Physiology and biotechnology of Embryonic stem cells
Our work aims to decipher cellular and molecular mechanisms controlling the establishment and the maintenance of pluripotency in several species including porcine, bovine and caprine but also Aves as a model of non-mammalian species and to compare them with those of the canonical mouse model, taken as the reference in the stem cell field. For this purpose, our research is based on three complementary axes.
Axis 1: Derivation and characterization of avian embryonic stem cells:
We were pioneers in the isolation, in vitro establishment and characterization of chicken embryonic stem cells (ESC) (Pain et al, 1996). These cells have been shown to be pluripotent since they display self-renewal and differentiation properties and contribute to chimera when they are re-injected into pregastrulatring embryos (Lavial et al., 2007). This long term establishment is achieved using various culture media, which leads to different pluripotent stem cells that are then characterized at the molecular, epigenetic and developmental levels (Kress et al., 2016). Primordial germ cells (PGC), which are closely related to ESCs but specified differently in avian species compared to mammalian ones, are also isolated, in vitro grown in long term cultures and analysed. Ongoing research is focused on the development of new tools based of fluorescent reporter genes to label and enrich cellular populations according to specific pluripotent states. Their developmental properties and their colonization potential are then investigated.
Axis2: Derivation of induced pluripotent stem cells from various mammalian species:
It has also been shown that somatic cells can be converted into pluripotent cells (iPSCs) by over-expressing 3 or 4 specific exogenous factors (Sox2, Oct4, Klf4 ± c-Myc). By using this “Yamanaka” cocktail combined with other factors, we derive new induced pluripotent stem (iPS) cell types from various mammalian rent animals (pig, sheep, goat, bovine, horse, etc…) or from more exotic ones such as bats and avian species (chicken, quail, duck). The recent mastering of the somatic reprogramming in avian species (Fuet et al., submitted) as well as the well-mastered approach in mammals, including the identification of new gene combinations (Jean et al., 2017, EP17305082.4, patent) opens the way to generate new cell types. Those different iPS cell types are characterized by immunochemistry (cell markers) and biochemistry (alkaline phosphatase and telomerase activity) and compared with the starting primary cells as well as with the early embryos by RNAseq and ChipSeq. All together those data provide a comparison basis for establishing the pluripotent networks in all those species with a phylogenetic perspective.
Axis3: Biotechnology of pluripotent cells:
Pluripotent stem cells are a unique source of cell plasticity to generate specific derivatives by controlling their differentiation process. By applying various protocols of differentiation to engage the pluripotent stem cells toward different lineages, a large spectrum of differentiated progenitors were able to be produced (Vautherot et al., 2013, N° FR1357346, patent, Couteaudier et al., 2015; Vautherot et al., 2017). Beside the fundamental research, for which obtaining and characterizing specific differentiated cells from pluripotent stem cells are a real challenge, one of the main interest of those pluripotent and iPS cells lies in their biotechnological potential. In particular, these cells and their differentiated derivatives are useful cellular substrates for viral and bacterial studies as well as vaccine production. Some of them are already used in industrial development and other are either under validation or developmental processes.