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Project title
Establishing 3-dimensional human organoids to study complex lymphatic anomaly
Supervisors names
Satyamaanasa Polubothu
David Long
Background
Complex lymphatic anomaly (CLA) is a rare, sporadic, congenital developmental anomaly characterised by multifocal proliferation of lymphatic vessels. This can occur in any tissue, leading to disruption of normal tissue architecture, organ dysfunction and often death in early childhood. There is no current cure and existing treatments are minimally effective with substantial side effects. A recent key advance in our understanding of the pathobiology of CLA was the identification of the genetic basis, namely somatic activating variants in the oncogenes PIK3CA and NRAS1. However, we are yet to fully understand how these somatic oncogenic mutations drive lymphangiogenesis and lead to gross destruction of surrounding tissues. The proposed research aims to shed light on these important questions by establishing a human lymphatic vascular organoid. This biomimetic organoid will enable the detailed study of the evolution of CLA. Delineation of the central pathways implicated in CLA pathogenesis is key to identifying novel therapeutic targets and a validated human CLA organoid will create an indispensable tool for future drug screening of novel targeted therapies.
Aims/Objectives and Associated Methods
1) To establish a 3- dimensional human lymphatic vascular organoid. To do this, we will adapt a protocol2 initially published by our collaborator Josef Penninger and now successfully established in Professor Long’s laboratory to generate a self-organising vascular organoid from stem cells. We will modulate the differentiation protocol to enable induction of lymphatic lineage3.
2) To establish a biomimetic human CLA organoid. Here, stem cells will be genetically engineered to express known causative mutations in either PIK3CA or NRAS prior to lymphatic differentiation, using an inducible PiggyBac transposon adapted from recently described protocols for generating inducible mutant heterocellular organoids4,5.
3) To functionally characterize a human CLA organoid with detailed analysis and quantification of disrupted lymphatic vessel formation in comparison to control wild-type organoids using high-resolution wholemount 3-dimensional imaging, vascular permeability assays and single-cell RNA-sequencing to assess molecular changes.
The student will learn cutting-edge techniques in maintenance and differentiation of induced pluripotent stem (IPS) cells, organoid modeling, and genetic engineering of IPS cell lines in a unique cross-cutting project across the themes of developmental biology and cancer biology.
Timeline
AIM 1: Month 0-9, AIM 2: Month 9-21, AIM 3: Month 21-30, Write-up, dissemination: Month 30-36
Contact
Maanasa Polubothu s.polubothu@ucl.ac.uk