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  Dr Paula Alexandre, Prof David Long  No more applications being accepted  Funded PhD Project (UK Students Only)

About the Project

Blood vessels play a pivotal role in brain development and function. They promote tissue growth and survival by supplying nutrients and oxygen to surrounding tissues, signals to progenitor populations, as well as guiding neuronal migration. Vascular defects are associated with brain disorders such as epilepsy and neurodegenerative diseases. However, we don’t fully understand how blood vessels influence brain development and, conversely, how brain cells can influence angiogenesis.

In Down Syndrome for example, abnormal vascularization is believed to be one of the potential factors that may contribute to the significantly lower incidence of solid brain tumours compared with the general population, as solid tumours typically require vascularization to grow (Osuna-Marco et al, 2021). Although this hypothesis could inform the design of novel tumour therapies, it is challenging to test because current brain organoids lack vasculature, and animal models do not fully replicate the human brain development and phenotype.

Alexandre’s lab has been investigating the development of the human hindbrain (Haldipur et al, 2019) in both healthy and Down syndrome. Their preliminary data suggests that the vascularization pattern is altered in Down Syndrome. Additionally, they have observed certain neural progenitor populations, which are not present in mice, closely interacting with blood vessels, indicating a potential link between blood vessels and neurogenesis. Furthermore, Alexandre’s lab is growing human hindbrain organoids derived from induced pluripotent stem cells (iPSCs) (Silva et al, 2020) and generating detailed single cell transcriptomic data from both healthy control and Down syndrome organoids that could complement the current study. Long’s lab brings extensive expertise in growing iPSC cell models and most recently, vascular organoids. His lab has proven expertise in tissue clearing methods, 3D imaging and quantification methods to study patterns of vascularisation (Jafree et al, 2019).

This project aims to vascularize hindbrain organoids and assess the influence of blood vessels on neurogenesis in both healthy and Down syndrome iPSC-derived organoids. Our initial aim is to test whether the defects in Down Syndrome vascularisation result from intrinsic signals or from the extrinsic factors provided by the brain cells.

The supervisory team has all the required expertise and reagents for the project.

Aims/Objectives:

Our aims are:

1) Promote vascularisation of healthy hindbrain organoids

2) Determine the impact of vascularisation in healthy derived hindbrain organoids.

3) Investigate the interactions between brain cells and vasculature in Down Syndrome.

The generation of vascularized brain organoids promises a ground-breaking impact across diverse disciplines. They enable precise modelling of human developmental brain disorders, delineate vascularised niches in brain tumours, and facilitate testing of novel therapies for diseases like brain degenerative disorders and paediatric brain tumours.

Methods:

Endothelial and neural cells originate from distinct embryonic layers, the mesoderm and ectoderm respectively, presenting a challenge in their simultaneous differentiation within organoid models. To address this, we propose a sequential approach wherein organoids are grown separately and then co-cultured post-differentiation to promote their healthy development and survival.

In Aim 1, we will explore both in vivo and in vitro strategies. In vivo, we aim to transplant GFP-expressing hindbrain organoids into human brain explants and culture these for 1-2 weeks. The GFP will allow us to distinguish between organoids and human brain cells. In vitro, we will culture vascular organoids within hydrogels containing various extracellular matrix components while hindbrain organoids are grown in neural media but in close contact with the vascular setup. If successful, we will progress to microfluidics in a microchip configuration.

In Aim 2, vascularized organoids will undergo immunohistochemistry clearing procedures and 3D imaging to investigate the extension and pattern of vascularization, cell proliferation, organoid growth, and cellular output when compared to non-vascularized organoids our ongoing work on the developing human hindbrain (CD34-blood vessel marker, mKi67-proliferative marker, ELAVL4-neuronal marker, etc).

Once optimal co-culture conditions are established, hindbrain and vascular organoids from both healthy and Down Syndrome subjects will be co-cultured in different combinations to delineate whether vascularization defects arise from blood vessels or neural cells (Aim3). Assessment of the phenotype will involve histological and comparative analysis between the different co-culture systems and human developing hindbrain structures.

Timeline:

Year1: focus on optimising the co-culture systems (aim1).

Year2: continue optimising the co-culture systems (aim1) and analysing the changes on cellular output (aim2). It also starts analysing the phenotype in Down Syndrome (aim3).

Year3: continue the analysis of Down Syndrome (aim3), write the manuscript and thesis.

Collaborations: 

Collaborators that can provide input into the project:

Professor Paolo de Coppi and Dr Giovanni Giobe at UCL GOS ICH, are experts in Stem cell and bioengineering approaches.

Professor Kathleen Milen and Dr Parthiv Haldipur are experts in human hindbrain development and brain malformations.

Plans for patient and public involvement and engagement for the project/student:

The student will have diverse opportunity to be involved in PPIE, including involvement in GOSH BRC events such as Family Fun Day and Playstreet. The Alexandre/Long groups are also involved in public engagement through outreach events for 16-18-year-olds interested in STEM, through the HDBI Inspire programme and the ICH Work Experience Scheme. Finally, the student will engage with patients through the LonDowns meetings and discuss the progresses in the field with clinicians, researchers and families from children with Down Syndrome.

Biological Sciences (4)

References

1- Osuna-Marco MP, López-Barahona M, López-Ibor B, Tejera ÁM. Ten Reasons Why People With Down Syndrome are Protected From the Development of Most Solid Tumors -A Review. Front Genet. 2021 Nov 5;12:749480. doi: 10.3389/fgene.2021.749480. PMID: 34804119; PMCID: PMC8602698.
2- Haldipur P, et al Spatiotemporal expansion of primary progenitor zones in the developing human cerebellum. Science. 2019 Oct 25;366(6464):454-460. doi: 10.1126/science.aax7526
3- Silva T. P., et al (2020). Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture. Frontiers in Bioengineering and Biotechnology, 8(Stem Cell Systems Bioengineering). https://doi.org/10.3389/fbioe.2020.000870
4- Daniyal J et al (2019) Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease eLife 8:e48183
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 About the Project