Derivation of induced pluripotent stem cells from an infertile boar carrying a reciprocal translocation

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Derivation of induced pluripotent stem cells from an infertile boar carrying a reciprocal translocation

Annabelle Congras, Harmonie Barasc, Florence Vignoles, Alain Pinton, Marielle Afanassieff, Bertrand Pain, Olivier Feraud, Kamila Canale-Tabet,

Martine Bouissou-Matet Yerle, Herve Acloque

To cite this version:

Annabelle Congras, Harmonie Barasc, Florence Vignoles, Alain Pinton, Marielle Afanassieff, et al.. Derivation of induced pluripotent stem cells from an infertile boar carrying a reciprocal translocation. Conference 2014 Epiconcept ”Epigenetics and Periconception Environment”, Oct 2014, Vilamoura, Portugal. COST Office European Cooperation in Science and Technology (Portugal), 2014, Proceed-ings of the EPICONCEPT Conference 2014 - COST Action FA1201. �hal-02739129�

Derivation of induced pluripotent stem cells from an infertile

boar carrying a reciprocal translocation

Annabelle Congras

, Harmonie Barasc

, Florence Vignoles

, Alain Pinton

, Marielle Afanassieff

, Bertrand Pain

, Olivier

Feraud

, Kamila Tabale-Canet

, Martine Bouissou-Matet Yerle

and Hervé Acloque

1. UMR1388 INRA/ENSAT/ENVT GenPhySE, Castanet-Tolosan, France 2. INSERM U846 and PrimaStem, SBRI, Bron, France

3. INSERM U935, Villejuif, France

EpiConcept Conference

Epigenetics and Periconception Environment

Vilamoura 01-03 October 2014

New insights in pig pluripotency by two approaches of cell reprogramming

Adult cell

Cocktail of reprogramming factors

Expression of pluripotency genes (1) and transcriptomic study (5) ES-like cell cycle (2) Teratomas formation (4) Karyotype stability (3) Embryoïd bodies formation (4) Chimaeras formation Silencing of exogenes (4) iPS cell INTRODUCTION

Derivation of germ cells from embryonic (ES) or induced (iPS) pluripotent stem cells in mouse and human is a great advance for the study of germ cell biology in the earlier step of development and provide an in vitro model for the analysis of new mechanisms leading to infertility.

Based on recent advances in pig iPS production, we propose here to create a library of porcine

iPS derived from fibroblasts of infertile boars carrying chromosomal rearrangements.

Using two systems of cell reprogramming, we produced and characterized iPS-like cell lines from fibroblasts with a t(Y;14) reciprocal translocation. Gene expression analysis, differentiation assays and cytogenetic analysis were used to assess the reprogramming efficiency of both protocols and the pluripotency level of the cell lines. This study gives new insights on pig pluripotency of which mechanisms have not been completely solved yet.

Two approaches :

Integrative viral infection  translocated lines I3 and I4 and

control line PB20

Non-integrative viral infection  translocated lines NI12,

NI13 and NI20

0 20 40 60 80 100 I3 p9 I3 p42 I3 p78 I3 p110 M étap h as es ( % ) SSC8 + SSC4 I4 0 20 40 60 80 100 I3 p9 I3 p42 I3 p78 I3 p110 Métaphas es ( % ) 8q+ der-Tri16 12p+ 9q+ 0 20 40 60 80 100 PB20 p15 PB20 p40 PB20 p73 Métaphas es ( % ) tri10 6q-

9q-NI13 (38 XY, t(Y;14))

NANOG OCT

4

SOX2 LIN28B CDH1 SSEA4

I3 I3 LIF+2i PB20 NI13 Brightfiel d 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% PEF I3 bFGF I4 bFGF PB20 bFGF I3 2i+LIF I4 2i+LIF PEF DH I3 bFGF DH I4 bFGF DH PB20 bFGF DH I3 2i+LIF DH I4 2i+LIF DH Cel lul es ( %) G2 S G1 Fluorescence Fluorescence Fluorescence Fluorescence Cell n u m b e r h OCT 4 hS OX 2 hK L F4 hC -M Y C S e V A CT B h OCT 4 hS OX 2 hK L F4 hC -M Y C S e V A CT B NI13 p9 NI13 p28 I3 I4

• Both I and NI lines express the core pluripotency genes.

• Integrative lines are maintained in LIF+2i culture media which promotes induction of naïve pluripotency.

• Pluripotency genes network in non integrative lines is more activated.

• Integrative lines accumulate chromosomal abnormalities and duplications among

passing in culture while non integrative line NI13 is stable at passage 45.

• Integrative lines have an ES-like cell cycle characterized by a short G1 phase and absence of the G1/S DNA damage checkpoint.

1. EXPRESSION OF PLURIPOTENCY GENES

2. ES-LIKE CELL CYCLE

4. DIFFERENTIATION ASSAYS : EBs and teratomas formation

5. TRANSCRIPTOMIC STUDY

3. KARYOTYPE ANALYSIS

• Integrative lines have a poor

differentiation potential : teratoma assay led to carcinoma formation. • NI lines form embryoid bodies resembling pig blastocysts in

morphology and gene expression. • Continuous transgene expression could prevent cells to complete

reprogramming and blocks cell differentiation.

• Clustering of iPS-like cell lines compared to fibroblasts.

• Up-regulation of cell-cycle related genes in reprogrammed lines.

• Differential expression of genes involved in

organogenesis, embryo development, cancer and pluripotency pathways between I and NI lines.

• Up-regulation of lipid metabolism in embryoïd bodies.

CONCLUSION AN PROSPECTS

We used two different reprogramming systems (I and NI) to produce iPS-like cells from t(Y;14) fibroblasts coming from an infertile boar. These cells lines share key pluripotency features with mouse and human iPS cells, like the expression of core pluripotency genes and the

specificity of their cell cycle. Depending on the culture conditions, I

cell lines express genes of both primed and naïve pluripotency. Those cells have a poor differentiation potential and accumulate easily

chromosomal abnormalities.

NI lines on the contrary are karyotypically stable over time, show a slow repression of exogenous reprogramming factors, seems to deeper activate the pluripotency network even in primed culture conditions and form blastocyst-like embryoïd bodies.

Further studies are needed to break down the barriers blocking the

complete reprogramming of pig somatic cells and access to ground

pluripotency : epigenetic barrier, loss of transgene expression, role of different signaling pathways (lipid metabolism) in pluripotency and requirement to extrinsic factors for cell differentiation.

PEF

I3 Cell cycle Cell cycle with

DNA damages Cell n u m b e r Cell n u m b e r Cell n u m b e r 0.002 0.004 0.008 0.016 0.031 0.063 0.125 0.250 0.500 1.000 2.000 4.000 8.000 16.000 32.000 64.000 F o ld C h a n g e I3 bFGF I3 LIF+2i I4 bFGF I4 LIF+2i ** *** *** *** * * *** * *** *** ** * *** * * ****** * *** ** *** *** * Blastocyst EB SOX2 and CDX2 expression in EB