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

我们目前对许多癌症的初始阶段的了解仍然有限。这是因为目前的人类癌症模型通常是基于永生化的细胞系和来自晚期肿瘤的细胞。虽然这些研究对评估成熟的肿瘤行为很重要，但它们对破译肿瘤形成和转移过程中的关键步骤帮助较小。由于我们知识上的这一差距，很难准确地确定致病事件，这些事件可能作为早期标志物具有潜在的临床价值。在这里，我们使用人类诱导的多能干细胞来模拟癌症的启动和迁移。我们特别感兴趣的是源于胚胎细胞的癌症，这种细胞被称为神经脊细胞。这些癌症包括神经母细胞瘤，一种罕见的儿科癌症。神经脊细胞是一种多能干细胞，可分化为多种组织，如黑素细胞、雪旺细胞、头面部骨骼和周围神经系统。在胚胎发育过程中，神经脊细胞从神经管中剥离，经历上皮-间充质的转变，并在类似于转移的过程中进行长距离迁移。我们的最终目标是建立一种方法，使我们能够使用患者来源的hPSCs来评估正常和疾病hPSCs的分化能力。为了实现这一点，我们需要定义可重复的方案来区分HiPSCs和神经脊。我们将描述发育转变，因为它们分化为包括交感神经系统和雪旺细胞在内的神经峰谱系，这将使我们能够准确地定位分化过程中的病理触发因素。在这个项目中，我们将开发一个体外平台，用于模拟神经脊发育过程中的发育转变。具体目标：1)比较人类PSCs来源的颅骨和躯干神经脊谱系的分子和细胞定义，这些谱系来自已建立的方案(干细胞技术)。2)在人干细胞中建立转基因PHOX2B报告基因系。3)建立交感神经元、肠神经元、雪旺细胞等主干神经脊衍生物的诱导分化方案。4)确定神经母细胞瘤相关遗传变异，包括ALK-F1174L，对主干神经脊衍生物分化的功能影响。总体目标是了解驱动神经脊谱系分离的细胞和分子事件。这将作为一个平台，功能分析疾病病因学潜在的发育事件，识别与神经母细胞瘤不同结果相关的细胞事件，并确定新的治疗途径。

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.