Getting true Pluripotent Stem Cells in Pigs: a key step for large scale ex-vivo "Genotype to Phenotype" studies – PluS4PiGs

To achieve its objectives, the project is organized around five scientific tasks, which are partially interdependent. They call on the skills and expertise of different partners in genomics, proteomics, bioinformatics, animal reproduction and stem cell biology.
Task 1: Molecular characterization of the embryonic microenvironment
We will generate and use single-cell expression (scRNA-seq) and chromatin accessibility (scATAC-seq) data from four stages of embryonic development (early, expanded, spherical and ovoid blastocysts). The proteome of uterine fluids will also be characterized by mass spectrometry. All of these data will be used to identify and characterize the cell populations present in the embryo as well as the molecular interactions between cells of the same population or between cells of different populations.
Task 2: Production of cell lines with reporter systems for pig pluripotency
Using CRISPR / Cas9 technology, we will integrate fluorescent reporter genes upstream of promoters of genes specifically active in pluripotent cells (OCT4, NANOG, SOX2, UCHL1). The combinatory of the different reporters will make it possible to select and visualize with precision the cells having activated the endogenous pluripotency networks.
Task 3: Definition of a combination of exogenous factors sufficient for reprogramming to a state of pluripotency
We will use the cell lines integrating fluorescent reporter systems for endogenous pluripotency to test the efficiency of new reprogramming factors identified in task 1 (transcription factors and epigenetic effectors). We will validate the combinations of reprogramming factors according to their ability to reactivate fluorescent reporters.
Task 4: Screening of small bioactive molecules capable of maintaining porcine pluripotency in vitro
We will use the lines produced in task 2 and integrating fluorescent reporter systems for endogenous pluripotency to screen for bioactive molecules. We will validate the combinatory of these molecules according to their ability to reactivate fluorescent reporters.
Task 5: Establishment of pluripotent pig cell lines
From the data from tasks 1 to 4, we will identify the molecules and factors necessary and sufficient to maintain porcine pluripotency in in vitro cultures. We will establish pluripotent lines from embryonic explants and from reprogrammed somatic cells. These lines will be characterized by their transcriptomic and epigenomic profile, their ability to differentiate both in vitro and in vivo.

Not concerned yet

PluS4PiGs is a collaborative project that aims to better understand the cellular and molecular mechanisms controlling embryonic pluripotency in the porcine species. The project also aims to transfer the knowledge acquired to facilitate the derivation and use of porcine pluripotent cells in G2P studies (from genotype to phenotype). This project offers promising prospects in different eras with a number of major breakthroughs:
Strong innovation potential for the animal breeding sector: pluripotent cells can be used to accelerate the identification of causal genetic variants controlling production, health and welfare traits. The genome of these cells can be easily modified to compare the effects of different alleles in the same haplotypes and in many different cell types. This information can then be used in prediction models for genomic selection or to introgress alleles of interest into a given population. Ultimately, the acquisition of porcine pluripotent cells will have major agronomic applications, in particular for improving the predictive power of genomic selection and the sustainability of the French and European pig industry.
A strategic model for ex vivo phenotyping and for reducing animal experimentation: Pluripotent cell lines represent an excellent biological system to study cell differentiation and organogenesis ex vivo thanks to the recent development of 3D cell culture systems. These protocols are now widely used to produce organoids from human pluripotent cells and to define ex vivo new treatments for so-called complex pathologies. The extension of these approaches to porcine pluripotent cells will allow large-scale phenotyping of traits that are difficult and / or expensive to measure in vivo, without increasing the use of animal experimentation, a major ethical and societal issue. Indeed, this project will actively contribute to the development of alternative methods to animal experimentation, which is a necessity today.
Cutting-edge fundamental research: pigs represent a valuable model for discovering new mechanisms and markers of pluripotency. Unlike humans and mice, embryonic development in pigs is characterized by a long pre- and peri-implantation phase in utero similar to that of most other mammals. Our study will lead to new discoveries on the molecular mechanisms controlling pluripotency and will highlight their conservation and / or divergences between mammalian species.

On-going project

The current increase in world meat demand together with global changes (climate changes, availability of agricultural resources, societal perception of breeding) require us to rethink our production systems. The pressure of livestock production on ecosystems needs to be reduced, food and health security need to be increased, as well as animal welfare. To achieve these objectives, a better knowledge of the link between genotype and phenotype is necessary.
Development of efficient cellular tools are key to address this new challenge because they are more suitable than living farm animals to generate biological data through high-throughput screens (HTS) and to perform functional analysis. Moreover they are more socially and ethically acceptable, safer regarding biosecurity and reduce the need of animal experimentation in agreement with the 3R (Replace, Reduce, Refine) rules. Within the different strategies that can be implemented so far, the production and use of pluripotent stem cells (PSCs) is in perfect agreement with those goals as these cells can differentiate in vitro and in vivo toward all the cell lineages. As such, these cells can be useful as novel cellular tools to evaluate the causality of genetic variants on quantitative traits and specific cell phenotypes.
However despite the progress made on mouse and rat models to produce PSCs, the establishment of PSCs in pig is still beset with problems and their pluripotent state still relies on the expression of exogenous factors. This impacts their differential potential and strongly restrict their use for ex vivo and in vitro phenotyping. The fact that producing fully pluripotent cells in pig remains impossible by standard procedures raises numerous concerns on the molecular mechanisms controlling pluripotency in pigs. One possibility is that the signaling pathways necessary to activate the core pluripotency network in mice and humans are inactive or insufficient in this species. Another hypothesis is that induced pluripotent stem cells generated in pigs carry genetic and epigenetic barriers, and are therefore not fully reprogrammed by conventional protocols. We propose to evaluate both hypotheses in pigs, by considering the specific microenvironment of pig embryonic PSCs.
We will characterize the epigenomic profiles of preimplantation pig embryos at the unicellular and tissue scales and the proteome of uterine fluids at the corresponding embryonic stages. Together with available transcriptomic profiles we will integrate this “multi-omics” information to infere regulatory networks driving pluripotency in pig blastocysts (Task 1). In parallel we will design and generate fluorescent tagged cell lines for tracing endogenous pluripotency (Task 2) and we will use these innovative tools (reporter cell lines and omics data) to perform a screening of small molecules and cytokines able to sustain pluripotency and cellular self-renewal (Task 4). Once defined, we will use this optimized culture system to improve the reprogramming process. Our aim is to overcome remaining epigenetic barriers through the use of new sets of reprogramming factors and epigenetic modifiers (identified through Task 1) to reach a fully reprogrammed state (Task 3). We finally propose to confirm the pluripotent state of the cell lines produced during this project through a complete molecular characterization and in particular by evaluating their ability to contribute to chimaeras (Task 5).
In conclusion, our project integrates different approaches (descriptive, functional and experimental) leading to significant progress towards the production of true pig pluripotent stem cells.