Dr_Labat_Stem_cells_10th_FEAMC_Meeting_Bratislava by yantingting



   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.



                          Embryonic germ         Multi
           Pluripotent       (EG) cells          potent
                          Tissue stem cells

                                   Adapted from David Prentice
Distinguishing Features of Progenitor/Precursors
              Cells and Stem Cells.
Human embryo at stage 8 cells
            (2 days)
     8 totipotent stem cells

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.

                          One cells
                          is taken
In vitro
culture                                One cell is sampled from each embryo
                  Embryo between          fertilized and cultured in vitro
                    6 to 12 cells

                                      Diagnostic test on each sampled cell

                                        Healthy embryos are selected for
In vitro fertilized ovocyte               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
• 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

- 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.
    Human embryonic stem cell lines approved by President
     Bush for federally funded research (August 9, 2001)

.   CyThera (California)                             9
•   ES Cell International (Australia)                 6
•   Geron Corporation (California)                    7
•   University of Göteborg (Sweden)                  19
•   Maria Biotech Co (Korea)                         3
•   Miz Medi Hospital (Korea)                         1
•   National Center for Biological Science (India)    3
•   Pochon CHA University (Korea)                    2
•   Reliance Life Sciences, Bombay (India)            7
•   Technion University, Haifa (Israel)              4
•   University of California SF (California)         2
•   Wisconsin Alumni Research Foundation             5
•   Bresagen, Inc (Georgia, USA)                     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, Itskovitz-
             Taylor 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
 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.



                        Embryonic germ         Multip
         Pluripotent       (EG) cells          otent
                        Tissue stem cells

                                 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
 Now, adult stem cells have been
found in every organ where it has
 been looked for them, including
   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
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
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
• they express CD14+
  and CD68+ markers
• They express
  constitutively HLA-DR
  Once they have adhered,they give
    rise to cells with different
phenotypes, among them fibroblast-
             like cells

They express collagen 1, collagen, 3, procollagen 1,
and fibronecti, while still expressing monocyte
  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
          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
Two major changes are observed when
the blood is drawned from patients with
fibrosis and/or malignant proliferations:
1. the organ stem cells proliferate
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
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
 Human therapeutic cloning was
proposed soon after Dolly the first
cloned sheep was presented to the
 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
                                                                                                   THERAPEUTIC CLONING
             Pipette                         ovocyte
                                                                                    In vitro

 Ovocyte               Enucleated                                Ovocyte with the
                        ovocyte                                   adult nucleus
                                                                                                    Embryonic stem cells
                                                                                                    under the influence of
                                                                                                    hormones or vitamins
                         of the adult
                             cell                        REPRODUCTIVE CLONING
 Adult        of the adult
  cell          nucleus


                                                           Implantation of the ovocyte                                       Neurons
                                                         containing the adult nucleus in
                                                          the womb of a foster mother

                                        Therapeutic and reproductive cloning
                             Sciences et avenir 2002 ; N° 130 Special Issue
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 immune-
  privileged 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
   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
• 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
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

Tse HF, Kwong Y-L, Chan JKF, Lo G, Ho C-L, Lau
Angiogenesis in ischaemic myocardium by
intramyocardial bone marrow mononuclear cell
 The Lancet; 2003: 47-49
 Transplantation of bone marrow
 stem cells for cutaneous wound

     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
  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
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
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
 The research on adult stem cells
despite its rapid progress is still in

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
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

- 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
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
        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)
The Wahington Fax is the official press organ of the NIHs

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