The rabbit preimplantation embryo as a paradigm to explore naive pluripotent stem cell derivation in non rodent species

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To cite this version:

Pierre Osteil, Yann Tapponnier, Anaïs Moulin, Thierry Joly, Pierre Savatier, et al.. The rabbit preimplantation embryo as a paradigm to explore naive pluripotent stem cell derivation in non rodent species. 11. ISCCR annual meeting, Jun 2013, Boston, United States. 1 p., 2013. �hal-02806396�

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Control/Tracking Number: 2013-P-1269-ISSCR Activity: Poster

Current Date/Time: 2/7/2013 5:04:00 AM

THE RABBIT PRE IMPLANTATION EMBRYO AS A PARADIGM TO EXPLORE NAIVE EMBRYONIC STEM CELL DERIVATION IN NON RODENT SPECIES

Author Block: OSTEIL, Pierre¹, TAPPONNIER, Yann¹, MOULIN, Anaïs¹, JOLY, Thierry², SAVATIER, Pierre¹, AFANASSIEFF, Marielle¹

1Stem cell and Brain Research Institute, INSERM U846, INRA USC1361, BRON, France, ²VetAgroSup, UPSP ICE, ISARA-Lyon, Lyon, France

Abstract:

The scarcity of pre-implantation embryos in human is a major limitation to the improvement of embryonic stem cell (ESC) derivation in non-rodent species. Rabbit is a good alternative as it can produce up to 30 embryos per super-ovulated females. Moreover, all rabbit ESC line produced so far showed the characteristics of primed pluripotency like human ESCs. In this work, we investigated the conditions suitable for the generation of naïve ESCs in rabbits. A first experiment aimed to study the effect of the pharmacological inhibitor of MEK signaling, PD0325901, on embryo development and ESC line derivation. We collected 576 eight-cell stage embryos, of which 317 were cultured in the presence of MEK inhibitor until they reached the early blastocyst stage. No difference in the rate and quality of embryo development between MEK inhibitor-treated and control embryos was observed. ICMs were isolated by immunosurgery and plated onto gelatin- or fibronectin-coated dishes in various media (DMEM/F12 + 20% KOSR, N2B27) supplemented with GSK3 inhibitor (CHIR99021), MEK inhibitor, and LIF (2i/LIF), or not. 80% of the ICMs plated, but none could be expanded beyond passage 2. We concluded that inhibition of MEK signaling fails to prevent spontaneous differentiation of

pluripotent stem cells in rabbit. A second experiment aimed to study the effect of LIF on ESC line derivation. We collected 262 ICMs, which were plated onto growth-inactivated mouse embryonic fibroblasts in DMEM/F12 supplemented with 20% KOSR (72), 20% KOSR + LIF (90), or 10% KOSR + 10% FCS + LIF (80). No outgrowth cultured in media lacking LIF could be expanded beyond passage 4. By contrast, 7 ESC lines were derived from outgrowths cultured and expanded in the presence of LIF, 2 in 20% KOSR + LIF, and 5 in 10% KOSR + 10% FCS + LIF. Two lines, designated rbES-LIF1 and rbES-LIF2, were expanded by gentle dissociation with collagenase until passage 40, and showed a normal karyotype. RbES-LIF1 and rbES-LIF2 displayed the cardinal features of pluripotent stem cells, i.e. expression of pluripotency markers, differentiation into derivatives of the 3 germ layers, and teratoma formation. However, they could not be cultured onto gelatin-coated dishes, they did not express markers associated with naïve pluripotency in rodents, and they did not survive in 2i/LIF medium. We concluded that LIF facilitates the derivation of ESCs but does not support naïve pluripotency in rabbits. When rbES-LIF1 and rbES-LIF2 cells were propagated for 20 passages in 10% KOSR + 10% FCS + LIF, and were enzymatically dissociated with Accutase into single cell suspensions, they acquired

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Secondary Topic: Embryonic Stem Cell Pluripotency

Keywords (Complete): Derivation ; Embryon ; Rabbit Additional Information (Complete):

*Does the research described in this abstract include a clinical trial?: No

*Is the research described in this abstract part of an academic-industry partnership or collaboration?: No

Awards (Complete):

*Would you like your submission to be considered?: Yes *I would like to apply for a travel grant.: Yes

Title: Junior Investigator Grad or Post Grad Student

Payment (Complete): Your credit card order has been processed on Thursday 7 February 2013 at 5:03 AM.

Status: Complete

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OCT4

0.00.20.40.60.81.0

NANOG

0.00.20.40.60.81.0

REX1

0.00.20.40.60.81.0

KLF4

0.00.20.40.60.81.0

TBX3

0.00.20.40.60.81.0

CLDN6

0.00.40.81.2

PIWIL2

0.00.20.40.60.81.0

DAZL

0.00.20.40.60.81.0

FBXO15

0123456

GBX2

020406080

ESRRB

0.00.20.40.60.81.0

CDH1

0.00.40.81.2

CDH2

0200400600800

0,00%

20,00%

40,00%

60,00%

80,00%

100,00%

0,00%

20,00%

40,00%

60,00%

80,00%

100,00%

ICM p0 p1 p2 p3 p4 p5 p6 p7 p8 p9 p10

KOSR + bFGF KOSR + LIF

KOSR + FCS + LIF + ACC KOSR 2i/LIF

Authors : OSTEIL, Pierre ¹ , TAPPONNIER, Yann ¹ , MOULIN, Anaïs ¹ , JOLY, Thierry ² , SAVATIER, Pierre ¹ , AFANASSIEFF, Marielle ¹

1 Stem cell and Brain Research InsTtute, INSERM U846, INRA USC1361, BRON, France, ² VetAgroSup, UPSP ICE, ISARA-Lyon, Lyon, France

INTRODUCTION: the scarcity of pre-implantaTon embryos in human is a major limitaTon to the improvement of embryonic stem cell (ESC) derivaTon in non-rodent species. Rabbit is a good alternaTve as it can produce up to 30 embryos per super- ovulated females. Moreover, all rabbit ESC line produced so far showed the characterisTcs of primed pluripotency like human ESCs. In this work, we invesTgated the condiTons suitable for the generaTon of naïve ESCs in rabbits.

RbESC-FGF RbESC-LIF RbESC-ALF ICM

0 10 20 30

40 41/42 41 41/42 42 42/43 43 43/44 44 44/45 45

LIF 1 P24 LIF 2 P25

Phase contrast AP staining Teratoma

Oct4 Karyotype

Phase contrast AP staining

48 hours post-injecTon -ALF4 -ALF8

48 hours post injecIon -ALF8

1 hour post-injecTon

0 0,2 0,4 0,6

40 41 42 43 44 45 46 47

ALF4 ALF8

GFP+

RbESC AnT SSEA1-

AF647

Cell sorTng InjecTon into 8- cell stage embryos Confocal analysis at blastocyst stage

Phase contrast

PI + GFP overlay Embryos at 1 hour post injecTon

Phase contrast

GFP

SSEA1

Merge

0 5 10

-505

PCA

Dim 1 (63.93%)

Dim 3 (4.41%)

LIF2LIF1 ES 4 TOT

ES 4 NEG ES 4 S1+

ES 8 TOT

ES 8 NEG ES 8 S1+

ES 18 TOT

ES 18 NEG ES 18 S1+

ES 19 TOT

ES 19 NEGES 19 S1+ ALF4 P10ALF4 P5ALF2 P37 ALF2 P39 ALF6 P6

ALF6 P9 ALF8 P5ALF8 P9

ALF10 P5ALF10 P8ALF12 P5ALF12 P8 ICMICM RbES_ALF

RbES_FGF

RbES_LIF

ICM RbES_ALF RbES_FGF RbES_LIF

Oct4 Phase contrast AP staining

Mesoderm

Oil Red O Desmine

GFAP

Ectoderm Endoderme

HPS

Teratoma

0 10 20 30 40 50

40 41/42 41 41/42 42 42/43 43 43/44 44 44/45 45

ES 4 P13 ES8 P17 ES18 P15 ES19 P21

GFP+

RbESC InjecTon into 8-cell stage embryo

II-RbESC-FGF

I- Pre-implantaIon embryo development, and ICM growth aRer plaIng onto feeders (+/- MEK inhibiIon with PD032)

III-RbESC-LIF IV-RbESC-ALF

V- Gene expression analysis

CONCLUSIONS:

1/ InhibiTon of MEK signaling prevents ICM outgrowths from generaTng ESCs in rabbit.

2/ Rabbit ESCs derived in Fetal Calf Serum on feeders, and dissociated to single cell suspension during passaging (RbESC-ALF), exhibit an elevated expression of genes whose expression is associated with naive pluripotency in rodents.

3/ RbESC-ALF cells exhibit a shorter G1 phase.

4/ RbESC-ALF cells can colonize the ICM of the rabbit blastocyst following injecTon into 8-cell stage embryos

RbESC-FGF cells display the cardinal features of embryonic stem cells. They express pluripotency markers, they form teratomas, and they have a stable karyotype (44XY).

Details are available in Osteil, Tapponnier, Markossian et al. Induced pluripotent stem cells derived from rabbits exhibit some characterisTcs of naïve pluripotency (2013) Biology Open

Karyotype

A) CharacterizaTon A) CharacterizaTon

G1 : 72.7%

S : 11%

G2/M : 16.3%

G1 : 55.9%

S : 20.2%

G2/M : 23.9%

G1 : 46.6%

S : 31.9%

G2/M : 21.5%

RbESC-FGF cells have a poor growthy rate and exhibit a long G1 phase.

G1 phase duraTon is an important feature of naïve cells. See Coronado et al. A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency. (2013) Stem Cell Research

B) Cell cycle analysis

C) ICM colonizaTon aeer injecTon into 8-cell stage embryos

One hundred and twelve 8-cell stage embryos were injected with 10-15 cells, and further cultured to the blastocyst stage. No blastocyst with GFP posiTve cells in the ICM could be observed.

259 252

203 317

271 254

0,0%

10,0%

20,0%

30,0%

40,0%

50,0%

60,0%

70,0%

80,0%

90,0%

100,0%

8 cells

(D0) Morula

(D1) Blastocyst

(D2)

KSOM KSOM + PD032

1-14

4-18

ICM plated

on feeders Day 1 Day 2

N o d i ff e r e n c e between PD032- t r e a t e d a n d untreated embryo i n t h e r a t e o f s u c c e s s f u l development was o b s e r v e d . I C M cultured in PD032 failed to generate ESC lines.

Serum : 20% KOSR

Growth factor : FGF2 Feeders: MEF (from OF1 mice)

Passaging: Clumps (Collagenase) Serum : 20% KOSR

Growth factor : LIF Feeders: MEF (from OF1 mice)

Passaging method: Clumps (Collagenase) Serum : 10% KOSR + 10%FCS

Growth factor : LIF Feeders: MEF (from OF1 mice)

Passaging method: Single cell suspension (Accutase)

Karyotype

A) CharacterizaTon

B) Cell cycle B) Cell cycle analysis

A) Barplots from qPCR data

B) Principal Component Analysis from qPCR data

R b E S C - L I F c e l l s display the cardinal f e a t u r e s o f embryonic stem cells. They express p l u r i p o t e n c y markers, they form teratomas, and they h a v e a s t a b l e karyotype (44XY).

RbESC-LIF cells exhibit a shorter G1 phase as compared to RbESC- FGF.

RbESC-ALF cells have a cell- cycle distribuTon characterized by a smaller fracTon of cells in G1 phase as compared to other rabbit ES cells.

0 0,1 0,2 0,3 0,4 0,5

40 41 42 43 44 45 46 47

ALF2 ALF6 ALF10 ALF12

RbESC-ALF cells are pluripotent (characterizaTon is ongoing). However, t h e y a c q u i r e d c h r o m o s o m a l abnormaliTes during passagings (43XX, 45XY)

Two RbESC-ALF lines have a relaTvely more stable karyotype (44XX)

RbESC lines ALF2 ALF4 ALF6 ALF8 ALF10 ALF12 TOTAL Injected embryos

(8-cell stage

embryos) 41 68 42 57 45 50 303

PosiTve embryos

+48h 2 12 10 7 12 5 48

PosiTve blastocysts

+48h 0 2 4 1 2 0 9

Of 303 embryos that developed to t h e b l a s t o c y s t stage, 9 had GFP posiTve cells in the ICM.

A s e l e c T o n o f pluripotency-associated genes was analyzed by q u a n T t a T v e R T - P C R . Followed by a PCA analysis According to the molecular signatures, the RbESC-FGF are the furthest from the ICM, whereas the RbESC- ALF cells are the closest.

RbESC-LIF cells exhibit an intermediate signature

Percentage of expanding ICM outgrowths during serial passaging (p0 to p10)

rbESC-FGF rbESC-LIF

rbESC-ALF rbESC-2i

AKNOWLEDGMENTS: This work was supported by research grants from ‘‘Agence NaTonale de la Recherche’’ (ANR) (project PLURABBIT no. PCS-09-GEM-08), European cooperaTon in Science and Technology (COST) AcTon (Rabbit Genome Biology (RGB)-Net no. TD1101), the HyPharm

C) ICM colonizaTon aeer injecTon into 8-cell stage embryos

The RbESC-ALF cells

acTvated the expression

of markers of naïve

pluripotency (Cdh1, Esrrb,

Rex1, Tbx3, Piwil2, Dazl)