Generation of functionally mature pancreatic organoids as a replacement strategy for animal-models of pancreatitis and pancreatic cancer

胰腺由两个器官合而为一。内分泌部分容纳控制血糖水平的胰岛素分泌细胞，外分泌部分产生控制消化的酶。影响外分泌胰腺的疾病，如胰腺炎和胰腺癌，死亡率很高，诊断为胰腺癌的患者死亡率高达95%。为了减轻这些疾病造成的社会负担，世界各地的研究团队都在努力更好地了解胰腺疾病，建立早期诊断和创新的治疗策略。为了开发和测试这些策略的有效性，复制大多数人类病理生理特征的基因工程小鼠模型经常在研究实验室中使用。尽管这些模型的使用对研究目的是必要的，但每年在世界范围内导致成千上万的动物被淘汰。类器官是由干细胞衍生的3D体外系统，它代表了一种非常有前途的动物模型替代品。类器官是器官的简化版本，它们应该概括生理和微观解剖学。不幸的是，迄今为止生成的胰腺类器官并不能忠实地再现胰腺的复杂性。这极大地限制了胰腺类器官作为胰腺疾病实验室中有价值的替代模型的采用。因此，本项目的目标是开发生理相关的类器官模型，用于体外研究胰腺炎和胰腺癌。胚胎中胰腺的形成受到其局部微环境的严格调控，但其精确组成尚不清楚。该项目的第一个目标是确定胚胎中观察到的胰腺微环境，并在体外人工繁殖以使类器官成熟。为此，我将在小鼠胚胎胰腺上使用尖端的测序技术，并精确定义构成其微环境的生化线索。然后，我将使用鉴定出的线索补充人工微腔，在其中培养类器官。培养的类器官将被监测以获得成熟的形态和生理胰腺标志物。这种方法将使我能够确定成熟胰腺类器官的最佳微腔组成。为了模拟胰腺炎，类器官将用小糖蛋白治疗，小糖蛋白是一种在动物模型中用于诱导胰腺炎的药物。为了模拟胰腺癌，我将使用过度激活已知在小鼠和人类中诱发胰腺癌的基因变体的转基因干细胞来生成类器官。我将通过比较在动物模型中观察到的病理生理特征来评估所获得的类器官是否为研究胰腺炎和胰腺癌的有价值的模型。该项目的完成必将大大减少动物实验，并为研究实验室提供新的工具来筛选下一代胰腺疾病治疗药物的效果。

The pancreas consists of two organs in one. The endocrine part houses insulin-secreting cells controlling blood glucose levels and the exocrine part produces enzymes controlling the digestion. Diseases affecting the exocrine pancreas, such as pancreatitis and pancreatic cancer, present dramatic mortality rates, as highlighted by the 95% mortality of patients diagnosed with pancreatic cancer. To alleviate the societal burden caused by these diseases, research teams all over the world are trying to better understand pancreatic diseases and establish early diagnosis and innovative therapeutic strategies. To develop and test the efficacy of these strategies, genetically engineered mouse models replicating most human pathophysiological features are routinely used in research labs. Although necessary for research purposes, the use of these models leads to the culling of thousands of animals worldwide every year.Organoids are emerging 3D in vitro systems derived from stem cells, which represent a very promising alternative to animal models. Organoids are simplified versions of organs, of which they should recapitulate the physiology and microanatomy. Unfortunately, the pancreatic organoids generated so far do not faithfully recapitulate the pancreas complexity. This strongly limits the adoption of pancreas organoids as valuable replacement models in the labs working on pancreatic diseases. The goal of this project is thus to develop physiologically relevant organoid models for studying pancreatitis and pancreatic cancer in vitro. The formation of the pancreas in the embryo is known to be tightly regulated by its local microenvironment, whose precise composition is still poorly known. The first goal of the project is to define the pancreas microenvironment observed in embryos and to reproduce it artificially in vitro for the maturation of organoids. To that end, I will use cutting-edge sequencing technologies on mouse embryonic pancreas and precisely define the biochemical cues that compose its microenvironment. I will then use the identified cues to supplement artificial microcavities in which organoids will be cultured. Cultured organoids will be monitored for the acquisition of mature morphological and physiologic pancreatic markers. This approach will allow me to identify the best microcavities composition to mature pancreatic organoids. To model pancreatitis, organoids will be treated with caerulein, a drug used to induce pancreatitis in animal models. To model pancreatic cancer, I will generate organoids using genetically modified stem-cells that over-activate a variant of a gene known to induce pancreatic cancer in mice and humans. I will assess whether the obtained organoids are valuable models to study pancreatitis and pancreatic cancer by comparing their pathophysiological features with those observed in animal models. The completion of this project will surely enable a significant reduction of animal procedures and provide research labs with new tools to screen the effect of next-generation drugs for pancreatic diseases treatment.