Development of human primordial germ cells towards the onset of sperm and egg differentiation in a novel model culture system

Periodic Reporting for period 1 - GERMINOID (Development of human primordial germ cells towards the onset of sperm and egg differentiation in a novel model culture system)

Reporting period: 2019-06-01 to 2021-05-31

What is the problem/issue being addressed?
The precise mechanisms regulating the origin and early development of human primordial germ cells (hPGCs) are not well understood, mainly because of technical reasons and the inaccessibility to early human post-implantation embryos (weeks 2–4 after conception). This lack of basic knowledge leads to protocols for hPGC-like cell (hPGCLC) specification and progression with low efficiencies.
To advance the knowledge in this key stage of human development, we proposed to combine efforts and create a robust in vitro system to model hPGC specification and development. This strategy was designed to benefit from the cell culture capabilities of the three-layer gradient system (3-LGS), a three-dimensional (3D) cell culture methodology that I invented during my PhD, as well as from the expertise of the Surani lab in creating artificial early stage hPGCs (hPGCLCs) from human pluripotent stem cells (hPSCs). We proposed to generate human gonadal organoids from embryonic primary cells, which we call “germinoids”, to explore support for developing hPGCLCs beyond the in vivo equivalent 2-wk stage.
Why is it important for society?
Understanding exactly how hPGCs are specified and how these cells progress is critical to recognize developmental issues and disease associated with the cell line responsible to carry genetic and epigenetic information to the next generations.
Fertility preservation: An increasing number of women and men are affected by fertility related problems. Among these, young boys and girls undergoing chemotherapy and radiotherapy treatments have a high risk to lose their germline and their chance to become biological parents. The use of hPSCs brings the opportunity to generate patient-tailored derived tissues, including hPGCLCs.
Livestock and endangered species: On the other hand, the application of similar principles to other animal species will contribute to design experimental pipelines for in vitro gametogenesis from species-specific PSCs. This will be of special relevance in reproductive assisted techniques for livestock and endangered species.
What are the overall objectives?
The objectives of this proposal were (1) to develop an in vitro embryonic gonadal organoid system (germinoid) to maintain hPGCs and support its maturation in long-term cultures and (2) to overcome the 2-week stage barrier and extend hPGCLC development to ~ week 5–9 supported by germinoid cultures. This would modelling the period after the hPGCs enter into the developing gonads.
Finally conclusion points
During this project, we reformulated and adjusted some of the initially proposed technics to achieve our main goals. From our findings we concluded that:
1. The hPGCLCs specified from the new protocols (developed during this project) have a higher propensity to progress beyond the nascently specified stage, at a tempo similar to that observed in vivo.
2. The germinoid co-culture demonstrated a beneficial role for hPGCLC progression in vitro. Therefore, our in vitro co-culture system has physiological relevance and shown to benefit hPGCLC progression in vitro.

During the period of this project we tested and optimized methodologies that lead us to the developments of novel protocols for hPGCLC specification and progression in vitro. The main results that I would like to highlight were:
1. Specification of hPGCLCs from a new class of in vitro precursors:
2. Generation of the first hESC-derived germinoid co-culture system to support hPGCLCs progression.
3. Demonstration that hPGCLCs specified from the new class of precursors could progress in vitro supported by germinoid co-cultures.
4. Demonstration that hPGCLCs specified from the new class of precursors have an enhanced competence to progress when compared with hPGCLCs specified from previous protocols.
Dissemination
During the period of this project, I put in place the plan dissemination strategy. More specifically:
1. I presented the initial plan and updated versions of this project to all members of the host lab in our weekly meetings.
2. I also presented this project and the results to the entire scientific community at the Gurdon Institute, twice.
3. I had the opportunity to present parts of this project and results at international conferences
a. At the 6th International Workshop on Molecular Andrology (06/12/2019, Giessen, Germany)
b. At Fertility annual meeting (04/01/2019, Birmingham, United Kingdom)
Exploitation
I am committed to carry on the exploitation of our results. More specifically:
1. We already established collaborations with other lab members and research groups for new research projects in order to build up research on our previous findings.

We demonstrate the specification of hPGCLCs from a new class of precursors, which show an enhanced competence to progress in vitro. The progression of these cells was supported by germinoid cultures and happened at a tempo similar to that observed in vivo.
Remarkably, the enhanced progression propensity of these new hPGCLCs paves the way for more efficient protocols for hPGCLC progression. Moreover, these new in vitro models will bring new opportunities not only to explore the mechanisms involved in hPGC specification and progression, but also to study associated diseases, including human infertility, as well as therapeutic approaches to minimize their impact in global health.

Summary of the GERMINOID action

Periodic Reporting for period 1 - GERMINOID (Development of human primordial germ cells towards the onset of sperm and egg differentiation in a novel model culture system)

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