Stem cell therapy: facts and myths 28/03/2017 Stem cell therapy: facts and myths Giuseppe Remuzzi Lyon, September 12, 2008 RemuzziStamPadova0508

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A 46-year-old man had an acute anterior myocardial infarction 28/03/2017 CASE REPORT - 1 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 RemuzziStamPadova0508

28/03/2017 CASE REPORT - 2 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 RemuzziStamPadova0508

28/03/2017 What options? RemuzziStamPadova0508

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28/03/2017 Adult stem cells couldn’t do much more than regenerate cell types of origin RemuzziStamPadova0508

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28/03/2017 Embryonic stem cells have the ability to self-renew and to differentiate into all three embryonic germ layers RemuzziStamPadova0508

ADULT STEM CELLS FOR CARDIAC REPAIR 28/03/2017 ADULT STEM CELLS FOR CARDIAC REPAIR Heart Cardiac stem cells Remodelling Angiogenesis Endogenous stem cells activation Differentiation into other cell types Paracrine effects Fat Mesenchymal stem cells Bone marrow Endothelial progenitor cells Blood RemuzziStamPadova0508

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TEIM: TransEndocardial IntraMyocardial injection 28/03/2017 Bone marrow mononuclear cells Circulating progenitor cells Skeletal myoblasts Mesenchymal stem cells TEIM: TransEndocardial IntraMyocardial injection RemuzziStamPadova0508

28/03/2017 Challenges RemuzziStamPadova0508

28/03/2017 Issues such as purity of cells, number and whether they have to be differentiated into cardiomyocytes before transplantation have never been addressed RemuzziStamPadova0508

Is this procedure safe in the long term? 28/03/2017 Is this procedure safe in the long term? To which extent these cells are retained into myocardial tissue? Is spatial distribution important? RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

Cardiac Cell Therapy - Mixed Results from Mixed Cells 28/03/2017 Corriere della Sera, GIOVEDI’, 4 APRILE 2002 The NEW ENGLAND JOURNAL of MEDICINE, SEPTEMBER 21, 2006 Cardiac Cell Therapy - Mixed Results from Mixed Cells Anthony Rosenzwieg, M.D. EDITORIAL RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

ACUTE KIDNEY INJURY - Cysplatin 28/03/2017 ACUTE KIDNEY INJURY - Cysplatin Mesenchymal stem cell injection 100 * * 80 Saline 60 BUN (mg/dl) 40 20 MSC 1 4 5 7 11 29 days Imberti et al., J Am Soc Nephrol, 2007 RemuzziStamPadova0508

28/03/2017 MESENCHYMAL STEM CELL ENGRAFT THE KIDNEY AND DIFFERENTIATE TO TUBULAR EPITHELIAL CELLS AT LOW EXTENT 1000X 4d Cisplatin female mouse + male MSC RemuzziStamPadova0508

Ki 67 positive nuclei / HPF 28/03/2017 MESENCHYMAL STEM CELLS ACCELERATE TUBULAR CELL REGENERATION AFTER CISPLATIN Cisplatin + saline Cisplatin + MSC 35 30 25 * * 20 Ki 67 positive nuclei / HPF 15 10 5 4 11 29 4 11 29 days Ki 67: nuclear proliferation marker Imberti et al., J Am Soc Nephrol, 2007 RemuzziStamPadova0508

INSULIN-LIKE GROWTH FACTOR-1 SUSTAINS STEM CELL-MEDIATED RENAL REPAIR 28/03/2017 INSULIN-LIKE GROWTH FACTOR-1 SUSTAINS STEM CELL-MEDIATED RENAL REPAIR cispl+ saline si-irrel MSC si-IGF-1 MSC 40 80 120 160 BUN (mg/dl) Imberti et al., J Am Soc Nephrol, 2007 RemuzziStamPadova0508

28/03/2017 EFFECT OF HUMAN STEM CELL TREATMENT ON CISPLATIN-INDUCED ACUTE KIDNEY INJURY IN NOD/SCID MICE Stem cell types Renal function Renal histology Survival 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 Morigi et al., Stem Cells, 2008 RemuzziStamPadova0508

28/03/2017 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 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 Patients Start with 3 patients: (5 x 106 MSC, 10 x 106 MSC, 15 x 106 MSC) If safe and successful: upgrade the number to 8 patients RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

Circulatory system Immune system Nervous system 28/03/2017 RemuzziStamPadova0508

28/03/2017 A lot of people think that scientists do not respect ethical and human values and consider science as something in conflict with faith RemuzziStamPadova0508

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Science and the ethics of science are two sides of the same coin 28/03/2017 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 RemuzziStamPadova0508

The NEW ENGLAND JOURNAL of MEDICINE 28/03/2017 The NEW ENGLAND JOURNAL of MEDICINE RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 According to the New York Times, Karl Rowe, head of the White House’s Office of Political Affairs, has declared that embryonic stem cells aren’t required because there is “far more promise from adult stem cells” RemuzziStamPadova0508

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28/03/2017 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 President’s Council on Bioethics, Prentice claimed that adult stem cells can effectively treat more that 65 diseases RemuzziStamPadova0508

28/03/2017 Not only this assertion is patently false, but the information purveyed on the Family Research Council’s Web site is pure hokum Schwartz, N Engl J Med, 2006 RemuzziStamPadova0508

“DE HUMANI CORPORIS FABRICA” Andrea Vesalius, 1543 28/03/2017 “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 author’s 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 RemuzziStamPadova0508

STEM CELLS, MIRACLE OR CURE? 28/03/2017 Disease FDA approved Effects/limitations Parkinson’s disease Spinal cord injury Amyotrophic lateral sclerosis Multiple sclerosis Rheumatoid arthritis Corneal regeneration Osteogenesis imperfecta Thalassemia major 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 Lack of long term data Improvement of symtpoms Efficacious (n = 2 patients) ANTONIO MISSIERI Smith et al., Science, 2006 RemuzziStamPadova0508

28/03/2017 To support the inclusion of Parkinson’s 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 Parkinson’s disease In fact, there is currently no FDA-approved adult stem cell treatments - and non cure of any kind - for Parkinson’s disease RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

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Injections of hope Washingtonpost.com 04 September 2008 28/03/2017 Washingtonpost.com 04 September 2008 Injections of hope Doctors Promote Offshore Stem Cell Shots, but Some Patients Cry Foul 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 RemuzziStamPadova0508

28/03/2017 Credit: iStockphoto RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

MAJOR CONCERNS - 1 The way they are - derived - characterized 28/03/2017 MAJOR CONCERNS - 1 The way they are - derived - characterized - established - maintained In vitro differentiation RemuzziStamPadova0508

MAJOR CONCERNS - 2 Epigenetic profiles Chromosomal aberrations 28/03/2017 MAJOR CONCERNS - 2 Epigenetic profiles Chromosomal aberrations Risk of tumors Genetic instability RemuzziStamPadova0508

28/03/2017 DISEASE MODELS WHERE HUMAN EMBRYONIC STEM CELLS HAVE BEEN SHOWN TO BE EFFECTIVE (mainly SCID mice) Cell type developed Animal model Reference 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 RemuzziStamPadova0508

28/03/2017 Immune-rejection RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

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28/03/2017 REPROGRAMMING OF SOMATIC CELLS TO A PLURIPOTENT STATE - Somatic cell nuclear transfer RemuzziStamPadova0508

28/03/2017 12 March 2004 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 RemuzziStamPadova0508

Corriere della Sera, 24 dicembre 2005 28/03/2017 Corriere della Sera, 24 dicembre 2005 RemuzziStamPadova0508

28/03/2017 MAJOR CONCERNS Mitochondrial transmission of aberrant epigenetic gene expression profiles Mitochondrial genome encodes a number of transplantation antigens that could trigger a immune response RemuzziStamPadova0508

We don’t know how an embryonic stem cell will behave in a human 28/03/2017 We don’t know how an embryonic stem cell will behave in a human We don’t known what will ultimately come out from research on embryonic stem cells RemuzziStamPadova0508

28/03/2017 DESPITE THE DRAWBACKS AND DEBATE, AN INTERNET SEARCH SHOWS SOME COMMERCIAL SOURCES OFFERING EMBRYONIC STEM CELL THERAPY Embryonic Tissues Center Ukraine Medra Inc., Dominican Republic NUTech Mediworld India RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 There is a need to increase public awareness and to manage the public expectations for human embryonic stem cell- based therapies RemuzziStamPadova0508

28/03/2017 Whether ethic is indeed part of science, why do not ask to scientists to solve ethical issues that they put themselves? RemuzziStamPadova0508

28/03/2017 CORRIERE DELLA SERA, 22 GENNAIO 2007 CORRIERE DELLA SERA, 15 DICEMBRE 2007 RemuzziStamPadova0508

Often, this does not happen and the embryo stops growing 28/03/2017 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 RemuzziStamPadova0508

This is also the case of the in vitro fecundation 28/03/2017 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 RemuzziStamPadova0508

Ospedale Peter Bent Brigham di Boston 28/03/2017 23 Dicembre 1954, ore: 8.00 Ospedale Peter Bent Brigham di Boston RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

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28/03/2017 Somehow, amniotic fluid cells are halfway between embryonic and adult stem cells In the laboratory they grow efficiently (at least in somebody’s hands) and they can become cells of the muscle, nervous system and liver RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 August 2006, August 2006, NATURE on line Human embryonic stem cell lines derived from single blastomeres Irina Klimanskaya, Young Chung, Sandy Becker, Shi-Jiang Lu & Robert Lanza Scientists from Massachusetts, led by Robert Lanza, applied a technique already used in fertilization clinics to obtain human embryonic stem cells RemuzziStamPadova0508

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28/03/2017 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 woman’s uterus and will give birth to a child RemuzziStamPadova0508

28/03/2017 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 embryo’s potential for life Not always the embryo survives but this does not happen even for natural fecundation RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

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Staminali “etiche”. Col trucco 28/03/2017 Pro-life official dismisses new stem-cell announcement as a sham By Nancy Frazier O’Brien Catholic News Service Avvenire Giovedì, 12 ottobre 2006 Staminali “etiche”. Col trucco RemuzziStamPadova0508

Date tempo alla scienza 28/03/2017 Corriere delle Sera, domenica 3 settembre 2006 “NATURE” E GLI EMBRIONI Date tempo alla scienza di Giuseppe Remuzzi RemuzziStamPadova0508

28/03/2017 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 Nature’s paper RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

January 2008 January 2008, Cell Stem Cell 28/03/2017 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

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GENERATION OF ISOGENIC PLURIPOTENT STEM CELLS 28/03/2017 GENERATION OF ISOGENIC PLURIPOTENT STEM CELLS Pluripotent cell types (can give rise to cells of the body) Reprogramming by 4 factor: - Oct4, - Sox2, - Klf4 - cMyc Fibroblast Mesechyme cell Hepatocyte Gastric epithelial cell Blastocyst stage embryo Bang et al., Science, 2008 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

Il padre di Dolly: inutile la clonazione 28/03/2017 Corriere della Sera Domenica 18 Novembre 2007 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” RemuzziStamPadova0508

Will this really be the case? Probably not, at least for now 28/03/2017 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 RemuzziStamPadova0508

These systems are low efficient 28/03/2017 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 RemuzziStamPadova0508

28/03/2017 David Cyranoski RemuzziStamPadova0508

iPS cells are the same as embryonic stem cells? 28/03/2017 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 RemuzziStamPadova0508

28/03/2017 Nature, 31 July 2008 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 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 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

PERSONALIZED PLURIPOTENT STEM CELL LINES 28/03/2017 PERSONALIZED PLURIPOTENT STEM CELL LINES 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 Nakatatsuji et al., Nature Biotech, 2008 RemuzziStamPadova0508

HLA-HAPLOTYPE BANKING OF PLURIPOTENT STEM CELL LINE 28/03/2017 HLA-HAPLOTYPE BANKING OF PLURIPOTENT STEM CELL LINE 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 Nakatatsuji et al., Nature Biotech, 2008 RemuzziStamPadova0508

WHO IS LEADING IPS TECHNOLOGY? 28/03/2017 WHO IS LEADING IPS TECHNOLOGY? 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 RemuzziStamPadova0508

28/03/2017 ‘IPS Academia Japan’ was set up by private company and a bank to manage Kyoto University’s iPS patent The central purpose of iPS academia is to prevent some groups or companies from monopolizing iPS technology RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 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 Transplant corrected hematopoietic progenitors back into irradiated mice Infect with Oct4, Sox2, Klf4 and c-Myc viruses Differentiate into hematopoietic progenitors Correct  sickle mutation by specific gene targeting iPS clones Corrected IPs *mouse  globin replaced with human  sickle globin gene Hanna et al., Science, 2007 RemuzziStamPadova0508

28/03/2017 Bone marrow was repopulated by normal cells and was able of producing normal erythrocytes So animals recovered RemuzziStamPadova0508

28/03/2017 NEURONS DERIVED FROM REPROGRAMMED FIBROBLASTS FUNCTIONALLY INTEGRATE INTO THE FETAL BRAIN AND IMPROVE SYPTOMS OF RATS WITH PARKINSON’S DISEASE Differentiation + inductive media (7 days) Fibroblast-derived iPS Mixed population of iPS cells/midbrain dopamine neuron precursors (efficiency < 80 %) FACS separation to minimize the risk of tumors Depletion SSEA-1 positive cells (undifferentiated tumorigenic iPS) Tx in brain Dopamine neurons (1-3 x 105 cells) Successful implantation and functional recovery Rat with Parkinson’s disease Werning et al., PNAS , 2008 RemuzziStamPadova0508

iPS CELLS AS MODELS FOR HUMAN GENETIC DISEASE 28/03/2017 iPS CELLS AS MODELS FOR HUMAN GENETIC DISEASE - Mechanism of disease - Effect of drugs - Teratology Generation of patient-specific iPS cells Differentation of various cell lineages from iPS cells 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 Nishikawa et al., Nature Rev Mol Cell Bio, 2008 RemuzziStamPadova0508

28/03/2017 INDUCED PLURIPOTENT STEM CELLS GENERATED FROM PATIENTS WITH ALS CAN BE DIFFERENTIATED INTO MOTOR NEURONS Adult ALS patient with L144F SoD1 mutation 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 Transcription factors Human fibroblasts Human induced pluripotent stem cells Embryoid bodies Treat cells with sonic hedgehog and retinoic acid Dimos et al., Science, 2008 Astrocytes Motor neurons RemuzziStamPadova0508

DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS 28/03/2017 DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS Disease Molecular defect Donor 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) 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 Fibrobast Bone marrow MSC Park et al., Cell, 2008 RemuzziStamPadova0508

28/03/2017 The use of embryonic stem cells is an only course to get someday to do without them RemuzziStamPadova0508

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Scientists will get there, they can do it 28/03/2017 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 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 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 RemuzziStamPadova0508

28/03/2017 iPS cell basic research would benefit enormously from effective continuing comparison with embryonic stem cells RemuzziStamPadova0508

28/03/2017 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” RemuzziStamPadova0508

Giuseppe Remuzzi, M.D. These slides are belonging to 28/03/2017 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 RemuzziStamPadova0508