adult stem cells,
• ASC are undifferentiated, found among
  differentiated cell types in a tissue or organ.
  ASC can renew themselves, and can differentiate
  to yield the major specialized cell types of the
  tissue or organ. Their primary role in a living
  organism is to maintain and repair the tissue in
  which they are found. There are approximately 20
  types of ASC in the human body.

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Classification

• totipotent cells
• pluripotent stem cells
• pluripotent "capable of becoming many things"
• multipotent stem cells
• progenitor (unipotent) stem cells
•  can produce only one cell type

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• each of early blastomeres (2,4,8 cell stage) is
  thought to be able to form an entire embryo,
  including the trophoblast
• isolated ICM cells probably cannot form the
  trophoblast cells

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ASC
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Embryonic Stem Cells
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Embryonic Stem Cells
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Possible Sources of Embryonic Stem Cells

• Surplus embryos
• Embryos made exclusively in order to receive ESC
• SCNT Therapeutic Cloning

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Dr. Woo Suk Hwang

• 24/11 2005 Dr. Hwang is forced to
  to step down
• female researchers in his own lab had supplied
  eggs for his research.
• two key scientific papers in 2005, both published
  in Science Magazine, were found to have been
  fabricated.

August 2005 Snuppy, an Afghan hound was cloned by
Dr. Hwang
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Dr. Hwang

• Dr Hwang was in March fired from his
  professorship at Seoul National University (SNU)
  and in May was charged with fraud and
  embezzlement. Dr Hwang had received millions of
  dollars' worth of funds from the state and
  private foundations for his research.
• The downfall of Dr Hwang came as a big shock to
  Koreans, who had taken great pride in what
  appeared to be the pioneering work of the stem
  cell researchers at Seoul National University.
• Dr Hwang at one point had 15,000 hard-core fans,
  who belonged to a "I love HWS" online community.

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Stem cells mimic human brainThe big surprise
was that it worked

• The blobs grew to resemble the brains of fetuses
  in the ninth week of development.
• the pea-sized neural clumps developed in this
  work could prove useful for researching human
  neurological diseases.
• The tissue balls lacked blood vessels, which
  could be one reason that their size was limited
  to 34 millimetres in diameter, even after
  growing for 10 months or more.

http//www.nature.com/news/stem-cells-mimic-human-
brain-1.13617
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Offspring from Oocytes Derived from in Vitro
Primordial Germ Celllike Cells in Mice

• Katsuhiko Hayashi, November 2012
• We show here that female (XX) embryonic stem
  cells and induced pluripotent stem cells in mice
  are induced into primordial germ celllike cells
  (PGCLCs), which, when aggregated with female
  gonadal somatic cells as reconstituted ovaries,
  undergo X-reactivation, imprint erasure, and cyst
  formation, and exhibit meiotic potential. Upon
  transplantation under mouse ovarian bursa, PGCLCs
  in the reconstituted ovaries mature into germinal
  vesicle-stage oocytes, which then contribute to
  fertile offspring after in vitro maturation and
  fertilization.

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legislativa

• Užitecný odkaz http//www.eurostemcell.org/stem-c
  ell-regulations

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history - USA
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History - USA

• 1975 The National Commission for the Protection
  of Human Subjects of Biomedical and Behavioral
  Research issued its report and recommendations
  for research on the human fetus
• no federal monies should be used for IVF of human
  eggs until a special Ethics Advisory Board
  reviewed the ethical issues
• 1994 NIH established the Human Embryo Research
  Panel
• some areas of human embryo research be acceptable
  for federal funding, including research on
  embryos created expressly for the purposes of
  research
• however, president Clinton directed NIH not to
  allocate resources to "support the creation of
  human embryos for research purposes"
• tough his directives said nothing about reseach
  involving spare embryos

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History - USA

• 1994
• Congress stopped the enterprise dead in its
  tracks by enacting an amendment to the omnibus
  appropriations bills that prohibited NIH from
  using federal funds for any and all research on
  human embryos.
• 1998
• NIH attorneys ruled that the prohibiting law
  actually permitted federal funding of research on
  the embryonic stem cell lines, provided that the
  researchers were not themselves responsible for
  the acts of embryo destruction needed to produce
  them.

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History - USA

• 2000
• After a study by the National Bioethics Advisory
  Commission supported such research, and after
  the NIH developed guidelines for it, President
  Clinton authorized such funding
• august 2001
• The federal government would agree to fund
  embryonic stem cell research only on already
  existing stem cell lines, but there would be any
  further destruction of human embryos

42
USA

• August 2000 scientists could apply for federal
  funding only for research utilizing 78 existing
  stem cell lines.
• the true number of available and suitable lines
  appears to be closer to twenty than the higher
  number.
• President Bush agreed to finance embryonic stem
  cell research, but limited federally financed
  research to 21 cell lines already in existence by
  2001.
• Kass, L.R., (2002) Life, Liberty and the Defense
  of Dignity. Encounter Books, New York, London. p.
  81 - 85

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USA

• the restriction do not hamper the use of private
  funds for the other lines, only federal funds
• 09/03/2009 president Obama releases federal
  funds
• http//edition.cnn.com/video//video/offbeat/2009/
  03/12/moos.sesame.street.layoffs.cnn
• 23/08/2010 A federal district judge blocked
  President Obamas 2009 executive order that
  expanded embryonic stem cell research, saying it
  violated a ban on federal money being used to
  destroy embryos.

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USA 23/08/2010

• With the case back in his court, Judge Lamberth
  ruled that the administrations policy violated
  the clear language of the Dickey-Wicker
  Amendment, a law passed annually by Congress that
  bans federal financing for any research in which
  a human embryo or embryos are destroyed,
  discarded or knowingly subjected to risk of
  injury or death.
• The Obama administration said that its rules
  abided by the Dickey-Wicker Amendment because the
  federal money would be used only once the
  embryonic stem cells were created but would not
  finance the process by which embryos were
  destroyed. The judge disagreed, writing that
  embryonic stem cell research necessarily depends
  upon the destruction of a human embryo.
• http//www.nytimes.com/2010/08/24/health/policy/24
  stem.html?pagewanted2_r1refscience

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USA 29/04/2011

• the appellate court said that because the law is
  written in the present tense, it does not extend
  to past actions.
• Judge Karen LeCraft Henderson, the dissenter in
  Fridays appeals court ruling, wrote that her
  colleagues perform linguistic jujitsu to arrive
  at their conclusion.
• The plain language of the law bans financing for
  all research that follows the destruction of
  embryos, she wrote, and it is meaningless to try
  to separate the process of destruction from the
  use of the stem cells that result from that
  destruction.
• http//www.nytimes.com/2011/04/30/health/30stemcel
  ls.html

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europe
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Research on embryos in vitro?Oviedo 1997

• Convention for the Protection of Human Rights and
  Dignity of the Human Being with regard to the
  Application of Biology and Medicine Convention
  on Human Rights and Biomedicine
• Oviedo, 4.IV.1997
• 1Article 18 Research on embryos in vitro
• Where the law allows research on embryos in
  vitro, it shall ensure adequate protection of the
  embryo.
• The creation of human embryos for research
  purposes is prohibited.

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Germany

• The German Parliament established the 2002 Stem
  Cell Act, which allows the import of hESCs for
  high-ranking research objectives.
• These must be evaluated by the Robert Koch
  Institute, a federal institute in Berlin, and its
  central ethics committee for stem-cell research
  (http//www.rki.de).
• Moreover, only hESC lines produced from surplus
  embryos from in vitro fertilization (IVF) before
  1 January 2002 can be legally imported. This key
  date was chosen to ensure that no hESC lines are
  directly produced for German research in other
  words, that no human embryos are destroyed 'on
  German order'

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Regulation of Research in  EU

• Germany prohibition to destroy embryos for
  obtaining hES. Allowance to work with hES if the
  lines were created before 01/01 2002
• 22/05 2008 this import and usage was shifted to
  01/05 2007

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Case C-34/10 Oliver Brüstle v Greenpeace
http//eur-lex.europa.eu/LexUriServ/LexUriServ.do?
uriCELEX62010CJ0034ENHTML

• Article 6
• 1.      Inventions shall be considered
  unpatentable where their commercial exploitation
  would be contrary to ordre public or morality
  however, exploitation shall not be deemed to be
  so contrary merely because it is prohibited by
  law or regulation.
• 2.      On the basis of paragraph 1, the
  following, in particular, shall be considered
  unpatentable
• (c)      uses of human embryos for industrial or
  commercial purposes

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Germany Case C-34/10 Oliver Brüstle v Greenpeace
http//eur-lex.europa.eu/LexUriServ/LexUriServ.do?
uriCELEX62010CJ0034ENHTML

• Under Paragraph 8(1) of the ESchG, an embryo is a
  fertilised human ovum capable of development,
  from the time of karyogamy, and any cell removed
  from an embryo which is totipotent, that is to
  say, able to divide and develop into an
  individual provided that the other conditions
  necessary are satisfied.
• A distinction must be made between those cells
  and pluripotent cells, which are stem cells
  which, although capable of developing into any
  type of cell, cannot develop into a complete
  individual.

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Germany Case C-34/10 Oliver Brüstle v Greenpeace
http//eur-lex.europa.eu/LexUriServ/LexUriServ.do
?uriCELEX62010CJ0034ENHTML http//

•   According to the referring court, having regard
  to the fact that Article 6(2) of the Directive
  does not allow the Member States any discretion
  as regards the fact that the processes and uses
  listed therein are not patentable (see Case
  C-377/98 Netherlands v Parliament and
  Council 2001 ECR I-7079, paragraph 39, and Case
  C-456/03 Commission v Italy 2005 ECR I-5335,
  paragraph 78 et seq.), the reference made in the
  second sentence of Paragraph 2(2) of the PatG to
  the ESchG, particularly to the definition of an
  embryo which Paragraph 8(1) of that Law gives,
  cannot be regarded as the fruit of the task left
  to Member States to put Article 6(2)(c) of the
  Directive into concrete terms in that regard,
  even though the Directive did not expressly
  define the concept of embryo. The only possible
  interpretation of that concept is European and
  unified. In other words, the second sentence of
  Paragraph 2(2) of the PatG and, in particular,
  the concept of embryo which it uses cannot be
  interpreted differently from that of the
  corresponding concept in Article 6(2)(c) of the
  Directive.

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Europa hESCs cannot be patented

• In the light of the foregoing considerations, the
  answer to the first question is that 
•         any human ovum after fertilisation, any
  non-fertilised human ovum into which the cell
  nucleus from a mature human cell has been
  transplanted and any non-fertilised human ovum
  whose division and further development have been
  stimulated by parthenogenesis constitute a human
  embryo within the meaning of Article 6(2)(c) of
  the Directive
•         it is for the referring court to
  ascertain, in the light of scientific
  developments, whether a stem cell obtained from a
  human embryo at the blastocyst stage constitutes
  a human embryo within the meaning of Article
  6(2)(c) of the Directive.

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Argumnets for and Against
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Definition of Human Embryo

• A human embryo is a discrete entity that has
  arisen from either
• (i) the first mitotic division when fertilization
  of a human oocyte by a human sperm is complete or
• (ii) any other process that initiates organized
  development of a biological entity with a human
  nuclear genome or altered human nuclear genome
  that has the potential to develop up to, or
  beyond, the stage at which the primitivestreak
  appears,
• and has not yet reached 8 weeks of development
  since the first mitotic division.

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Arguments for obtaining Embryonic Stem Cells from
embryos

• The prospects are beyond our scope
• Only 30-50 zygote implants. If Mother Nature is
  so prodigal, we may not to bother with embryos.
• individual is in-dividium, non-divisible.
• Human embryo is nothing than cluster of cells
• if we approved abortions, there is no discussion
  about status of embryos
• utilitarianism we have to consider the ethical
  calculation, gains and losts happy and healthy
  patient/destroyed embryo

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Arguments for obtaining Embryonic Stem Cells from
embryos

• If abortions are legal, protection of embryos is
  inproportionate, inadequate
• ES cells are not embryos. These cells are no
  more totipotent. If we already have these lines,
  they are cells, not embryos.
• individuum is etymologically indivisible
• MZ twins could arise until the end of the second
  week. Until this time we cannot speak about
  individuum

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Arguments for obtaining Embryonic Stem Cells from
embryos

• Argument of utilitarianism on the one side of
  the ethical scale is healthy happy patient, on
  the other side of the scale embryo which can not
  feel neither pain, nor pleasure

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Arguments against obtainig Human Embryonic Stem
Cells

• Embryo is not, embryos are. Every embryo has a
  unique genetic design
• An Embryo has a potentiality to become an adult
  human person, so we must treat an embryo as a
  person.
• every embryo has two living beings, parents
• Aristotle four causes
• Immanuel Kant
• the problem of freedom
• difference between cost/value and dignity

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Aristotle

• causa materialis
• causa formalis
• causa efficiens
• causa finalis

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Aristotle

• causa materialis cluster of atoms, cluster
  of cells, C,H,O,N, etc.
• causa formalis form of living matter is a soul
• causa efficiens where from I came?
• causa finalis what is goal of my life?

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Cluster of cells Five day old embryo is
composed from150 200 cells, adult human from 5.
1014 cells
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http//www.nytimes.com/2008/12/04/us/04embryo.html
?_r1
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• Ms. Best said her nine embryos have the
  potential to become beautiful people.
• The thought of giving them up for research
  conjures all sorts of horrors, from Frankenstein
  to the Holocaust, she said, adding that
  destroying them would be preferable.
• Her teenage daughter favors letting another
  couple adopt the embryos, but, Ms. Best said, she
  would worry too much about what kind of parents
  they were with, what kind of life they had.

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Solutions that typically are not offered

• Smaller numbers of patients wished for solutions
  that typically are not offered.
• Among them were holding a small ceremony during
  the thawing and disposal of the embryos, or
• having them placed in the womans body at a time
  in her cycle when she would probably not become
  pregnant, so that they would die naturally.

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• Some people pay storage fees for years and years.
• Others stop paying and disappear, leaving the
  clinic to decide whether to maintain the embryos
  free or to get rid of them.
• They would rather have you pull the trigger on
  the embryos,
• Its like, I dont want another baby, but I
  dont have it in me I have too much guilt to
  tell you what to do, to have them discarded. 

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SCNT

• this technique is too elaborate, and hence too
  expensive for routine use
• technique might be more properly called research
  cloning

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• In 2001, research group at a biotechnology
  company in Massachusetts observed a few early
  cell divisions in such an experiment
• A few years later, researchers at Seoul national
  University, in South Korea, reported cloning
  embryos to the blasocyst stage...
• ...but the scientists were later found guilty of
  research misconduct and data fabrication.

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Reproductive and Therapeutic Cloning
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Cybrids

• applications have been made by three teams of UK
  researchers to the HFEA for licences to use
  rabbit, cow and goat eggs to create cytoplasmatic
  hybrid embryos, so-called cybrids
• the proposal, which has found widespread support
  in the bioscience community, was the subject of a
  public consultation exercise, which resulted in
  the HFEA giving in principle approval for the
  use of the technique in 2007

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Cybrids
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Many thanks Marek Vácha