Periodic Reporting for period 1 - germcells (A platform for the generation of human gametes in vitro)
Reporting period: 2016-03-01 to 2018-02-28
Among farm animals, the pig is recognised as an excellent model for studying multiple human diseases because of similar development and physiology to humans. In this project we use the pig to investigate early lineage decisions that lead to the formation of the gamete precursors.
The overall objective of this project is to characterize the molecular program of pluripotency and the germ cell lineage in the pig by analysing the emergence of pluripotency during early embryo development, and characterizing germ cells specification in post-implantation embryos. This knowledge will allow the establishment of culture conditions for the derivation of pluripotent cells and gametes in vitro.
In order to reveal the gene regulatory network involved in the emergence of pluripotency and in the specification of germ cells, single cell RNA sequencing was performed in individual cells collected from in vivo produced pig embryos at 6 embryonic stages. I generated a transcriptional map of pig embryo development, showing gradual segregation of lineages. I also revealed the transcriptional circuitry of pluripotency and showed that in females, dosage compensation and X chromosome inactivation is accomplished in the late epiblast before lineage priming. Additionally, the molecular profile, signalling pathways and the epigenetic landscape of pre-migratory and gonadal PGCs were analysed.
Work package 2:
Based on information derived from objective 1, I investigated how modulation of key signalling pathways can affect early lineages segregation and promote epiblast differentiation over hypoblast.
Pig PGCs were recovered from day 22 – 30 pig foetuses and cultured under the conditions that resulted successful in previous experiments with Tcam2 cells, a human seminoma cell line. Evidence of reprogramming was obtained by gene expression analysis, however no stable cell lines could be expanded during this conversion.
Pig embryonic stem cells (ESC) derivation was attempted in collaboration with Dr. Jennifer Nichols at the Stem Cell Institute (Cambridge). ESC lines could be established under different conditions modulating ERK and WNT signalling.
Work package 3:
I focused on the early regulation of germ cells specification and aimed to functionally address the importance of SOX17 through a knock-out model based on the recently developed CRISPR-Cas9 technique. This technology was not available when the proposal was written. Due to the limited time I was only able to produce SOX17 knock-out blastocysts. In future, these embryos will be transferred to surrogate sows and recovered at day 14 of pregnancy, when PGCs are already specified.
Furthermore, IL6 knockout embryos were generated by CRISPR-Cas9 embryo editing and showed impaired development.
• Milestone 1: A transcriptional map of pig pre-implantation embryo development and germline specification was generated.
• Milestone 2: Pig pluripotent cells from different origins were cultured under different conditions: embryonic stem cells from pig blastocysts in collaboration with the Stem Cell Institute and embryonic germ cells from foetal gonads at The University of Nottingham.
• Milestone 3: I set up a system for the efficient generation of knock-out pig embryos to functionally prove the importance of specific genes in pluripotency and germline specification.
Exploitation and dissemination of the results
The data generated from objective 1 (transcriptional map of pig preimplantation embryos by scRNAseq), 2 (effect of key signalling pathways on early lineages segregation and pluripotency) and 3 (effect of IL6 knock-out on the emergence of pluripotent cells) has led to the preparation of one manuscript that is being submitted to a high impact journal. This novel transcriptome atlas of pig pre-implantation embryo will have a remarkable impact in the field of pluripotent cells and chimera generation from farm animals.
The data generated on PGCs will lead to the preparation of another manuscript that will be submitted to a high impact journal in the field. This knowledge will be crucial for the development of strategies for the generation of human germ cells in vitro.
I have already published a book chapter and I have presented 2 posters and one short talk in international conferences. I have also participated in several outreach activities for the general public, scientists and school students.
Inhibition of specific pathways with small molecules resulted in embryos more suitable for embryonic stem cells derivation, a new approach that had never been used before in the pig. Furthermore, I have applied the recently discovered conditions to derive human and monkey naïve embryonic stem cells to pig embryos for the first time.
Gene editing in large animals has remained very challenging for decades. I have developed and set up the cutting-edge technology CRISPR-Cas9 in the lab in order to dissect the early events that lead to the formation of germ cells and pluripotent cells in the embryo. By implementing this revolutionary technology in the host lab, I have opened a broad window of possibilities to design experiments based on genetic modification of pigs that can be applied to multiple projects, putting the host group at the forefront of gene editing technologies applied to large animals.