HAL Id: hal-02746874
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Submitted on 3 Jun 2020
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Molecular and functional characterization of rabbit embryonic stem cell lines
Pierre Osteil, Suzy Markossian, Murielle Godet, Thierry Joly, Pierre Savatier, Marielle Afanassieff
To cite this version:
Pierre Osteil, Suzy Markossian, Murielle Godet, Thierry Joly, Pierre Savatier, et al.. Molecular and functional characterization of rabbit embryonic stem cell lines. 4. International Rabbit Biotechnology Meeting, Jun 2011, Budapest, Hungary. 2011, 4th International Rabbit Biotechnology Meeting, 30th June - 1st July, Budapest, Hungary. �hal-02746874�
MOLECULAR AND FUNCTIONAL CHARACTERIZATIONOF RABBIT EMBRYONIC STEM CELL LINES
OSTEIL P.1, MARKOSSIAN S.1, GODET M.1, JOLY T.2, SAVATIER P.1, AFANASSIEFF M.1
1AgroBioStem, USC INRA/INSERM/UCB Lyon1 2008, Stem Cell and Brain Research Institute, INSERM U846, Bron, France.
2Unité CRYOBIO, UPSP ENVL/ISARA-Lyon, ISARA, Lyon.
Lines mESc RbFc EScRh RbES-4 RbES-8 RbES- 18 RbES-19
Day of culture 1 3 3 1 3 1 3 3 1 3
OCT4 + - ++ + + + + + + +
Nanog + - ++ + + + + + + +
SSEA1 + - - + - + - + + +
SSEA4 - - ++ - - - - - - +
CD90 - - ++ - - - - - - -
TRA-1-60 - - ++ - - - + - - -
N-Cadh - - + + + + + + + +
E-Cadh + - + + + + + + + +
2. Pluripotency markers
AntibodyHoechst
OCT4 SSEA-1 E-Cadh N-Cadh Tra1-
SSEA4 60 CD90
7. Injection of SSEA1+RbESCs into 8-cell stage rabbit embryos
6. Interspecies injection of mESCs and RbESCs into 8-cell stage embryos
GFP+ RbESC Anti SSEA1-
AF647
Cell sorting
Injection into 8- cell embryo
CAG Lentiviral Vector EOS
Lentiviral Vector GAE
(2) (3)
DE-Oct4+ETP
GAE
Control EOS
Wight
BL BL
BL
BL BL BL
BL BL
GFP
GFP GFP GFP
GFP GFP
GFP
GFP GFP
48h PI
69h PI
6D PI
ICM infection and analysis of
GFP expression during RbESC derivation
4. Expression of the distal Enhancer of mouse Oct4 gene in RbESCs
Infection of 16-cell embryos and analysis at blastocyst stage
Bright light
Green fluorescence
GFP
immuno-labelling
GAE
EOS
ICM
trophectoder m
zona pellucida
Hotta et al., Nat Methods 2009, 6:370-6
control EOS
Mouse ES Human ES (H1)
Mouse EpiSC Rabbit ES
GFP expression in EOS-infected RbESC
G1 G2
S
M
Pluripotent cell cycle
Check point
G2 G1
S M
Somatic cell cycle
Check point Check
point
5. Cell cycle of RbESCs
controladriamycine
Mouse ES Mouse EpiSC (cl.1) Mouse EpiSC (cl.2) Rabbit fibroblasts
controladriamycine
RbESC-8 RbESC-19
RbESC-4 RbESC-18
Mésoderm
Red O Oil Desmine
GFAP
Ectoderm Endoderm
Teratoma on SCID mouse kidney
HPS
Embryoïd bodies from
RbESC
3. Pluripotency
RbESC-4 RbESC-18
1 2 3 4
Injection into 8-cell embryo
Confocal analysis at blastocyst stage
Bright light
PI + GFP overlay
Conclusion
Our results indicate that rabbit ESCs exhibit a heterogeneous profile of pluripotency markers and do not all show the cell-cycle cardinal features of pluripotent stem cells. Furthermore, they are unable to participate in embryo development in vivo.
GFP-Heterozygote male Second generation morula
50% wild type
50% GFP positive
1. Establishment of rabbit embryonic stem cell (RbESC) lines
Green fluorescenceBright light
Wild female PCR against SRY on RbESC
Karyotype of RbESC-4
Morphology (x40) Alkaline phosphatase (x40)
44XY
Nanog
Bright light
GFP
SSEA1
Merge
Embryos just after GFP+/SEEA1+ RbESC injection
Introduction
We have derived four Rabbit Embryonic Stem Cell (RbESC) lines from New Zealand GFP-transgenic and wild-type embryos. Inner cell masses (ICMs) were isolated from 25 rabbit blastocysts by immunosurgery, and plated onto growth-inactivated murine embryonic fibroblasts in a medium supplemented with 13ng/ml FGF2. Fifty percents were able to form an outgrowth, and 16% produced a population of highly proliferating cells that could be regularly passaged.
GFP+ ESCs
RbESCs
mESCs hESCs
SSEA-1 SSEA-1
SSEA-1
SSEA-4 SSEA-4
SSEA-4
1 2 3 4
Bright light
GFP
Merge
Confocal Overlay
