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1 Stem cell therapy: facts and myths Giuseppe Remuzzi Lyon, September 12, 2008.

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Presentation on theme: "1 Stem cell therapy: facts and myths Giuseppe Remuzzi Lyon, September 12, 2008."— Presentation transcript:

1 1 Stem cell therapy: facts and myths Giuseppe Remuzzi Lyon, September 12, 2008

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3 3 A 46-year-old man had an acute anterior myocardial infarction He had a revascularization procedure, but more than 12 h post the beginning of symptoms The occluded vessel was reopened, but the myocardial tissue was irremediably damaged CASE REPORT - 1

4 4 Because of irreversible myocardial damage he will have a diminished quality of life He is a modern individual, who is well informed about novel alternative options CASE REPORT - 2

5 5 What options?

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7 7 Adult stem cells couldnt do much more than regenerate cell types of origin

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9 9 Embryonic stem cells have the ability to self-renew and to differentiate into all three embryonic germ layers

10 10 ADULT STEM CELLS FOR CARDIAC REPAIR Heart Blood Bone marrow Fat Cardiac stem cells Endothelial progenitor cells Mesenchymal stem cells Differentiation into other cell types Paracrine effects Remodelling Angiogenesis Endogenous stem cells activation

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12 12 TEIM: TransEndocardial IntraMyocardial injection Bone marrow mononuclear cells Circulating progenitor cells Skeletal myoblasts Mesenchymal stem cells

13 13 Challenges

14 14 Issues such as purity of cells, number and whether they have to be differentiated into cardiomyocytes before transplantation have never been addressed

15 15 Is this procedure safe in the long term? To which extent these cells are retained into myocardial tissue? Is spatial distribution important?

16 16 Many scientists say that they are reluctant to undertake further clinical trials until they hear more definitive answers about the risks and benefits of adult stem cell therapies

17 17 The NEW ENGLAND JOURNAL of MEDICINE, SEPTEMBER 21, 2006 Cardiac Cell Therapy - Mixed Results from Mixed Cells Anthony Rosenzwieg, M.D. EDITORIAL Corriere della Sera, GIOVEDI, 4 APRILE 2002

18 18 In a mouse model of myocardial infarction, infused bone marrow stem cells differentiated only into blood cells, not cardiac myocytes, and they failed to contribute to myocardial regeneration The results of all studies do not promote the use of intracoronary infusions of autologous bone marrow to improve ventricular function Schwartz, N Engl J Med, 2006

19 19 ACUTE KIDNEY INJURY - Cysplatin days Mesenchymal stem cell injection BUN (mg/dl) * * Saline MSC Imberti et al., J Am Soc Nephrol, 2007


21 21 MESENCHYMAL STEM CELLS ACCELERATE TUBULAR CELL REGENERATION AFTER CISPLATIN Ki 67 positive nuclei / HPF days Cisplatin + saline Cisplatin + MSC * * Ki 67: nuclear proliferation marker Imberti et al., J Am Soc Nephrol, 2007

22 22 0 cispl+ saline cispl+ si-irrel MSC cispl+ si-IGF-1 MSC BUN (mg/dl) INSULIN-LIKE GROWTH FACTOR-1 SUSTAINS STEM CELL-MEDIATED RENAL REPAIR Imberti et al., J Am Soc Nephrol, 2007

23 23 EFFECT OF HUMAN STEM CELL TREATMENT ON CISPLATIN-INDUCED ACUTE KIDNEY INJURY IN NOD/SCID MICE Morigi et al., Stem Cells, 2008 Stem cell typesRenal functionRenal histologySurvival at 7 days Bone marrow Cord blood Amniotic fluid Protected Preserved 50 % 86 % Currently testing None of the untreated mice survived cisplatinum at 7 days

24 24 To evaluate the rate of renal function loss up to 15 days post-MSC infusion, as assessed by serial measurements of serum creatinine concentration (primary outcome variable) and of glomerular filtration rate (GFR) estimated by prediction equation THE HUMAN PILOT STUDY This is a pilot, explorative study to test the feasibility and safety of systemic infusion of donor ex-vivo expanded MSCs to repair the kidney and improve function in patients with solid organ cancers who develop acute renal failure after chemotherapy with cisplatin Primary aim Patients Start with 3 patients: (5 x 10 6 MSC, 10 x 10 6 MSC, 15 x 10 6 MSC) If safe and successful: upgrade the number to 8 patients

25 25 Frozen-thawed donated human embryos produced by in vitro fertilization were cultured to the blastocyst stage Inner cell masses were isolated by immunosurgery and plated on irradiated mouse embryonic fibroblast feeder After 9 to 15 days, inner cell mass- derived outgrowth was dissociated to obtain embryonic stem cell clumps that were replated on fibroblast feeder

26 26 Circulatory system Nervous system Immune system

27 27 A lot of people think that scientists do not respect ethical and human values and consider science as something in conflict with faith

28 28

29 29 NATURE, 11 september 2003 Science and the ethics of science are two sides of the same coin Scientists must take the lead in ensuring the the progress of science is both ethical and as free from political intervention as possible, if for no other reason than that only they can do so


31 31 In his veto message, the President explained that stem cells… can be drawn from children, adults, and the blood in umbilical cords with no harm to the donor, and these stem cells are currently being used in medical treatments

32 32 According to the New York Times, Karl Rowe, head of the White Houses Office of Political Affairs, has declared that embryonic stem cells arent required because there is far more promise from adult stem cells

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34 34 It seems that the White House received this idea from David Prentice, a senior fellow for life sciences at the Family Research Council and an advisor to Republican members of Congress In a report of the Presidents Council on Bioethics, Prentice claimed that adult stem cells can effectively treat more that 65 diseases

35 35 Not only this assertion is patently false, but the information purveyed on the Family Research Councils Web site is pure hokum Schwartz, N Engl J Med, 2006

36 36 DE HUMANI CORPORIS FABRICA Andrea Vesalius, 1543 When Andrea Vesalius first published his radical De Humani Corporis Fabrica, the ancient texts of Aristotle and Galen were still judged authoritative in the medical school of Europe By performing his own dissections, Vesalius discovered errors in the ancient authors teachings The De Humani Corporis Fabrica, which drew attention to these flaws, initially threatened the academic medical establishment but ultimately won Vesalius admiration and a post as court physician to Charles V

37 37 Smith et al., Science, 2006 DiseaseFDA approvedEffects/limitations NO YES No benefit No rigorous study Safe Feasible No or low effects chemotherapy side effects Modest effect Concern of durability of the effects Partial improvement of vision Feasible Lack of long term data Improvement of symtpoms Efficacious (n = 2 patients) ANTONIO MISSIERI Parkinsons disease Spinal cord injury Amyotrophic lateral sclerosis Multiple sclerosis Rheumatoid arthritis Corneal regeneration Osteogenesis imperfecta Thalassemia major STEM CELLS, MIRACLE OR CURE?

38 38 To support the inclusion of Parkinsons disease on his list, Prentice cites congressional testimony by a patient and a physician, a meeting abstract by the same physician, and two publications that have nothing to do with stem cell therapy for Parkinsons disease In fact, there is currently no FDA-approved adult stem cell treatments - and non cure of any kind - for Parkinsons disease

39 39 For spinal cord injury, Prentice cites personal opinions expressed in Congressional testimony by one physician and two patients There is currently no FDA-approved adult stem cell treatment or cure for spinal cord injury

40 40 By promoting the falsehood that adult stem cell treatments are already in general use for 65 diseases and injuries those who repeat his claims mislead laypeople and cruelly deceive patients Smith et al., Science, 2006

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42 42 Brain-damaged three-year-old patient was treated in a clinic of Bahamas with fetal stem cells for 25,000$ When he returned home the child began have seizures and was still unable to walk and talk Desperate patients, spending high amount of money, risk their life with offshore stem cell injections provided by doctors promising false hopes 04 September 2008 Injections of hope Doctors Promote Offshore Stem Cell Shots, but Some Patients Cry Foul

43 43 Credit: iStockphoto

44 44 The use of human embryonic stem cells in cell replacement therapies has been limited due to several technical and ethical issues There has been an extensive debate about the benefits and drawbacks of adult vs embryonic stem cell use in therapies

45 45 The way they are - derived - characterized - established - maintained In vitro differentiation MAJOR CONCERNS - 1

46 46 Epigenetic profiles Chromosomal aberrations Risk of tumors Genetic instability MAJOR CONCERNS - 2

47 47 DISEASE MODELS WHERE HUMAN EMBRYONIC STEM CELLS HAVE BEEN SHOWN TO BE EFFECTIVE (mainly SCID mice) Cell type developedAnimal modelReference Oligodendrocyte progenitors Cardiomyocytes Hepatocytes Chondrocytes Endothelial cells Neural precursors Pancreatic cells Neuroepithelial precursors and dopaminergic neurons Human embryonic stem cells Spinal cord injury Myocardial infarction Toxic-liver injury Spinal fusion Surgical induction of hind limb ischemia Quinolinic acid-induced Huntington Streptozotocin diabetes Parkinson Open neural tube defect Keirstead et al., 2005 Nakamura et al., 2005 Laflamme et al., 2007 Leor et al., 1996 Kehat et al., 2004 Caspi et al, 2007 Seo et al., 2005 Muschler et al., 2003 Cho eet al., 2007 Song et al., 2007 Shim et al., 2007 Sontag et al., 2007 Ben-Hur et al., 2004 Lee et al., 2006

48 48 Immune-rejection

49 49 Generation of patient specific human nuclear transfer embryonic stem cells lines may circumvent the problem of immuno-rejection, the greatest challenge in cell replacement therapy

50 50


52 52 Derivation of a pluripotent embryonic stem cell line from a cloned human blastocyst that displays typical embryonic stem cell morphology and cell surface markers (somatic cell nuclear transfer of 242 oocytes, 20 inner cell masses, 1 embryonic stem cell line) Human embryonic stem cells obtained from nuclear transfer maintain after 70 passages normal karyotype and are genetically identical to the somatic nuclear donor cells 12 March 2004

53 53 Corriere della Sera, 24 dicembre 2005

54 54 Mitochondrial transmission of aberrant epigenetic gene expression profiles Mitochondrial genome encodes a number of transplantation antigens that could trigger a immune response MAJOR CONCERNS

55 55 We dont know how an embryonic stem cell will behave in a human We dont known what will ultimately come out from research on embryonic stem cells


57 57 As there are no peer reviewed publications on Medline from these groups, nothing is known about how they prepare the cells, how the safety and side effects are evaluated, and how credible their claims are

58 58 There is a need to increase public awareness and to manage the public expectations for human embryonic stem cell- based therapies

59 59 Whether ethic is indeed part of science, why do not ask to scientists to solve ethical issues that they put themselves?


61 61 One possibility is to isolate cells from embryos that are not growing anymore Indeed, in nature, only a small part of fertilized embryos finds the proper conditions to develop Often, this does not happen and the embryo stops growing

62 62 This is also the case of the in vitro fecundation Seven times out of ten the embryo stops growing and is non implanted into uterus Those embryos are dead

63 63 23 Dicembre 1954, ore: 8.00 Ospedale Peter Bent Brigham di Boston

64 64 However, the death of the embryo does not mean the death of all the embryonic cells Cells isolated from dead embryos have the ability to grow in vitro. Therefore, it should be possible using them for transplantation exactly as it is done with organs obtained from cadavers

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66 66 Somehow, amniotic fluid cells are halfway between embryonic and adult stem cells In the laboratory they grow efficiently (at least in somebodys hands) and they can become cells of the muscle, nervous system and liver

67 67 The article by De Coppi et al., fails to provide any convincing evidence to support the claim that amniotic fluid-derived stem cells are able to generate neurons The evidence supports the conclusion that amniotic fluid stem cells are capable of entering the neuroectodermal lineage We agree that it remains to be proven that amniotic fluid stem cells can differentiate to yield mature neurons and did not make such a claim in our published report Toselli et al., Nature Biotechnology, 2008 De Coppi et al., Nature Biotechnology, 2008

68 68 Certainly, if it was possible to use these cells for curing diseases, ethical issues would be solved However, it would be ingenuous to drop the research on the embryonic stem cells for focusing only on the fluid amniotic cells

69 69 Human embryonic stem cell lines derived from single blastomeres Irina Klimanskaya, Young Chung, Sandy Becker, Shi-Jiang Lu & Robert Lanza August 2006, NATURE on line August 2006, Scientists from Massachusetts, led by Robert Lanza, applied a technique already used in fertilization clinics to obtain human embryonic stem cells

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71 71 When an hereditary disease is suspected, one cell can be extracted from a two-days old embryo, formed by eight-ten cells, in order to perform DNA analysis In the case no anomalies are found, the embryo is transplanted into the womans uterus and will give birth to a child

72 72 Single-cell biopsy from two-days old embryos for pre-implantation genetic diagnosis represents a routine procedure More than thousands five hundred of these interventions have been performed without interfering with the embryos potential for life Not always the embryo survives but this does not happen even for natural fecundation

73 73 From a single cell of a two-days old embryo, Robert Lanza derived pluripotent embryonic stem cells with the potential to give rise to any tissue and organ Initially, embryonic stem cells were obtained from the blastocyst (composed of about 150 cells) with a procedure that required the destruction of the embryo

74 74

75 75 Staminali etiche. Col trucco Avvenire Giovedì, 12 ottobre 2006 Pro-life official dismisses new stem-cell announcement as a sham By Nancy Frazier OBrien Catholic News Service

76 76 Date tempo alla scienza di Giuseppe Remuzzi Corriere delle Sera, domenica 3 settembre 2006 NATURE E GLI EMBRIONI

77 77 Irina Klimankaya first has separated the cells from the embryo and then she has grown them in culture up to eight months without loosing their embryonic features This is what has been done and this is what is reported on the Natures paper

78 78 Those embryos did not survive, however this was not the aim of Robert Lanza What he intended to prove was that you can obtain many embryonic stem cells from one cell extracted from a eight-ten cells embryo, exactly the same procedure carried out after in vitro fertilization in the clinics for fertility

79 79 January 2008, Cell Stem Cell January 2008

80 80 About one year has been passed before the same researchers demonstrated that the embryo subjected to the single cell biopsy for cell line derivation can survive and develops normally up to the end of the pregnancy

81 81

82 82 GENERATION OF ISOGENIC PLURIPOTENT STEM CELLS Bang et al., Science, 2008 Reprogramming by 4 factor: - Oct4, - Sox2, - Klf4 - cMyc Fibroblast Mesechyme cell Hepatocyte Gastric epithelial cell Pluripotent cell types (can give rise to cells of the body) Blastocyst stage embryo

83 83 Retrovirus and lentivirus, highly risky for tumour induction, are used to deliver genes into the cells and to allow their integration into DNA Moreover: one of the four genes transferred into the cells is an oncogene, associated to cancer development

84 84 Cambio di rotta Dopo la scoperta di uno scienziato giapponese: i geni per far ringiovanire le cellule adulte Il padre di Dolly: inutile la clonazione Ian Wilmut: Create staminali senza embrioni, scelgo questa via Corriere della Sera Domenica 18 Novembre 2007

85 85 One would have said (and written) that using this technique no more embryonic stem cells will be needed Will this really be the case? Probably not, at least for now

86 86 These systems are low efficient To deliver genes for reprogramming adult cells, Japanese researchers performed gene transfection thousands of times to get one cell expressing them Clearly, such an approach has no chances to be used in the clinical practice

87 87 David Cyranoski

88 88 The efficiency is low (less than 1 %) and expertise in human embryonic stem cell culture is absolutely needed Viral vectors used to transfer the genes into cells, as well as some of the genes themselves may cause cancer Whether cultured differentiated cells still retain undifferentiated embryonic stem and iPS cells is a critical point because of risk of tumorigenesis iPS cells are the same as embryonic stem cells? Even iPS cell lines seem to differentiate into the cell of choice, some variation between cell lines is unavoidable Ethical issues about the possible use of iPS cell to derive human gametes

89 89 Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors Jeong Beom Kim, Holm Zaehres, Guangming Wu, Luca Gentile, Kinarm Ko, Vittorio Sebastiano, Marcos J. Arauzo, David Ruau, Dong Wook Han, Martin zenke and Hamd R. Scholer Nature, 31 July 2008 Adult mouse neuronal stem cells that constitutively express higher endogenous level of Sox2 and c-Myc than embryonic stem cells, can be reprogrammed into iPS cells by introducing only two factor (Oct-4 and Klf-4) This procedure avoids the problem of ectopic expression of the transcription factor cMyc that causes tumorigenicity in offspring

90 90 It will be crucial to understand whether embryonic stem cells derived from adult cells behave the same as embryonic stem cells, for how long they can maintain pluripotency and, finally how to address them to become cells needed for organ repair Up to that time, The Lancet wrote, research on embryonic stem cells has to go on

91 91 The time and the cost necessary for the production of clinical grade iPS cell lines and production of differentiated cell types for transplantation could limit wide adaptation in clinical practice PERSONALIZED PLURIPOTENT STEM CELL LINES Nakatatsuji et al., Nature Biotech, 2008

92 92 It was estimated the 50 iPS cell lines obtained from homozygous donor with HLA-A, HLA-B, HLA-DR haplotypes could provide closed immunological matches for more that 90 % of Japanese population HLA-HAPLOTYPE BANKING OF PLURIPOTENT STEM CELL LINE Nakatatsuji et al., Nature Biotech, 2008

93 93 In Japan, as in Europe, patent is awarded to the researchers who file first, in the United Stated the patent goes to the grand that can show it invented the technology first Kyoto University stalled over developing a strategy to protect its patents because of a lack of a legal expertise on involvement with industry WHO IS LEADING IPS TECHNOLOGY?

94 94 IPS Academia Japan was set up by private company and a bank to manage Kyoto Universitys iPS patent The central purpose of iPS academia is to prevent some groups or companies from monopolizing iPS technology

95 95 The company Izumi Bio set up by Sakurada N. (a researcher who claimed to have generated iPS cells in April 2007) and funded by high powered-venture capital companies announced a major research collaboration and licensing agreement to focus on application for iPS cells with Gladstone Institute in S. Francisco where dr. Yamanaka has a joint position

96 96 TREATMENT OF SICKLE CELL ANEMIA MOUSE MODEL WITH iPS CELLS GENERATED FROM AUTOLOGOUS SKIN Humanized sickle cell anemia mouse model (h S/h S)* Tip fibroblasts iPS clones Infect with Oct4, Sox2, Klf4 and c-Myc viruses Corrected IPs Correct sickle mutation by specific gene targeting Differentiate into hematopoietic progenitors Transplant corrected hematopoietic progenitors back into irradiated mice Hanna et al., Science, 2007 *mouse globin replaced with human sickle globin gene

97 97 Bone marrow was repopulated by normal cells and was able of producing normal erythrocytes So animals recovered

98 98 NEURONS DERIVED FROM REPROGRAMMED FIBROBLASTS FUNCTIONALLY INTEGRATE INTO THE FETAL BRAIN AND IMPROVE SYPTOMS OF RATS WITH PARKINSONS DISEASE Fibroblast-derived iPS Mixed population of iPS cells/midbrain dopamine neuron precursors (efficiency < 80 %) Dopamine neurons (1-3 x 10 5 cells) Differentiation + inductive media (7 days) FACS separation to minimize the risk of tumors Depletion SSEA-1 positive cells (undifferentiated tumorigenic iPS) Rat with Parkinsons disease Successful implantation and functional recovery Werning et al., PNAS, 2008 Tx in brain

99 99 iPS CELLS AS MODELS FOR HUMAN GENETIC DISEASE Nishikawa et al., Nature Rev Mol Cell Bio, 2008 Patient-derived iPS cells can be used to examine the disease process at a cellular level iPS cells can be used to test response to possible drugs and might be used to develop patient-specific cell therapy - Mechanism of disease - Effect of drugs - Teratology Differentation of various cell lineages from iPS cells Generation of patient- specific iPS cells

100 100 INDUCED PLURIPOTENT STEM CELLS GENERATED FROM PATIENTS WITH ALS CAN BE DIFFERENTIATED INTO MOTOR NEURONS Generation skin fibroblasts iPS cells from a 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS) These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiated into motor neurons Dimos et al., Science, 2008 Adult ALS patient with L144F SoD1 mutation Human fibroblasts Transcription factors Human induced pluripotent stem cells Embryoid bodies Treat cells with sonic hedgehog and retinoic acid Motor neuronsAstrocytes

101 101 DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS Park et al., Cell, 2008 DiseaseMolecular defectDonor cell ADA + SCID Gausher disease type III Duchenne muscolar dystrophy Becker muscolar dystrophy Down syndrome Parkinson disease Juvenile diabetes mellitus Swachman-Bodlan-Diamond syndrome Huntington disease Lesch-Nyhan syndrome (carrier) Fibrobast Bone marrow MSC Fibrobast GGG>AGG, exon 7 and Del(GAAGA) exon 10, ADA gene AAC>AGC, exon 9, G-insertion, nucleotide 84 of cDNA, GBA gene Deletion of exon 45-52, dystrophin gene Unidentified mutation in dystrophin Trisomy 21 Multifactorial IV2+2T>C ans IV3-1G>A, SBDS gene 72 CAG repeats, hungtinton gene Heterozygosity of HPRT1

102 102 The use of embryonic stem cells is an only course to get someday to do without them

103 103

104 104 The finishing line will be crossed but nobody can tell now if this will happen within months or years Scientists will get there, they can do it

105 105 The dissemination of iPS cell technology is likely to encourage the development of animal models, in order to investigate the behaviour of these differentiated cells in vivo

106 106 It is still premature to predict what future new worlds will become available with iPS cell technology, because of our limited knowledge of the molecular process that occurs during reprogramming

107 107 iPS cell basic research would benefit enormously from effective continuing comparison with embryonic stem cells

108 108 NATURE 4 September 2008 By changing one little word, the committee drafting the Republican 2008 election platform calls for banning all human embryo research in the United States, whether publicly or privately funded It changed and to or so that the platform now calls for a ban the creation of or experimentation on human embryos for research purposes

109 109

110 110 These slides are belonging to Giuseppe Remuzzi, M.D. Mario Negri Institute for Pharmacological Research, Bergamo, Italy. Using these slides is only authorized by mentioning the source

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