Stem cell therapy:

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Stem cell therapy facts and myths Giuseppe
Remuzzi Lyon, September 12, 2008
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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
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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
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What options?
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Adult stem cells couldnt 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
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
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Bone marrow mononuclear cells
Circulating progenitor cells
Skeletal myoblasts
Mesenchymal stem cells
TEIM TransEndocardial IntraMyocardial injection
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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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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
Mesenchymal stem cell injection
100


80
Saline
60
BUN (mg/dl)
40
20
MSC
0
0
1
4
5
7
11
29 days
Imberti et al., J Am Soc Nephrol, 2007
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MESENCHYMAL STEM CELL ENGRAFT THE KIDNEY AND
DIFFERENTIATE TO TUBULAR EPITHELIAL CELLS AT LOW
EXTENT
Cisplatin female mouse male MSC
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MESENCHYMAL STEM CELLS ACCELERATE TUBULAR CELL
REGENERATION AFTER CISPLATIN
Cisplatin saline
Cisplatin MSC
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30
25


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Ki 67 positive nuclei / HPF
15
10
5
0
4
11
29
4
11
29 days
Ki 67 nuclear proliferation marker
Imberti et al., J Am Soc Nephrol, 2007
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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
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
Morigi et al., Stem Cells, 2008
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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
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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 system
Immune system
Nervous 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 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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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
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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
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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 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
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STEM CELLS, MIRACLE OR CURE?
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 Feasible Lack of long term
data Improvement of symtpoms Efficacious (n 2
patients)
Parkinsons disease Spinal cord
injury Amyotrophic lateral sclerosis Multiple
sclerosis Rheumatoid arthritis Corneal
regeneration Osteogenesis imperfecta Thalassem
ia major
ANTONIO MISSIERI
Smith et al., Science, 2006
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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
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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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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
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Credit iStockphoto
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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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MAJOR CONCERNS - 1
The way they are - derived - characterized -
established - maintained In vitro
differentiation
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MAJOR CONCERNS - 2
Epigenetic profiles Chromosomal
aberrations Risk of tumors Genetic instability
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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 Hep
atocytes 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 Op
en 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
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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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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
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Corriere della Sera, 24 dicembre 2005
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MAJOR CONCERNS
Mitochondrial transmission of aberrant epigenetic
gene expression profiles Mitochondrial genome
encodes a number of transplantation antigens that
could trigger a immune response
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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
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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
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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 8.00 Ospedale 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 somebodys
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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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
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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 womans
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 embryos
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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Pro-life official dismisses new stem-cell
announcement as a sham By Nancy Frazier
OBrien Catholic News Service
Avvenire Giovedì, 12 ottobre 2006
Staminali etiche. Col trucco
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Corriere delle Sera, domenica 3 settembre 2006
NATURE E GLI EMBRIONI
Date tempo alla scienza di Giuseppe Remuzzi
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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 Natures 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
January 2008, Cell Stem Cell
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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
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
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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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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
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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
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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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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
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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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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
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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
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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
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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
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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
(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
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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 PARKINSONS DISEASE
Differentiation inductive media (7 days)
Fibroblast-derived iPS
Mixed population of iPS cells/midbrain dopamine
neuron precursors (efficiency lt 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 Parkinsons disease
Werning et al., PNAS , 2008
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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
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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
Motor neurons
Astrocytes
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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-Bodl
an-Diamond syndrome Huntington
disease Lesch-Nyhan syndrome (carrier)
Fibrobast Fibrobast Fibrobast Fibrobast Fibr
obast Fibrobast Fibrobast Bone marrow
MSC Fibrobast Fibrobast
GGGgtAGG, exon 7 and Del(GAAGA) exon 10, ADA
gene AACgtAGC, exon 9, G-insertion, nucleotide 84
of cDNA, GBA gene Deletion of exon 45-52,
dystrophin gene Unidentified mutation in
dystrophin Trisomy 21 Multifactorial Multifact
orial IV22TgtC ans IV3-1GgtA, SBDS gene 72 CAG
repeats, hungtinton gene Heterozygosity of HPRT1
Park et al., Cell, 2008
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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
107
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