Establishing a patient-derived hiPSC differentiation protocol to model development of neural crest cancers

Our current understanding of the initial stages of many cancers is still limited. This is because current human cancer models are often based on immortalised cell lines and cells derived from late-stage tumours. While these studies are important for assessing mature tumour behaviour, they are less helpful for deciphering critical steps during tumour formation and metastasis. Because of this gap in our knowledge, it is difficult to pinpoint the causative events, which may be of potential clinical value as early markers. Here, we use human induced pluripotent stem cells to model cancer initiation and migration. We are specifically interested cancers that arisefrom embryonic cells called neural crest cells. These cancers include neuroblastoma, a rare paediatric cancer. Neural crest cells are multipotent stem cells that give rise to diverse tissues such as melanocytes, Schwann cells, craniofacial skeleton and peripheral nervous system. During embryogenesis, neural crest cells delaminate from the neural tube, undergo epithelial-mesenchymal transition and migrate long distances in a process akin to metastasis. Our ultimate aim is to establish an assay allowing us to use patient-derived hiPSCs to assess the differentiation capacity of normal and diseased hiPSCs. To achieve this, we need to define reproducible protocols for differentiation of hiPSCs to neural crest. We will profile the developmental transitions as they differentiate to neural crest lineages including sympathetic nervous system and Schwann cells, which will allow us to pinpoint pathological triggers during differentiation.

n this project, we will develop an in vitro platform for modelling development transitions during neural crest development. Specific aims:1)Compare the molecular and cellular definition of human PSCs-derived cranial and trunkneural crest lineages generated fromestablished protocols (STEMCELL Technologies). 2)Generate a transgenic Phox2b reporter line in human stem cells.3)Establish a differentiation protocol for generating trunk neural crest derivatives such assympathetic neurons, enteric neurons and Schwanncells. 4)Determine the functional consequence of neuroblastoma-associated genetic variants, including ALK-F1174L, on differentiation of trunk neural crest derivatives.The overallgoal will be to understand the cellular and molecular events driving neural crest lineage segregation.This will serve as a platform for functional analysis of the developmental events underlying disease aetiology, the identification of cellular events associated with variable outcomes in neuroblastoma, and identify novel therapeutic avenues.