A novel in vitro reporter system for blood stem cell activity

血液干细胞(或称造血干细胞，HSC)是涉及血液和免疫系统的再生医学应用的基本组成部分。造血干细胞移植在癌症医学和基因治疗领域有着悠久的历史，并越来越多地被用于包括病毒感染(HIV)和自身免疫(多发性硬化症)在内的一系列疾病。然而，尽管付出了巨大的努力和投入，研究人员在很大程度上未能在相当长的一段时间内保持完全功能的造血干细胞。最近在小鼠HSC扩增方面的进展使研究人员处于突破这一数十年来的障碍的边缘(Wilkinson等人，《自然》2019)。28天的方案具有令人难以置信的功能造血干细胞的扩增能力(~200倍)，但造血干细胞仍然代表绝大多数细胞，并且单细胞扩增培养之间存在显著差异。进一步的优化将增强HSC的扩增能力，允许对纯化的扩增的HSC进行分子和细胞分析，并导致人类HSC扩增的关键改进。这是一个快速发展的领域，该系统已经在世界各地得到使用。目前精确识别“真正的”HSC的金标准功能分析需要体内HSC移植，这意味着它们需要非常密集的小鼠，只需一份手稿，经常涉及数百只受体动物来展示功能活性。这就迫切需要更强大的体外分析来检测功能强大的造血干细胞。这个博士项目旨在验证这样的体外系统，并将努力使其在小鼠品系中普遍适用，理想情况下，将其应用于人类血液干细胞系统。总的来说，这个项目将分两个阶段进行。第一个是验证体外报告系统，并对含有HSC的培养物进行分子图谱分析，以提高我们对扩增的HSC的理解，并确定替代报告基因(Fgd5)的候选分子。第二个阶段是从功能上验证这些候选标记物取代或改善FGD5表达的能力，从而为将该策略转化为人类血液干细胞扩增系统奠定基础。

Blood stem cells (or Haematopoietic Stem Cells, HSCs) are the fundamental component of regenerative medicine applications involving the blood and immune system. HSC transplantation has a long history in the fields of cancer medicine and gene therapy and is increasingly being explored in a wide array of diseases including viral infections (HIV) and autoimmunity (multiple sclerosis). However, despite significant efforts and investment, researchers have largely failed to maintain fully functional HSCs for substantial periods of time. Recent advances in mouse HSC expansion have put researchers on the cusp of breaking through this decades-old barrier (Wilkinson et al., Nature 2019). The 28-day protocol possesses incredible expansion capacity (~200-fold) of functional HSCs, but the HSCs still represent the vast minority of cells and substantial variability exists between single cell expansion cultures. Further optimisation will enhance HSC expansion capacity, permit molecular and cellular analyses of purified expanded HSCs, and lead to critical improvements in human HSC expansion. This is an area of rapid development with the system already being utilised worldwide. Current gold standard functional assays to precisely identify 'true' HSCs demand in vivo HSC transplantation meaning they are extremely mouse intensive with single manuscripts regularly involving hundreds of recipient animals to demonstrate functional activity. This creates an urgent need for more robust in vitro assays to detect functional HSCs.This PhD project aims to validate such an in vitro system and will strive to make it universally applicable across mouse strains and, ideally, translate its utility to the human blood stem cell system. Broadly speaking, this project will proceed in two phases. The first is to validate the in vitro reporter system and perform molecular profiling on HSC-containing cultures to both improve our understanding of expanded HSCs and to identify candidate molecules to replace the reporter gene (Fgd5). The second stage is to functionally validate these candidate markers for their ability to replace, or imporve upon, Fgd5 expression, thereby setting the stage for translation of the strategy to human blood stem cell expansion systems.