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。与其他内脏器官不同的是，当肝脏受损时，它能够进行多轮再生。最近，被称为有机类化合物的3D细胞聚集体被描述为可以在体外模拟肝脏再生的模型。再生信号如何整合细胞内外的信号，包括细胞的能量平衡，以引导成年干细胞退出静止、增殖和分化，目前尚不清楚。该项目将使用从小鼠和人类成人肝脏中提取的有机化合物来阐明肝脏再生背后的代谢信号功能。代谢组学的进展揭示了代谢底物和副产物通过糖酵解和氧化磷酸化平衡来调节干细胞命运的多个系统。类有机物提供了有形的系统，用于在独立于全身代谢或其他信号信号影响的受控环境中，评估特定代谢信号通路在分化过程中的活动和效果。本项目的目标是：1)使用完全化学定义的介质和合成水凝胶作为3D支架，获得肝脏有机物培养的完全代谢控制。2)利用基于基因编码的FRET代谢通量传感器的酶分析和有机物成像来表征再生过程中的代谢状态。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的可获得性，给予代谢抑制剂，以及通过对限速糖酵解酶的遗传操作，将实现扰动。更深入地了解肝脏再生是基础生物学的一个关键兴趣，最终可能有助于为患有慢性肝损伤的患者提供治疗方法。重要的是，了解肝脏前体细胞的代谢需求将有助于更好地总结体外功能肝脏模型，用于个性化药物、药物发现和毒性研究等治疗目的。

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.