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Research project

DC10 Haemogenic gastruloids: a novel approach to model GATA2 deficiency in vitro with embryonic organoids


Haemogenic gastruloids: an in vitro novel approach to model blood differentiation and malignancies with 3D embryo-like structures derived from Embryonic Stem Cells.

Keywords: Gastruloids, embryo models, embryonic and induced pluripotent stem cells, embryonic haematopoiesis, GATA2 deficiency 

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In mouse embryos, blood formation occurs in several spatially and temporally distinct waves, and only the last wave, which takes place in the dorsal aorta, generates definitive haematopoietic stem cells (HSCs) (Panel a in Annex; Dzierzak 2018 Cell Stem Cell). In humans, the process of blood formation is less well characterized. This is mostly due to technical, ethical, and legal limitations associated with studying in vivo human embryos in the developmental stages where blood formation takes place. To address these challenges, 3D embryo models that allow in vitro studies into mouse and human post-implantation development have been generated in the last decade (Sozen 2022 Sem. In Cell and Dev. Biology). In this project, we will make use of the gastruloids embryo model (van den Brink 2021 Trends in Cell Biology).

Gastruloids are 3-dimensional embryo-like structures generated from mouse (van den Brink 2014 Development) or human (Moris 2020 Nature) embryonic stem cells that can be used to study post-implantation development in vitro. A key advantage of gastruloids over previous in vitro models (such as embryoid bodies or 2D stem cell cultures) is that they recapitulate the shape and tissue organization of in vivo embryos with spatial and temporal precision (Beccari 2020 Nature). Recently, we together with Cristina Pina (Brunel University London) developed a haemogenic blood-generating version of the mouse gastruloids protocol (Ragusa 2022 bioRxiv; panel b in Annex). This haemogenic mouse gastruloids (Haem-mGas) model recapitulates blood vessel formation, generates the different waves of haematopoiesis, and can be used to model MNX1-driven infAML in vitro (Ragusa 2022 bioRxiv). However, human versions of such haemogenic gastruloid models do currently not yet exist.

GATA2 deficiency is a pediatric group of disorders that result from inactivating mutations in one of the two parental GATA2 alleles (Kotmayer 2022 Br J Haematol). The transcription factor GATA2 plays essential roles in embryonic haematopoiesis, and precise levels of it are needed for the maintenance of functional blood and lymphatic stem cells. In patients with GATA2 deficiency, cellular levels of GATA2 are decreased, resulting in a precancerous condition that commonly progresses to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The exact mechanisms by which GATA2 deficiency results in MDS/AML are unknown, but (pre)malignant transformations are thought to already occur in utero, during early embryonic development of the blood system.

Research aim and objectives: In this project, we will develop the first human haemogenic gastruloids (Haem-hGas) model. We will then use this model to study both healthy and GATA2 deficient human haematopoiesis in vitro in 3D. By comparing GATA2 deficient Haem-hGas with isogenic controls at various timepoints in gastruloid development using state of the art technologies we will identify 1) transcriptomic and epigenetic effects of GATA2 deficiency during human development 2) which cell populations are affected by the GATA2 deficiency and 3) the developmental stage in which defects start to appear. Altogether, this project will contribute to a mechanistic understanding of the effects of GATA2 deficiency during human (blood) development. Haemogenic gastruloids have already successfully been generated from mouse ESCs (Ragusa bioRxiv 2022), and we have preliminary data that shows that it is possible to generate haemogenic gastruloids from human ESCs and iPSCs.

Potential to enhance the career perspectives and employability of the DC: The student will gain extensive knowledge of embryo models, embryology, haematopoiesis and leukemia, and will receive training in 2D and 3D cell culture, reporter line generation, confocal and high-throughput Widefield microscopy, flow cytometry, HCR (RNA) and antibody stainings, single-cell RNA sequencing, ATAC sequencing, and data-analysis. In addition, the DC will receive training in soft skill such as supervising students, writing publications, and science outreach.

Potential for scientific, technological, economic, or societal impact: We will develop a first human blood gastruloid model, which can be used to study blood development and diseases therein in vitro. Furthermore, we will gain mechanistic insights in the role of GATA2 deficiency in embryonic haematopoiesis. Our model will also allow to screen for compounds or environmental stressors that decrease or increase the penetrance of the disease.

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Non-genetic Heterogeneity and Leukaemia Evolution Lab, Brunel University (London, the UK)