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Stem cell therapy: facts and myths

Giuseppe Remuzzi

Lyon, September 12, 2008

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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

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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

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What options?

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Adult stem cells couldn’t do much more than regenerate cell types of origin

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

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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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12TEIM: TransEndocardial IntraMyocardial injection

Bone marrow mononuclear cells

Circulating progenitor cells

Skeletal myoblasts

Mesenchymal stem cells

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Challenges

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

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Is this procedure safe in the long term?

To which extent these cells are retained into myocardial tissue?

Is spatial distribution important?

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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

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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

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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

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ACUTE KIDNEY INJURY - Cysplatin

0

20

40

60

80

100

0 1 4 5 7 11 29 days

Mesenchymal stem cell injection

BU

N (

mg/

dl)

**

Saline

MSC

Imberti et al., J Am Soc Nephrol, 2007

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MESENCHYMAL STEM CELL ENGRAFT THE MESENCHYMAL STEM CELL ENGRAFT THE KIDNEY AND DIFFERENTIATE TO TUBULAR KIDNEY AND DIFFERENTIATE TO TUBULAR EPITHELIAL CELLS AT LOW EXTENTEPITHELIAL CELLS AT LOW EXTENT

Cisplatin female mouse + male MSC

1000X

4d

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MESENCHYMAL STEM CELLS ACCELERATE MESENCHYMAL STEM CELLS ACCELERATE TUBULAR CELL REGENERATION AFTER CISPLATINTUBULAR CELL REGENERATION AFTER CISPLATIN

Ki 6

7 po

sitiv

e nu

clei

/ H

PF

0

5

10

15

20

25

30

35

4 11 29 4 11 29 days

Cisplatin + saline Cisplatin + MSC

* *

Ki 67: nuclear proliferation marker

Imberti et al., J Am Soc Nephrol, 2007

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0cispl+saline

cispl+si-irrel MSC

cispl+si-IGF-1 MSC

40

80

120

160

BU

N (

mg/

dl)

INSULIN-LIKE GROWTH FACTOR-1 SUSTAINS STEM CELL-MEDIATED RENAL REPAIR

Imberti et al., J Am Soc Nephrol, 2007

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EFFECT OF HUMAN STEM CELL TREATMENT ON CISPLATIN-INDUCED ACUTE KIDNEY INJURY IN NOD/SCID MICE

Morigi et al., Stem Cells, 2008

Stem cell types Renal function Renal histology Survival at 7 days

Bone marrow

Cord blood

Amniotic fluid

Protected

Protected

Preserved

Preserved

50 %

86 %

Currently testing

None of the untreated mice survived cisplatinum at 7 days

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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 106 MSC, 10 x 106 MSC, 15 x 106 MSC)

If safe and successful: upgrade the number to 8 patients

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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

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Circulatory systemNervous system

Immune system

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

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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

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The NEW ENGLAND JOURNAL of MEDICINE

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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

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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”

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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

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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

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“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

37 Smith et al., Science, 2006

Disease FDA approved Effects/limitations

NO

NO

NO

NO

NO

NO

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

FeasibleLack of long term dataImprovement of symtpoms

Efficacious (n = 2 patients)

AN

TO

NIO

MIS

SIE

RI

Parkinson’s disease

Spinal cord injury

Amyotrophic lateral sclerosis

Multiple sclerosis

Rheumatoid arthritis

Corneal regeneration

Osteogenesis imperfecta

Thalassemia major

STEM CELLS, MIRACLE OR CURE?

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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

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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

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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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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

Washingtonpost.com04 September 2008

Injections of hopeDoctors Promote Offshore Stem Cell Shots, but Some Patients Cry Foul

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Cre

dit:

iSto

ckph

oto

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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

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The way they are - derived- characterized- established - maintained

In vitro differentiation

MAJOR CONCERNS - 1

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Epigenetic profiles

Chromosomal aberrations

Risk of tumors

Genetic instability

MAJOR CONCERNS - 2

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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

HepatocytesChondrocytesEndothelial cells

Neural precursors

Pancreatic cellsNeuroepithelial precursors and dopaminergic neurons

Human embryonic stem cells

Spinal cord injury

Myocardial infarction

Toxic-liver injurySpinal fusionSurgical induction of hind limb ischemia

Quinolinic acid-induced Huntington

Streptozotocin diabetesParkinson

Open neural tube defect

Keirstead et al., 2005Nakamura et al., 2005

Laflamme et al., 2007Leor et al., 1996Kehat et al., 2004Caspi et al, 2007

Seo et al., 2005Muschler et al., 2003Cho eet al., 2007

Song et al., 2007

Shim et al., 2007

Sontag et al., 2007 Ben-Hur et al., 2004

Lee et al., 2006

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Immune-rejection

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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

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REPROGRAMMING OF SOMATIC CELLS TO A PLURIPOTENT STATE - Somatic cell nuclear transfer

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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

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Corriere della Sera, 24 dicembre 2005

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Mitochondrial transmission of aberrant epigenetic gene expression profiles

Mitochondrial genome encodes a number of transplantation antigens that could trigger a immune response

MAJOR CONCERNS

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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

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DESPITE THE DRAWBACKS AND DEBATE, AN INTERNET SEARCH SHOWS SOME COMMERCIAL SOURCES OFFERING EMBRYONIC STEM CELL THERAPY

Medra Inc., Dominican Republic

Embryonic Tissues Center Ukraine

NUTech MediworldIndia

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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

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

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

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CORRIERE DELLA SERA, 22 GENNAIO 2007

CORRIERE DELLA SERA, 15 DICEMBRE 2007

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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

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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

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

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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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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

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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

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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

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Human embryonic stem cell lines derived from single blastomeresIrina 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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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

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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

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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

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Staminali “etiche”. Col trucco Avvenire Giovedì, 12 ottobre 2006

Pro-life official dismisses new stem-cell announcement as a sham

By Nancy Frazier O’BrienCatholic News Service

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Date tempo alla scienza

di Giuseppe Remuzzi

Corriere delle Sera, domenica 3 settembre 2006

“NATURE” E GLI EMBRIONI

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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

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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

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January 2008, Cell Stem Cell

January 2008

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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

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GENERATION OF ISOGENIC PLURIPOTENT STEM CELLS

Bang et al., Science, 2008

Reprogramming by 4 factor:- Oct4, - Sox2, - Klf4 - cMycFibroblast

Mesechyme cellHepatocyteGastric epithelial cell

Pluripotent cell types(can give rise to cells of the body)

Blastocyst stage embryo

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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

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Cambio di rotta Dopo la scoperta di uno scienziato giapponese: i geni per far “ringiovanire” le cellule adulte

Il padre di Dolly: inutile la clonazioneIan Wilmut: “Create staminali senza embrioni, scelgo questa via”

Corriere della Sera Domenica 18 Novembre 2007

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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

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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 David Cyranoski

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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

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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

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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

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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

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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

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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?

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‘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

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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

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TREATMENT OF SICKLE CELL ANEMIA MOUSE MODEL WITH iPS CELLS GENERATED FROM AUTOLOGOUS SKIN

Humanized sickle cell anemia mouse model (hS/hS)*

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

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Bone marrow was repopulated by normal cells and was able of producing normal erythrocytes

So animals recovered

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NEURONS DERIVED FROM REPROGRAMMED FIBROBLASTS FUNCTIONALLY INTEGRATE INTO THE FETAL BRAIN AND IMPROVE SYPTOMS OF RATS WITH PARKINSON’S DISEASE

Fibroblast-derived iPS

Mixed population of iPS cells/midbrain dopamine neuron precursors (efficiency < 80 %)

Dopamine neurons(1-3 x 105 cells)

Differentiation + inductive media

(7 days)

FACS separation to minimize the risk of tumors

Depletion SSEA-1 positive cells (undifferentiated tumorigenic iPS)

Rat withParkinson’s disease

Successful implantation and functional recovery

Werning et al., PNAS , 2008

Tx in brain

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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

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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

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DISEASE-SPECIFIC INDUCED PLURIPOTENT STEM CELLS

Park et al., Cell, 2008

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)

Fibrobast

Fibrobast

Fibrobast

Fibrobast

Fibrobast

Fibrobast

Fibrobast

Bone marrow MSC

Fibrobast

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

Multifactorial

IV2+2T>C ans IV3-1G>A, SBDS gene

72 CAG repeats, hungtinton gene

Heterozygosity of HPRT1

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

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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

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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

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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

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

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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”

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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