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STEM CELLS AND THE MYTH
OF ETERNAL YOUTH: ADULT
VERSUS EMBRYONIC STEM
CELLS
Dr. Marie - Louise Labat
The myth of eternal youth
has always haunted humanity.
Today, what is at stake
is regenerative medicine
using stem cells.
Stem cells are undifferentiated
cells that given the right signals
can differentiate to the many
(more than 200) specialized cells
that make up the organism.
Stem cells have the ability to
self-replicate for the lifetime
of the organism.
Different types of stem cells
are involved during human
development and adult life.
HUMAN DEVELOPMENTAL CONTINUUM
Fetus
Totipotent
Stem
Cells
Embryonic germ
Pluripotent
(EG) cells
Pluripotent
Tissue stem cells
Pluripotent/Multipotent
Pluri/
Multi
potent
Adapted from David Prentice
Distinguishing Features of Progenitor/Precursors
Cells and Stem Cells.
http://stemcells.nih.gov/info/scireport/chapter4.pdf
Human embryo at stage 8 cells
(2 days)
8 totipotent stem cells
Inner
Mass
Human embryo at blastocyst stage
200-250 cells (4 to 5 days)
(Pluripotent stem cells)
From G. Baker: http://www.city.ac.uk/cs/lecturenotes/neuro_biomedsciences.pdf
Then, gastrulation leads to
the establishment of definitive germ
layers (ectoderm, endoderm, and
mesoderm) that harbour
multipotent cells.
From Human Embryology, Larsen, DeBoeck University Ed, Bruxelles, Belgium, 1996
According to the classical dogma,
pluripotency was thought to be lost
after the blastocyst stage.
Therefore, so-called pluripotent embryonnic stem cells are
extracted before gastrulation, from
the inner mass of the blastocyst
From ‘Science et Avenir’ 2002, n° 130, special issue
Martin G;
Isolation of pluripotent cell lines
from early mouse embryos.
Proc Natl Acad Sci USA 1981;
78: 7634-7838.
Thomson JA, Istkovitz-Eldor J,
Shapiro SS, Waknitz MA, Swiergel JJ,
Marshall VS, Jones JM.
Embryonic stem cell lines derived
from the human blastocyst.
Science 1998; 282: 1145-1147
The authors concluded that such
human embryonic stem cells could
be used for transplantation medicine,
in order to cure:
- Alzheimer’s disease
- Parkinson’s disease
- Juvenile type diabetes
- Spinal cord injury
However, because extraction
of the stem cells from the blastocysts
leads to the destruction of a human
embryo, a worldwide debate both
ethical and political started.
From the time that the ovum is
fertilized, a life is begun that is
neither that of the father nor
the mother, it is rather the life of
a new human being with his own
growth, it would never become
human, if it were not human already.
John-Paul II (Evangelium vitae)
The study by Thomson’s team
was made using ‘supernumerary
embryos’ donated by the parents
after informed consent
and after institutional review
board approval.
But the question is:
Whom do these embryos
belong to, if not to themselves?
This question is linked to another one:
When does human life begin ?
At an April 2002 press conference,
Senator A. Specter (Penn) was asked
by a reporter, within the context
of embryonic stem cell research,
when life begins.
Senator Specter replied:
« I haven’t found it helpful
to get into the details. »
What are exactly these human
embryonic stem cells?
First of all, their isolation
is the direct consequence
of in vitro fertilization and
preimplantation diagnosis.
Pipet
One cells
is taken
In vitro
culture
Embryo between
6 to 12 cells
One cell is sampled from each embryo
fertilized and cultured in vitro
Diagnostic test on each sampled cell
In vitro fertilized ovocyte
Healthy embryos are selected for
implantation in the uterus
Preimplantation genetic diagnosis
Sciences et Avenir, 2002; 130, Hors-série
• 1978: birth of the first baby by in vitro fertilization
(IVF) in Great Britain.
• 1982: birth of a baby by in vitro fertilization in
France.
• During the past 20 years, 100 000 babies were born
in France by IVF. In 1998, more than 13 000 babies
were born by IVF (1,8% of birth).
This number is in constant increase, due to:
• 1. greater efficiency of ovary stimulation,
• 2. cryopreservation of embryos.
As a result, the human embryo
became available
for experimentation.
Human embryo (3 days old, 16 cells)
Science et Avenir 2002; 130
Special issue
In fact, embryonic stem cells,
at the heart of the present debate
are not present as such
in the embryo,
they result from manipulations
in the laboratory.
The pluripotent cells extracted from the inner mass of the blastocyst are
manipulated in the laboratory to give rise to cell lines that proliferate indefinitely
Science et Avenir 2002, n°130, special issue
Manipulation of embryonic stem cells
in the laboratory
- After dissociation of the inner mass of
human blastocysts, the cells are first grown on
a feeder layer of irradiated mouse embryonic
fibroblasts,
- after 9 to 15 days of culture, the outgrowth
are harvested and replated,
- cell lines are selected by their prolonged
undifferentiated proliferation characteristics
while retaining the ability to differentiate into
the three embryonic germ-layers.
http://stemcells.nih.gov/info/scireport/appendix.pdf
Human embryonic stem cell lines approved by President
Bush for federally funded research (August 9, 2001)
.
CyThera (California)
•
•
•
•
•
•
•
•
•
•
•
•
ES Cell International (Australia)
Geron Corporation (California)
University of Göteborg (Sweden)
Maria Biotech Co (Korea)
Miz Medi Hospital (Korea)
National Center for Biological Science (India)
Pochon CHA University (Korea)
Reliance Life Sciences, Bombay (India)
Technion University, Haifa (Israel)
University of California SF (California)
Wisconsin Alumni Research Foundation
Bresagen, Inc (Georgia, USA)
9
6
7
19
3
1
3
2
7
4
2
5
4
These cells lines, created for
possible future disease treatments
were grown on mouse fibroblasts.
That could expose humans to
animal virus their immune
system couldn’t fight, the U.S.
medical ethics panel said on
November 2003.
Attempts are made to grow human embryonic stem
cell lines without using a feeder layer of mouse
fibroblasts and without using fetal calf serum
Amit M, Shariki C, Margulets V, Itskovitz-Eldor J
Feeder and serum-free culture
of human embryonic stem cells.
Biol Reprod 2004; 70: 837-45
According to the US National
Institutes of Health (NIH),
only 11 cell lines can really be
considered (some were discarded
because contaminated by viruses,
or cross-contaminated,
or because they were not really
stem cells, or for ethical reasons).
One of these cells lines, that had
received the agreement of the NIH, that
was considered as stable and devoid of
chromosome abnormalities was used as a
reference. It was widely distributed in
150 laboratories all around the world.
This reference cell line was recently
shown by Draper and Smith, to be
instable and to present an excess of
chromosome 12 and 17.
Draper JS, Smith K, et al
Recurrent gain of chromosome
17 q and 12 in cultured human
embryonic stem cells.
Nat Biotechnol 2004; 22: 53-54
108th congress
of Office of Legislative Policy and Analysis
Ronald McKay
National Institute of Neurological Disorders
and Stroke
May 22, 2003
« There is clear evidence that human ES cells
will form teratomas, complex mixtures of
different cells, but much less is known about
efficiently generating specific cell type … »
In fact, it is difficult to differentiate
embryonic stem cells toward one
direction only:
Schuldiner M, Yanuka O, ItskovitzTaylor J et al.
Effect of eight growth factors on the
differentiation of cells derived from
human embryonic stem cells.
Proc Natl Acad Sci 2000; 97: 11307
Even the pioneer of human
embryonic stem cell research,
J. A. Thomson, does not believe any
more in the therapeutic use of human
embryonic stem cells.
In december 1998, J. A. Thomson
testified before the American Senate
that human embryonic stem cells
were going to cure:
- Alzheimer’s disease
- Parkinson’s disease
- Juvenile type diabetes
- Spinal cord lesions
On June 2002, J. D. Thomson
stated at the
« Workshop on the Basic Biology of Mammalian Stem Cells »
organized by
the National Institute of General Medical Sciences :
‘in the next decade, relatively few clinical trials
in « cell therapy » will be initiated with human
embryonic stem cells, due to safety concerns
and possible immune interference …
Forecasting that stem cells would have a greater
impact on understanding the roots of disease
rather than on treatment’
Indeed, that last proposition:
‘…stem cells would have a greater
impact on understanding the roots of
disease rather than on treatment’ could
not have motivated the associations of
patients to back research on human
embryonic stem cells.
A similar evolution can be noted in
the statements of the other pionnier
J. Gearhardt who derived in 1998
pluripotent cell lines from germ cells
from human embryos.
November 19, 2002, J. Gearhart
stated in the Washington Fax, the
official press organ of the National
Institutes of Health:
« Embryonic and fetal stem cells
likely will never be used as part of
disease therapy, but research into
both embryonic and fetal stem cells
is critical and will provide scientists
with vital informations that will
enable treatments.
However, the worlwide ethical
and political debate continues
without taking into account these
new informations.
Most of the politicians are
convinced that embryonic stem
cells are, for sure, going to cure
Alzheimer’s disease, diabetes,
Parkinson’s disease, etc. …
« Research …if conducted in a true
scientific way and if it respects moral
law , cannot be in conflict with faith. »
John-Paul II (Gaudium et spes, n 36)
Concerning research on
embryonic stem cells, clearly the
research has not been well
conducted.
While this debate on human
embryonic stem cells was going
on, major changes in our
knowledge about stem cells
present in adults occured.
According to the classical dogma, it
was thought, that pluripotent stem
cells were completely lost after the
blastocyst stage.
Now it is know that pluripotent stem
cells are still present in adult life.
They are called organ stem cells
or adult stem cells, because being
present in the adult.
HUMAN DEVELOPMENTAL
CONTINUUM
Fetus
Totipotent
Stem
Cells
Embryonic germ
Pluripotent
(EG) cells
Pluripotent
Tissue stem cells
Pluripotent/Multipotent
Pluri/
Multip
otent
Adapted from David Prentice
Until these past few years,
adult stem cells were known only
in permanently renewing tissues,
such as blood, skin,
gastrointestinal tract, and bone.
They were thought to have
the same embryological origin as
the tissue in which they are
hidden.
Now, adult stem cells have been
found in every organ where it has
been looked for them, including
brain.
They are also present bone
marrow, blood and cord blood.
Adult organ stem cells are not
involved in organogenesis.
They are responsible for normal
tissue renewal and for
regeneration following damage.
Organ (adult) stem cells are reserve
stem cells which keep embryonic
characteristics during all the life of
the individual.
They seed the different organs,
where they normally remain
quiescent until, in response to
precise signals, they proliferate and
differentiate to insure the integrity of
the organism.
Their origin is the neural crest.
(Labat et al. Biomed and
Pharmacotherapy, 2000)
Organ stem cells present in the
adult share similarities with
embryonic stem cells: both are
able to proliferate and to
differentiate into all kinds of
cellular types i.e. they are
pluripotent.
But, there are major differences
between adult and embryonic
stem cells:
contrarily to embryonic stem
cells, adult stem cells are
mastered cells that the adult
organism knows how to control.
Adult stem cells show their
potential of proliferation and
differentiation only in response
to precise signals.
Their role is tissue repair,
that, like every immunological
reaction, has to be initiated, to
develop and to know how to end.
That means that adult stem cells
are tightly controlled.
Many mechanisms are probably
involved in that control.
One of them involves a particular
subset of T lymphocytes called
phagic T lymphocytes because of
their unique mode of action.
This has been evidenced studying
organ stem cells present in adult
blood.
Adult stem cells present in blood
constitute, together with stem
cells hidden in the organs,
a single pool of pluripotent stem
cells in homeostatic equilibrium.
In vitro Characterization of the
normal circulating stem cells
Before adhesion, they
look like monocytes:
• they stain for non
specific esterases
activity
• they express CD14+
and CD68+ markers
• They express
constitutively HLA-DR
molecules
Once they have adhered,they give
rise to cells with different
phenotypes, among them fibroblastlike cells
They express collagen 1, collagen, 3, procollagen 1,
and fibronecti, while still expressing monocyte
markers.
The normal stem cells are almost
quiescent. Once they have adhered
and started to differentiate, their
time-life is short. They activate a
special subpopulation of CD4+T
lymphocytes called phagic T
lymphocytes (PTLs). Phagic T
lymphocytes adhere to the
differentiated stem cell, penetrate
inside them. As a result, the
differentiated stem cell is
destroyed.
A
B
A. Adhesion of phagic T lymphocytes to the stem
cell that started to differentiate.
B. Penetration of a phagic T lymphocyte
into the stem cell.
Phagic T lymphocyte adhering to the stem cell
Phagic T ymphocytes present inside the stem cell
that has started to differentiate
‘Explosion of the stem cell under the action of phagic T lymphocytes’
Labat et al. Biomed and Pharmacother 2001;
Only normal stem cells are
destroyed by phagic T
lymphocytes when they start to
differentiate out of a repair
purpose (for instance, when
tissue repair is completed).
It is a beneficial exception to self
tolerance, in order to terminate
the repair process and to avoid
the development of diseases such
as fibrosis or malignant
proliferation.
Two major changes are observed when
the blood is drawned from patients with
fibrosis and/or malignant proliferations:
1. the organ stem cells proliferate
indefinitely,
2. they escape phagic T lymphocytes
control and as a consequence they
accumulate in vitro giving rise to a tissue
that evokes the pathology.
These findings have consequences
in terms of transplantation:
• Donor adult stem cells have to match
the tissue type of the host:
- not to avoid rejection, as is the case
for specialized cells,
- but to allow their control
by phagic T lymphocytes.
In fact, recent studies showed
that adult stem cells are not
rejected when transplanted into
mismatching recipient.
Adult organ stem cells are said to
be ‘invisible’ to the immune
system of the host.
Hori J, NG TF, Shatos M, Klassen H,
Streilein JW, Young MJ.
Neural progenitor cells lack
immunogenicity and resist
destruction as allografts.
Stem Cells 2003; 21: 405-416
Saito T, Kuang J-Q, Bittira B,
Al-Khaldi A, Chiu RC-J.
Xenotransplant Cardiac Chimera:
Immune tolerance of adult stem
cells.
Ann Thorac Surg 2002; 74: 19-24
This immune ‘invisibility’ is also a
property of embryonic stem cells.
This is an important finding since
human therapeutic cloning was
precisely proposed to avoid the
supposed inevitable rejection of
stem cells coming from
incompatible supernumeray
embryos.
Human therapeutic cloning was
proposed soon after Dolly the first
cloned sheep was presented to the
public.
Dolly was presented to the public on February 1997.
It was the first animal cloned with the genetic material
taken from adult cells.
From David Prentice
Nucleus extracted from the
ovocyte
Pipette
THERAPEUTIC CLONING
In vitro
culture
Ovocyte
Enucleated
ovocyte
Ovocyte with the
adult nucleus
Embryonic stem cells
under the influence of
Nucleus
of the adult
cell
hormones or vitamins
REPRODUCTIVE CLONING
Muscle
cells
Adult
adult
cell
Isolation
of the adult
nucleus
Skin
cells
Blood
cells
Implantation of the ovocyte
containing the adult nucleus in
Pipette
the womb of a foster mother
Therapeutic and reproductive cloning
Sciences et avenir 2002 ; N° 130 Special Issue
Neurons
When such ‘therapeutic cloning’ was
proposed, it was simply assumed
that transplantation rules for
embryonic stem cells should be the
same as for specialized cells.
It is now known that it is not the case:
Fändrich F, Dresske B, Bader M and
Schulze M
Embryonic stem cells share immuneprivileged features relevant for
tolerance induction .
J Mol Med 2002; 80: 343-350
These findings were confirmed by
studies from the Robarts Research
Institute in Canada:
Li L, Baroja ML, Majumdar A,
Chadwick K, Rouleau A, Gallacher L,
Ferber I, Lebkowski J, Martin T,
Madrenas J, Bhatia M
Human embryonic stem cells possess
immune-privileged properties
Stem Cells 2004; 22: 448-456
In addition, the adult organism cannot
control stem cells coming from a 5 day
old embryon (either ‘supernumerary
embryo or cloned embryo).
The ability to be controlled by phagic T
lymphocytes is, indeed, acquired during
the development of the immune system.
It is a property of adult stem cells that
distinguish them from embryonic stem
cells.
That explain, in part, why
embryonic stem cells grow into
teratoma and/or teratocarcinoma
when they are transplanted in an
adult organism.
That is also in agreement with the
fact that when these mouse
teratocarcinoma cells are reinjected
in a five day old normal mouse
embryo, they mix with the cells of
the inner mass and behave normally.
Reimplanted in a mouse uterus, this
5 day old embryo develops to give
birth to a normal mouse.
• Because embryonic stem cells are not
rejected when transplanted into
mismatching recipients, the reason why
human therapeutic cloning was proposed
disappears. Logically the debate should
have stopped.
• Despite these new findings, the political
debate still goes on.
Contrarily to embryonic stem
cells, adult stem cells have
already been successfully used in
clinical trials.
Horwitz EM, Prockop et al.
Transplantability and therapeutic
effects of bone marrow-derived
mesenchymal cells in children
with osteogenesis imperfecta.
Nat Med 1999; 309-313
The beneficial effect was
confirmed in 2002
Horwitz EM et al.
Isolated allogeneic bone marrow-derived
mesenchymal cells engraft and stimulate
growth in children with osteogenesis
imperfecta: implications for cell therapy
of bone. Proc Natl Acad Sci USA 2002;
99: 8932-8937
Succesful treatment of infarcted myocardium by bone
marrow graft
Germany
• Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S,
Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B,
Messingr D, Arseniev L, Hertenstein B, Ganser A, Drexler
H. Intracoronary autologous bone-marrow cell transfer after
myocardial infarction: the BOOST randomised controlled
trial. Lancet 2004; 364: 141-8
• Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C,
Klinge H, Schumichen C, Nienaber CA, Freund M,
Steinhoff G. Autologous bone-marrow stem-cell
transplantation for myocardial regeneration. Lancet 2003;
361: 45-46.
Perin EC, Dohamnn HF, Borojevic R, Silva SA,
Sousa AL, Mesquita CT, Rossi MI, et al.
Transendocardial, autologous bone marrow cell
transplantation for severe, chronic ischemic heart
failure.
Circulation 2003; 107: 2294-302
Strauer BE, Brehm M, Zeus T, Kostering M,
Hernandez A et al.
Repair of infarcted myocardium by autologous
intracoronary mononuclear bone marrow
transplantation in humans.
Circulation 2002; 106: 1913-8
China
Tse HF, Kwong Y-L, Chan JKF, Lo G, Ho C-L, Lau
C-P
Angiogenesis in ischaemic myocardium by
intramyocardial bone marrow mononuclear cell
implantation.
The Lancet; 2003: 47-49
Transplantation of bone marrow
stem cells for cutaneous wound
healing
Badavias EV, Falanga V
Treatment of chronic wounds with
bone-marrow derived cells.
Arch Dermatol 2003; 139: 510-516
Corneal graft with highly promising clinical
capabilities have been grown from limbal
stem cells.
Nishida K, Yamato M, Hayashida Y,
Watanabe K, Maeda N, Watanabe H et al.
Functional bioengineered corneal epithelial
sheet grafts from corneal stem cells expanded
in vivo on a temperature-responsive cell
culture surface
Transplantation 2004; 77: 379-385
Transplantation of limbal stem cells
in blind patients
Wylegala E, Tarnawska D,
Wroblewska EM
Limbal stem cell transplantation
from HLA-compatible living donor.
Long term observation.
Klin Oczna 2003; 105: 378-83
Implantation of autologous neural
stem cells in a patient with
Parkinson disease by Dr. Michel
Levesque, Los Angeles
A beneficial effect of 83% was reported in
September 2002, in the absence of additional
medication.
Treatment of spinal cord injury by
transplantation of stem cells from the
olfactory mucosa:
In Portugal (Dr. Carlos Lima),
11 patients around the world have
been already treated by such
procedure with some functional
improvement.
The cord blood : a rich source of
stem cells
(from the baby)
Cord blood contains hematopoietic
and organ stem cells that both belong
to the category of adult stem cells.
Dr. Nice Gardini from the ‘Centro de
Criopreservazione’ (Forli, Italy)
reported in July 2004 that for the
first time a french woman was
successfully treated for leukemia by
transfusion of cord blood from an
italian baby.
For other diseases than leukemia,
preliminary results from animal
studies, using cord blood organ stem
cells, are promising.
-We have to discover what these adult
stem cells really are.
.- We have to learn how they are
controlled. The mechanism shown here
is probably only one among many others.
- We have to understand why these
control mechanisms fail in pathological
situations of fibrosis and
chondrosarcoma
CONCLUSIONS
The research on adult stem cells
despite its rapid progress is still in
infancy.
It remains to discover what stem
cell therapy really is.
That is necessary to insure an
efficient and safe therapeutic use.
At the present time, it appears
that only adult stem cells can be
used for stem cells therapy
because the organism knows how
to control them.
If the research had been well
conducted, the rules governing
embryonic stem cells
transplantation should have been
studied in mice before claiming
a potential therapeutic use.
Peter Medawar, Nobel Price 1958
studied on mice the
transplantation rules for
specialized tissues, so helpful for
humankind.
In 1998, in his testimony in front of the
American Senate, Dr John Thomson,
proposed 3 main utilisations for the human
embryonic stem cells he just isolated:
- 1. to cure degenerative diseases
- 2. to better understand human embryo
development
- 3. to test new drugs.
« The biomedical industry is critical and
growing component of New Jersey’s
economy, and would be significantly
diminished by limitations imposed on
stem cell research »
Senate of New Jersey (USA) September
30, 2002
Embryonic stem cells lines, either
coming from ‘supernumerary
embryos’ or cloned embryos are
patentable intellectual property.
In the case of adult stem cells, in
most cases autologous grafts are
performed.
Money or Ideology
Ideology can be a driving force as
potent as money.
Humankind claims for himself the
creator’s right to interfere in the
mystery of life
John Paul II (august 2002)
Humankind claims for himself the
creator’s right to interfere in the
mystery of life
John Paul II (august 2002)
Ideology does not necessarily
lead to scientific truth.
When Louis Pasteur discovered
microorganisms, he had against
him all the ones who wanted to
believe in spontaneous
generation.
The multiplication of
interdisciplinary exchanges along
with philosophical and theological
reflection will foster the work of
truth and respect for the mystery of
human being
John Paul II (november 2001)
Addenda
The Wahington Fax is the official press organ of the NIHs