Cellules souches embryonnaires et reprogrammation: quelles ...€¦ · • Cellule souche (Stem...

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)


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


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)


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


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


� 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: les facteurs influençant la reprogrammation

Stadtfeld & Hochedlingler, 2010

Stem Cells: the iPSC: les facteurs influençant la reprogrammation:

Stadtfeld & Hochedlingler, 2010

MEF miPS (AP+)

20µm


A- hiPS B

C- AP+ D AP+


B- hiPS


CEF


ciPS – AP+


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).


Embryo

Somatic cells

CEF, ….

PGC

ES

iPS

Somatic

Reprogramming

Differentiation

Direct

Reprogramming


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


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.


Thank you for your attention

Stem Cells

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