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

肝脏胆汁淤积性疾病的特征是胆汁流动受损和胆汁酸在肝脏中有害堆积。遗传学是胆汁淤积性疾病风险的关键因素，转运蛋白基因的罕见突变是胆汁淤积的公认原因。最近的研究强调，肝脏转录增强因子中的常见遗传变异也有助于胆汁淤积。然而，与编码序列不同，增强子在其他物种中的保守性很差，使得动物模型在研究胆汁淤积遗传因素方面的有效性至多是有限的。用人类模型系统取代动物模型研究胆汁淤积风险变异的一个主要障碍是缺乏适当特征的人体肝脏体外模型。人诱导多能干细胞(IPSC)来源的肝细胞(IHEPS)是研究人类基因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，在极化诱导条件下培养的iHEs显示出增强的功能。我们预计，更好地描述IPSC来源的肝脏体外系统的分子特征将有助于使用它们来研究肝病遗传学，并能够在本项目和未来的研究中取代动物模型。在这个项目中，一个多学科团队加入了人类胆汁淤积症、调节基因组学、干细胞建模和脂质组学的专业知识，将研究两种培养系统(经典2D模型与优化胆汁淤积模型的新三明治模型)，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)更好地概括人类肝细胞的相关特征，以及(2)更适合从功能上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，以突出啮齿动物不适合作为模型的基因座。然后，我们将对胆汁淤积症基因座进行CRISPR激活筛选，以确定非编码变体的目标基因，因为增强子可以与基因在不同的距离上相互作用。最后，我们将关注一个基因座，它将通过结合IPSC中的基因组编辑和胆汁淤积症优化的iHEPS协议来建模。然后，编辑后的iHEPS将被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体的特征。我们期望证明iHEPs在研究与胆汁淤积相关的非编码变异方面具有更好的适用性，为iHEPs风险基因变异到功能的研究提供了一个框架。更广泛地说，这些数据集和分析框架将适用于在未来的研究中调查其他肝病的风险基因。

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.