1. 2014 Marek Vácha There is a growing gulf between what medicine can do and what the health service can afford. Richard Gardner, chair of the Royal Society´s working group on stem cells and therapeutical cloning

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)

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 Alzheimer´s 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

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.html?_r=1&ref=science&or ef=slogin

14. Shinya Yamanaka and John Gurdon

15.  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  It´s 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.  "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 Fellow Oxford Uehiro Centre for Practical Ethics University 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.  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, Parkinson´s disease, and at least a dozen other diseases  a patient´s 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.

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

31. Possible Sources of Embryonic Stem Cells  Surplus embryos  Embryos made exclusively in order to receive ESC  SCNT = Therapeutic Cloning

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 brain „The 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 3–4 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 Cell–like 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 cell–like 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. Užitečný odkaz: http://www.eurostemcell.org/stem-cell-regulations

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 http://edition.cnn.com/video/#/video/offbeat/2009/03/12/moos.sesame.street.layoffs.cnn  23/08/2010 A federal district judge blocked President Obama’s 2009 executive order that expanded embryonic stem cell research, saying it violated a ban on federal money being used to destroy embryos.

45. USA 23/08/2010  With the case back in his court, Judge Lamberth ruled that the administration’s 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/24stem.html?pagewanted=2&_r=1&ref=science

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 Friday’s 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/30stemcells.html http://www.nytimes.com/2011/04/30/health/30stemcells.html

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?uri=CELEX:62010CJ0034:EN:HTML  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?uri=CELEX:62010CJ0034:EN:HTML  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?uri=CELEX:62010CJ0034:EN:HTML http:// http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:62010CJ0034:EN:HTML  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.

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 ccausa materialis ccausa formalis ccausa efficiens ccausa finalis

62. ccausa materialis: „cluster of atoms“, „cluster of cells“, „C,H,O,N,“ etc. ccausa formalis – form of living matter is a soul ccausa efficiens „where from I came?“ c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. 10 14 cells

64. http://www.nytimes.com/2008/12/04/us/04embryo.html?_r=1

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 woman’s 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,”  “It’s like, ‘I don’t want another baby, but I don’t 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