Cellules souches embryonnaires et reprogrammation:
quelles différences entre espèces ?.
Berder 2013
Corrélation, causalité et régulation en biologie
Bertrand Pain
SBRI - Stem Cell & Brain Research Institute,
U846 INSERM, USC1361 INRA , UCB Lyon 1
De la poule ou de l’oeuf….
Les Holocéphales : les Callorhynchidés, les Chiméridés, les Rhinochiméridés.
Une chimère….
Une chimère….
Animal mythologique – Chimère d’Arruzzo – Bronze étrusque
Une chimère….
Une chimère….
Chimère génétique
Chimère génétique
Une chimère….
SSEA1
SSEA1
EMA1
EMA1
The GFP::cES cells are contributing to chimeras when injected into recipient Stage X embryos
Une chimère….
• Cellule souche (Stem cell - ‘SC’): une cellule capable de
s’autorenouveller, de différencier et donner des cellules spécialisées
différenciées
• Cellule souche induite (induced Pluripotent Stem cells – ‘iPS’) : unecellule souche obtenue par reprogrammation moléculaire d’une cellulesomatique
Stem Cells
Somatic cell
Nuclear
Transfert Self
renewal
Multipotent Stem
cell
Embryonic stem
- ES -cell
Stem Cells
Progenitor
Differentiation
potential
Ground/naïve
state
Somatic cell
Progenitors
Primed state
Nuclear
Transfert Somatic
Reprogramming
Self
renewal
Multipotent SCPluripotent ESC
mESCmEpiSC
hESC, RhESC
Stem Cells
Direct
Reprogramming
EC: Embryonic
Carcinoma(Kleinsmith et al. 1964)
ESC: Embryonic Stem Cells(Evans & Kaufman, 1981; Martin, 1981,
EG : Embryonic
Germ Cells(Matsui et al., 1992;
Resnick et al.;1992)
iPS: Induced
Pluripotent Stem(Takahashi & Yamanaka, 2006;
Okita et al., 2007
Werning et al., 2007)
EpiSC: Epiblast
Stem Cells(Brons et al., 2007
Tesar et al., 2007)
GSC: Germ Stem cells(Kanatsu-shinohara et al., 2004)
SSC: Spermatogonial Stem cells
TS: Trophoblast Stem Cells
(Tanaka et al., 1998)
XEN: Extra Embryonic
endoderm Stem Cells(Kunath et al., 2005)
Stem Cells: the mouse Embryonic Stem (mES) cells
• 1964: carcinomes embryonnaires
• 1975: cellules de carcinomes embryonnaires (cellules EC)
• 1978: anticorps SSEA1
• 1981: premières cellules souches embryonnaires (cellules ES)
• 1984: maîtrise de la recombinaison homologue
• 1992: obtention de cellules EG murines in vitro
• 1998: clonage murin
• 2000: cellules ES issues de clones
• 2003: cellules germinales issues de cellules ES in vitro
• 2004: obtention des GSC in vitro
• 2006: cellules iPS murines
• 2007: cellules EpiSC……
• 2008: cellules iPS humaines
• 2009: état naïf-induit
• 2010: reprogrammation directe
• ….
• � Obtention de nombreux modèles avec perte ou gain de fonction (knock out / knock in)
Stem Cells: the mouse Embryonic Stem (mES) cells
Stem Cells: the mouse Embryonic Stem (mES) cells
Stem Cells: the mouse Embryonic Stem (mES) cells
Eomes
Ascl2
Gata3
Tead4
Sox2 Nanog
Cdx2
Gata6
Gcnf
Oct4
Activation
Repression
Pluripotent stem cell
Trophectoderm
Extraembryonic endoderm
Eomes
Ascl2
Gata3
Tead4
Sox2 Nanog
Cdx2
Gata6
Coup-TFs
Gcnf
Elf5
Oct4
Activation
Repression
Pluripotent stem cell
Trophectoderm
Extraembryonic endoderm
� Schematic Transcription Factor network controlling ES cells self- renewal and
differentiation in the mouse model (adapted from Niwa , 2007).
Stem Cells: the control of pluripotency
Stem Cells: the control of pluripotency
Schematic representation of core pluripotency transcription factor circuit with parallel input
from LIF/Stat3 and GSK3 inhibition/Tcf3 derepression (adapted from Martello et al., 2012)
Two Phases of Pluripotency: Ground state naive pluripotency is established in the epiblast of the
mature blastocyst and may be captured in vitro in the form of ESCs. Shortly after implantation, the
epiblast transforms into a cup-shaped epithelium and becomes primed for lineage specification and
commitment in response to stimuli from the extraembryonic tissues. EpiSCs are the in vitro
counterpart of primed epiblast.. (adpated from Nichols & Smith, 2009)
Stem Cells: the control of pluripotency
4.5 dpc blastocyst
mESC mEpiSC
6.5 dpc blastocystLIF
FGF/Activin
Klf4
FGF/Activin
In vivo
In vitro
• How to establish and control pluripotency ?.
• Nuclear reprogramming by somatic cell nuclear transfer (SCNT)
– Wilmut et al., 1997; Wakayama et al., 1998;
• Cell fusion
– Cowan et al., 2005; Tada et al., 2001;
• Cell modification � génération des iPS (induced pluripotent Stem cells) (2006)
– Oct4: l’orchestrateur de la pluripotence et de la reprogrammation
– Sox2: le partenaire
– Klf4: le facilitateur
– c-Myc: le moteur
– Nanog: le gardien
– Lrh1, Lin28, Essrb, : les remplaçants
Stem Cells: the iPSC
Stem Cells: the iPSC
The Nobel Prize recognizes two scientists who discovered that mature, specialised cells can be reprogrammed to become immature cells capable of
developing into all tissues of the body. Their findings have revolutionised our understanding of how cells and organisms develop.
John B. Gurdon discovered in 1962 that the specialisation of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an
egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell
still had all the information needed to develop all cells in the frog.
Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem
cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are
able to develop into all types of cells in the body.
These groundbreaking discoveries have completely changed our view of the development and cellular specialisation. We now understand that the
mature cell does not have to be confined forever to its specialised state. Textbooks have been rewritten and new research fields have been
established. By reprogramming human cells, scientists have created new opportunities to study diseases and develop methods for diagnosis and
therapy.
The Nobel Prize in Physiology or Medicine 2012
jointly to
John B. Gurdon and Shinya Yamanaka
for the discovery that mature cells can be reprogrammed
to become pluripotent
http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/press.html
Stem Cells: the iPSC
� Première démonstration par Takahashi & Yamanaka, Cell 126, 663 (2006).
� Identification de Oct4, Sox2, Klf4 et c-Myc comme acteurs ‘essentiels’ = combinaison ‘OSKM’
Stem Cells: the iPSC
Stem Cells: the iPSC: les facteurs influençant la reprogrammation
Stadtfeld & Hochedlingler, 2010
Stem Cells: the iPSC: les facteurs influençant la reprogrammation:
Stadtfeld & Hochedlingler, 2010
MEF miPS (AP+)
20µm
Stem Cells: the iPSC
A- hiPS B
C- AP+ D AP+
Stem Cells: the iPSC
B- hiPS
Stem Cells: the iPSC
CEF
Stem Cells: the iPSC
ciPS – AP+
Stem Cells: the iPSC
mES AP+ cES AP+
ciPS AP+
Expression of mouse OSK factors in tadpole
muscle leads to GFP loss and cell mass
appearance
Generation of Xenopus-iPS-like cells in vivo.
(from Vivien et al., 2012).
Stem Cells: the iPSC
Stem Cells: the iPSC
Embryo
Somatic cells
CEF, ….
PGC
ES
iPS
Somatic
Reprogramming
Differentiation
Direct
Reprogramming
Stem Cells: the iPSC
hES cells SCNT ES cells iPS cells Adult stem cells
Derivation method Removal of cells from
ICM of blastocyst
embryo from IVF
Transfer of somatic cell
nucleus to enucleated
egg, development to
blastocyst,
removal of ICM
Reprogramming of
somatic cells by
introduction of specific
regulatory factor genes
Isolation from
adult tissues
Characteristics Differentiate into all
cell types
Differentiate into all
cell types
ES cell like
characteristics
Successful treatments
demonstrated
Excess of IVF
embryos exist
Stem cells can be
matched to patient
Stem cells can be
matched to patient
Stem cells can be
matched to patient
Limitations Immune rejection
tissues
No human SCNT
cell lines exist
Unknown if cells can
differentiate into all
cell types
Cells not found in all
tissues Produce a limited
number of cell types
Risk of tumors
(teratomas) from
transplanting
undifferentiated cells
Risk of tumors
(teratomas) from
transplanting
undifferentiated cells
Risk of tumors
(teratomas) from
transplanting
undifferentiated cells and
from expression of
introduced genes
Difficult to identify,
isolate and
grow
Stem Cells: the iPSC
Fig. 1 Schematic depiction of various aspects of regenerative medicine.
Cells from humans (in some cases from the patients themselves) can be harvested, cultured ex vivo and
differentiated to desired cell types, which can then be used for transplantation. In addition, human ES cells and
iPS cells may in the future be used for cell therapy. Human ES cells and iPS cells are also valuable resources for
in vitro studies, for example for toxicity screenings of pharmaceutical products or for learning more about the
disease process. In some cases, endogenous stem cells may be activated in vivo for tissue repair.
Stem Cells: the iPSC
Thank you for your attention
Stem Cells
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