Chuva de Sousa Lopes Group – Human Development and Gametogenesis

Oogenesis spotlighted: making mature human oocytes

Women who survive childhood cancer often fail to conceive because their eggs are damaged by (gonadotoxic) chemotherapy. A major breakthrough has been the possibility to cryopreserve cortical strips of their ovarian tissue for autologous transplantation later in life. This has led to over 100 successful pregnancies worldwide, including several in the Netherlands. However, the risk of reintroducing cancer cells with the ovarian graft in patients with previous hematopoietic malignancies is too great and alternatives are needed.

In a project funded by the European Research Council (Consolidator grant) we propose to build on the groups expertise in gametogenesis in mice and humans and perform a detailed study of the cellular networks and molecular pathways that control development and maturation of the oocyte within the human ovary. We have access and ethical approval for research on human foetal tissue and postnatal ovarian biopsies over a wide age range. We will use this rare material to systematically benchmark the transcriptional profile of cells in the human ovary (oocytes as well as somatic cells) during development and adulthood using 10XGenomics, a novel cost-efficient single-cell technology that allows the transcriptomics profiling of thousands of cells in a matter of hours. Thereafter, we will apply mathematical algorithms to reveal cellular identities, developmental trajectories and signalling networks that control oogenesis. With this knowledge, I plan to engineer a human follicular niche creating a “mini-ovary” in vitro that could support the formation and maturation of the oocyte (using patient-specific cells) and to explore mechanisms of follicle maturation through a xenotransplantation mouse model. The cellular outcomes of these assays will be sequenced using 10XGenomics and directly compared to their in vivo counterparts.

Our approach will lead to more effective personalized-therapy for fertility preservation and contribute to the development of an in vitro mini-ovary organoid model to use in human reproductive toxicology and disease modelling.

Gametogenesis in the dish: Can human pluripotent stem cells differentiate to germ cells of the opposite sex?

There are presently no in vitro assays for human sex-determination. Such assays would be welcomed by clinicians both as disease models for infertility (to study causes and develop treatments) and possibly to develop gamete-replacement strategies, and by industry to replace/reduce the number of compulsory animal reproductive toxicity tests required by regulatory authorities prior to new drug use in humans and clinical trials.

In research funded by NWO-ZonMW VICI we propose to combine our expertise in human gametogenesis and pluripotent stem cells (hPSCs) to address this major challenge of assay development. Taking advantage of our unique regular access to human fetal tissue, which has ethical approval, we aim to optimise a proof-of-principle gametogenesis (co-culture) organoid-model to direct hiPSCs towards male or female germ cell-like fates. In addition, we will determine the genetic factors present on the sex chromosomes that regulate differentiation to male- and female germ cell-like fates. For this, we plan to use gene-editing technology in XY, XX and XO hiPSCs to either remove or add Y-linked and X-linked genes and systematically investigate how this influences the differentiation capacity to male and female germ cell fates.

Our results will lead to novel insights into gametogenesis and sex determination in humans, highlighting both the molecular niche necessary to support germ cell development and understanding of how sex-chromosomes regulate germ cell sex-specific differentiation. The project will provide an important step towards a long-sought gametogenesis assay for disease modelling and reproductive toxicology. Moreover, the possibility of generating functional stem cell derived-gametes starting from patient-specific PSCs and the additional possibility to answer couples of the same sex regarding own genetic children will open an important societal debate that could alter the way we approach (in)fertility in the clinic.

In vitro generation of human induced pluripotent stem cell-derived gametes to model human embryogenesis

Gamete formation is a long and complex process, involving significant remodelling of the cytoplasm and nucleus of germ cells in a sex-specific manner. After fertilization, a totipotent cell (or zygote) is optimally prepared to enter the developmental program from which new individuals eventually emerge. We know very little about the first steps of human development, as in the Netherlands and many other countries generation of human embryos for scientific research is prohibited by law.

This project, funded by ZonMW PSIDER, has two main aims: 1) to develop methods to differentiate human induced pluripotent stem cells (hiPSCs) into male and female gametes in vitro, while ensuring a built-in genetic defect (to prevent hiPSC-gametes derived-embryos from being viable; according to the Dutch law, these are not considered embryos, and could be used in the future for valuable research) and 2) to explore ethical, legal and societal aspects of this approach, providing insight in whether, and under what conditions, the use of such alternative embryo-model could be considered acceptable or preferable.

This multidisciplinary project contributes to the development of suitable and acceptable alternative human embryo-models to study human early embryogenesis and transmission of hereditary diseases.

Understanding human gametogenesis and early development to improve fertility

Many organ-specific diseases display an onset or result from a disruption that occurs during pregnancy. Access to comprehensive tissue-wide transcriptional data from as many developing human tissues as possible will provide a comparative baseline for in vitro models of human tissue. Within this project, part of reNEW and funded by the Novo Nordisk Foundation, we will focus on understanding the earliest stages of human development, mapping the temporal and spatial development of the embryo and extraembryonic tissues, including the placenta. We will generate datasets from a wide number of tissues and developed bioinformatic tools with which to directly compare these profiles with in vitro models. The continued acquisition and analysis of early human development will form a critical underpinning resource for groups across reNEW, particularly supporting studies of gametogenesis, fertility, urogenital development, and diseases of sex development. We have a particular interest in how the gametes form in both males and females, on the biology of the sex chromosomes (X and Y), genomic imprinting and X-inactivation. Within reNEW, we will investigate the process of female (oocyte) and male (sperm) gametogenesis, with a focus on cellular networks and molecular pathways that control development and maturation. Ultimately, we aim to develop assays using human pluripotent stem cells (PSCs) to mimic relevant aspects of human female and male gametogenesis and to develop a human-PSC system that could be used as drug-screen platform for human reproductive toxicology.