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Stem Cells

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Title: 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.
5
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.
6
  • 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)
7
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)

8
iPS - Induced pluripotent stem cells
9
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

10
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

11
  • 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

12
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13
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

14
2012 Shinya Yamanaka wins Nobel Prize
Shinya Yamanaka and John Gurdon
15
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

16
  • 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.

17
  • Shinya Yamanaka with his postdoctoral research
    associate Kazutoshi Takahashi

18
2012 Shinya Yamanaka wins Nobel Prize
  • "He deserves not only a Nobel Prize for Medicine,
    but a Nobel Prize for Ethics."

19
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)

20
  • 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

21
  • 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)

22
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

23
This technique has already been used succesfully
to treat sickle-cell disease in a mouse that had
been genetically engineered to have the disease
24
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.

25
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26
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

27
  • 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

28
ASC
29
Embryonic Stem Cells
30
Embryonic Stem Cells
31
Possible Sources of Embryonic Stem Cells
  • Surplus embryos
  • Embryos made exclusively in order to receive ESC
  • SCNT Therapeutic Cloning

32
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33
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
34
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.

35
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
36
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.

37
legislativa
  • Užitecný odkaz http//www.eurostemcell.org/stem-c
    ell-regulations

38
history - USA
39
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

40
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.

41
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

43
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.

44
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45
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

46
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

47
europe
48
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.

49
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'

50
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

51
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

52
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.

53
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.

54
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.

55
Argumnets for and Against
56
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.

57
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

58
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

59
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

60
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

61
Aristotle
  • causa materialis
  • causa formalis
  • causa efficiens
  • causa finalis

62
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?

63
Cluster of cells Five day old embryo is
composed from150 200 cells, adult human from 5.
1014 cells
64
http//www.nytimes.com/2008/12/04/us/04embryo.html
?_r1
65
  • 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.

66
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.

67
  • 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. 

68
SCNT
  • this technique is too elaborate, and hence too
    expensive for routine use
  • technique might be more properly called research
    cloning

69
  • 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.

70
Reproductive and Therapeutic Cloning
71
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

72
Cybrids
73
Many thanks Marek Vácha
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