Liver organoid systems to identify metabolic signalling functions underlying liver regeneration and enhance drug discovery.

肝脏执行许多对我们的健康至关重要的功能，包括蛋白质合成，消化和药物代谢/解毒。引人注目的是，与其他内脏器官相反，当受损时，肝脏能够进行多轮再生。最近，3D细胞聚集体，称为类器官，已经被描述为可以在体外模拟肝脏再生。再生信号如何整合细胞内和细胞外的信号，包括细胞的能量平衡，以指导成体干细胞退出静止，增殖和分化尚不清楚。该项目将使用来自小鼠和成人肝脏的类器官来阐明肝脏再生的代谢信号功能。代谢组学的进展已经揭示了多个系统，其中代谢底物和副产物通过糖酵解和氧化磷酸化的平衡来调节干细胞的命运。类器官提供了有形的系统，在该系统中，在与全身代谢或其他信号线索的影响分开的受控环境中，评估分化期间特定代谢信号传导途径的活性和效果。该项目旨在：1）使用完全化学成分确定的培养基和合成水凝胶作为3D支架获得肝脏类器官培养的完全代谢控制。2)使用酶促测定法表征再生期间的代谢状态，并使用基于基因编码的FRET代谢通量传感器对类器官进行成像。3)通过代谢扰动研究再生过程中代谢状态的功能相关性。通过改变氧气和营养物质的可用性、给予代谢抑制剂以及通过对限速糖酵解酶的遗传操作来实现扰动。对肝再生的更深入了解是基础生物学的一个关键兴趣，最终可能有助于为慢性肝损伤患者提供治疗。重要的是，了解肝祖细胞的代谢要求将导致更好地概括体外功能性肝脏模型的方案，用于治疗目的，如个性化医疗，药物发现和毒性研究。

The liver performs many functions vital to our health, including protein synthesis, digestion and drug metabolism/detoxification. Strikingly and in contrast to other internal organs, when damaged the liver is capable of multiple rounds of regeneration. Recently, 3D cell aggregates, known as organoids, have been described that can model liver regeneration in vitro. How regenerative signals integrate intra- and extra-cellular cues, including the energy balance of the cell, to guide adult stem cells to exit quiescence, proliferate and differentiate is not clear. This project will use organoids derived from mouse and human adult liver to elucidate the metabolic signalling functions underlying liver regeneration. Advances in metabolomics have revealed multiple systems in which metabolic substrates and byproducts regulate stem cell fate via a balance of glycolysis and oxidative phosphorylation. Organoids provide tangible systems in which to assess the activity and effect of specific metabolic signalling pathways during differentiation in a controlled environment separate from the influence of whole-body metabolism or other signalling cues. This project aims to; 1) Gain full metabolic control of liver organoid culture using fully chemically-defined media and a synthetic hydrogel as 3D scaffold. 2) Characterise metabolic state during regeneration using enzymatic assays and imaging of organoids with a genetically encoded FRET-based metabolic flux sensor. 3) Investigate the functional relevance of metabolic state during regeneration through metabolic perturbations. Perturbations will be achieved by alterations to oxygen and nutrient availability, administration of metabolic inhibitors, and through genetic manipulation of rate-limiting glycolytic enzymes. Greater insight into liver regeneration is a key interest for basic biology and ultimately may help derive therapies for patients suffering from chronic liver injury. Importantly, understanding the metabolic requirements of liver progenitors will lead to protocols to better recapitulate functional liver models in vitro for therapeutic purposes such as personalised medicine, drug discovery and toxicity studies.