Establishment of a variant-to-function framework for cholestatic diseases using human liver in vitro models

肝性胆汁淤积性疾病的特征是胆汁流动受损和胆汁酸在肝脏内的有害积聚。遗传是导致胆汁淤积症风险的关键因素，转运体基因的罕见突变已被证实是胆汁淤积症的原因。最近的研究强调，肝脏转录增强子内常见的遗传变异也有助于胆汁淤积。然而，与编码序列不同的是，增强子在其他物种中的保守性很差，这使得研究胆汁潴留遗传因素的动物模型的有用性充其量是有限的。用人类模型系统替代用于研究胆汁淤积风险变异的动物模型的一个主要障碍是缺乏适当特征的人类肝脏体外模型。人类诱导多能干细胞（iPSC）衍生的肝细胞（iHeps）是研究人类遗传变异对肝脏转录程序和最终疾病相关细胞输出的影响的一个有吸引力的模型。特别是，在诱导极化条件下培养的iHeps显示出增强的功能。我们设想，ipsc衍生的肝脏体外系统的分子特征的更好表征将促进其用于研究肝脏疾病遗传学，并使其能够在本研究和未来的研究中替代动物模型。在这个项目中，一个多学科团队将结合人类胆汁淤积、调控基因组学、干细胞建模和脂质组学方面的专业知识，研究两种培养系统（经典2D vs.针对胆汁淤积建模优化的新三明治模型），生成全球表达和染色质活性图。这些系统将以人类肝脏数据集为基准，以确定哪个系统(1)更好地概括了人类肝细胞的相关特征，以及(2)更适合于功能性地研究与人类胆汁淤积相关的遗传位点。这些数据还将与从啮齿动物获得的数据进行系统比较，突出显示啮齿动物不适合作为模型的基因位点。然后，我们将对胆汁沉积位点进行crispr激活筛选，以确定非编码变异的靶基因，因为增强子可以在不同距离上与基因相互作用。最后，我们将重点关注一个位点，该位点将通过将iPSC中的基因组编辑与胆汁淤积优化的iHeps方案相结合来建模。然后，编辑后的iHeps将用于研究与胆汁淤积相关的细胞输出，包括小管的形成和脂质组学特征。我们希望证明iHeps在研究与胆汁淤积相关的非编码变异方面具有优越的适用性，为iHeps中风险位点的变异-功能调查提供一个框架。更广泛地说，这些数据集和分析框架将适用于未来研究中其他肝脏疾病的风险位点。

Hepatic cholestatic diseases are characterised by impaired bile flow and harmful accumulation of bile acids in the liver. Genetics is a key contributing factor to cholestatic disease risk, with rare mutations in transporter genes being well-established causes of cholestasis. Recent studies have highlighted that common genetic variants within liver transcriptional enhancers also contribute to cholestasis. However, unlike coding sequences, enhancers are poorly conserved in other species, making the usefulness of animal models for the study of cholestasis genetic factors limited at best.A major hurdle to replace animal models for the study of cholestasis risk variants with human model systems is the lack of appropriately characterised human liver in vitro models. Human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) are an attractive model to study the effects of human genetic variants on hepatic transcriptional programs and ultimately disease-relevant cellular outputs. In particular, iHeps cultured under polarisation-inducing conditions show enhanced functionality. We envision that a better characterisation of the molecular features of iPSC-derived liver in vitro systems will facilitate their use to investigate liver disease genetics and enable the replacement of animal models in this and future studies.In this project, a multidisciplinary team joining expertise in human cholestasis, regulatory genomics, stem cell-based modelling and lipidomics, will investigate two culture systems (classic 2D vs. new sandwich model optimised for cholestasis modelling) generating global expression and chromatin activity maps. These systems will be benchmarked against human liver datasets to identify which system (1) better recapitulates relevant features of human hepatocytes, and (2) is better suited to functionally investigate genetic loci associated with human cholestasis. These data will also be systematically compared against data obtained from rodents, highlighting loci for which rodents are not suitable models. We will then carry out a CRISPR-activation screen of cholestasis loci to identify the target genes of noncoding variants, as enhancers can interact with genes at varying distances. Finally, we will focus on one locus, which will be modelled by combining genome editing in iPSC with the cholestasis-optimised iHeps protocol. Edited iHeps will then be used to investigate cholestasis-relevant cellular outputs, including formation of canaliculi and lipidomic characterisation. We expect to demonstrate the superior suitability of iHeps to study noncoding variants associated with cholestasis, providing a framework for variant-to-function investigation of risk loci in iHeps. More broadly, these datasets and analysis framework will be applicable to investigate risk loci for other liver diseases in future studies.