Periodic Reporting for period 4 - PLASTINET (Plasticity of the Pluripotency Network)
Période du rapport: 2023-07-01 au 2024-12-31
A few days after fertilisation mammalian embryos form a blastocyst comprised of three tissues; trophoblast and hypoblast are the forebears of extraembryonic structures, while naive epiblast cell are the pluripotent source of the embryo proper. Classical mouse embryological studies indicate that lineage potencies are determined concomitant with segregation of the three founder tissues. Textbook definitions of pluripotency thus exclude extraembryonic potential. Consistent with this paradigm, mouse embryonic stem cells are generally ineffective in producing trophoblast or hypoblast derivatives. However, we have discovered that human naïve pluripotent cells have high intrinsic competence for trophoblast formation. Furthermore, unlike in mouse, extraembryonic transcription factors are present in human epiblast in vivo. These findings challenge the dogma of early lineage restriction but may be compatible with the ancestral origin of pluripotency. We hypothesise that extraembryonic plasticity underlaid by entwined regulatory networks is the evolutionary template of pluripotency. Consequently, signal modulation to suppress extraembryonic specification may be crucial for capture of stem cells representative of naïve epiblast in most mammals. We will examine human and non-human primates, farm animals in which embryos undergo extended development before implantation, and a marsupial in which pluripotent cells are generated from the trophoblast. In a cross-disciplinary approach we will employ transcriptomics, embryo and stem cell experimentation, and formal computational modelling to uncover the core biological program moulded by evolution into different forms. We aim to establish hitherto elusive chimaera-competent embryonic stem cells from species of importance for research, biomedical applications and livestock improvement. We will obtain fresh insight into the molecular logic governing early development, lineage plasticity, pluripotent identity, and stem cell self-renewal.
Work during the first and second period has been severely disrupted and delayed by the COVID pandemic. However, we have succeeded in deriving new pluripotent stem cells from pig, sheep and cattle. We further showed by transcriptome analyses that these pluripotent stem cells are closely related to the pre-gastrulation embryonic disc stage of development in these animals. Several companies in the emerging cellular agriculture field have taken licenses to these cell lines.
In the third period we have established and characterised naive pluripotent stem cells from non-human primates. The non-human primate naive stem cells are very similar to human naive stem cells and likewise related to pre-implantation embyro epiblast. Interestingly, however, they require a subtle but essential modification of the culture system to sustain propagation. We have also refined a mathematical method for feature selection from single cell sequencing data to obtain higher resolution of cell states and trajectories in the developing embryo and thence to better assign identity to ex vivo stem cells.
In the fourth period we completed the characterisation of naive pluripotent stem cells from chimpanzee, demonstrating that they are competent to form early embryo-like structures.
In the third period we have established and characterised naive pluripotent stem cells from non-human primates. The non-human primate naive stem cells are very similar to human naive stem cells and likewise related to pre-implantation embyro epiblast. Interestingly, however, they require a subtle but essential modification of the culture system to sustain propagation. We have also refined a mathematical method for feature selection from single cell sequencing data to obtain higher resolution of cell states and trajectories in the developing embryo and thence to better assign identity to ex vivo stem cells.
In the fourth period we completed the characterisation of naive pluripotent stem cells from chimpanzee, demonstrating that they are competent to form early embryo-like structures.
We have established for the first time a fully defined culture condition for livestock pluripotent stem cells that moreover is the same for the three economically important species, pig, sheep and cattle.
We have established and validated the first naive pluripotent stem cells from non-human primates.
We have established and validated the first naive pluripotent stem cells from non-human primates.