Human Intestinal Organoids: Pre-Clinical Models of Non-Inflammatory Diarrhea

描述（由申请人提供）：由于缺乏可用且易于使用的人体肠道模型，对人体肠道生理、腹泻疾病的病理生理和抗腹泻药物治疗的认识一直受到阻碍。最近有两种方法（来自LGR5阳性肠道干细胞和来自分离的完整肠隐窝）被开发出来，使人类小肠类器官能够形成正常出现的绒毛/隐窝轴，其中包含所有正常发生的上皮细胞（Na吸收细胞、Cl分泌细胞、杯状细胞、肠内分泌细胞和Paneth）。我们建议比较这两种类型的人类小肠类器官，并利用它们来促进对正常肠道消化生理和几种重要的人类腹泻疾病的病理生理的理解。我们将1)比较两种类型的人类小肠类器官，a)它们是如何随着时间的推移而发展极性和分化的，并定义参与Na吸收和Cl分泌的运输蛋白的分布和随类器官分化的变化，这些过程在腹泻中变得异常。b)在模拟正常消化生理并在腹泻中变为异常的基础和调节条件下，检测NHE3和CFTR两种对腹泻病理生理起重要作用的转运蛋白在类器官中的功能。c)利用类器官和对Na吸收和Cl分泌的了解，增加对霍乱毒素、轮状病毒和肠出血性大肠杆菌引起的三种重要人类腹泻疾病的认识。类器官的其他重要潜在用途是：a)基于NHE3和CFTR荧光测定的高通量药物筛选；b)个性化医疗，因为类器官可以从个体患者身上开发。

DESCRIPTION (provided by applicant): Understanding of human intestinal physiology, pathophysiology of diarrheal diseases and development of anti-diarrheal drug therapy has been held back by lack of an available and easy to use model of human intestine. Recently two methods (from LGR5 positive intestinal stem cells and from isolated intact intestinal crypts) have been developed to allow development of human small intestinal organoids that form normal appearing villus/crypt axes which contain all normally occurring epithelial cells (Na absorptive, Cl secretory, goblet, enteroendocrine and Paneth). We propose to compare the two types of human small intestinal organoids and use them to advance understanding of normal intestinal digestive physiology and the pathophysiology of several important human diarrheal diseases. We will 1) compare the two types of human small intestinal organoids for a) how they develop polarity and differentiation over time and define the distribution and changes with organoid differentiation of transport proteins involved in Na absorption and Cl secretion, processes which become abnormal in diarrhea. b) Test the function in the organoids of two transport proteins that are important for the pathophysiology of diarrhea, NHE3 and CFTR under basal and regulated conditions which mimic normal digestive physiology and become abnormal in diarrhea. c) Use the organoids and understanding of Na absorption and Cl secretion to increase understanding of three important human diarrheal diseases caused by cholera toxin, rotavirus and enterohemorrhagic E. coli. Other important potential uses of the organoids are for a) high throughput drug screening based on the fluorescence assays for NHE3 and CFTR and b) personalized medicine, since the organoids can be developed from individual patients.