Developing preclinical human iPSC-based HTS assays to identify therapeutic agents for biliary atresia

项目摘要 这项研究的目的是开发基于IPSC的患者HTS检测方法，以便于 治疗胆道闭锁(BA)纤维化的治疗发现。BA是引起儿科疾病最常见的原因 在美国是终末期肝病，如果不治疗，在生命的头两年内不可避免地会致命。 值得注意的是，BA是人类中最迅速的纤维化肝病，与显著的 儿童发病率和死亡率。与其他肝病逐渐进展为 几十年来，BA婴儿在几周到几个月内典型地发展为纤维化/硬化 出生后。虽然有一种姑息性的外科手术，但还没有已知的治疗方法来阻止 进行性纤维化；大多数出生时患有这种疾病的婴儿将需要肝移植以 活下去。开发有效的抗纤维化药物的一个主要挑战是缺乏一种模型 人类疾病。 人类诱导多能干细胞(IPSC)技术提供了一种替代 使用患者组织进行疾病建模的功能性、可再生和相关的细胞源。基于 我们在体外疾病建模方面的专业知识，我们最近成功地开发出了BA患者- 并已证明这些细胞产生显著更多的胶原和其他 纤维化标志物和胆汁分化不足(BA的主要疾病特征)，与 健康儿童的ipscs。这一关于BA患者IPSCs的新研究路线使其具有可行性 在更贴近人类的环境中评估潜在药物的有效性和安全性。因此，我们相信 这种BA的人体细胞模型可以作为一种理想的系统来鉴定有效的抗纤维化药物 复方丹参治疗肝纤维化的研究。 在目前的研究中，我们建议：1)建立一种新的高通量检测方法来评估抗肝纤维化 用COL1A1报告基因BA-IPSC细胞株研究化合物对BA纤维化的影响。我们将确定 小型化分析和稳健的分析自动化的条件。2)执行引导筛选以 使用临床药库验证和优化分析，以确保自动化、可靠性和 测定重现性。3)开发独立的二级检测方法，通过进一步确定命中选择的优先顺序 验证HITS的抗肝纤维化作用并评价其保护/不良反应 取自患者IPSCs的肝胆组织。在这项研究结束时，我们将拥有 开发了一种强大的、基于患者细胞的HTS检测方法，能够识别新的疾病靶点和线索 为BA患者开发新的治疗方法。此外，该项目的成功将是向前迈出的一步 在将IPSC的基本发现转化为治疗应用方面，帮助实现他们在 发展再生医学。

Project Summary The objective of the research is to develop patient iPSC-based HTS assays that can facilitate therapeutic discovery for treating biliary atresia (BA) fibrosis. BA is the most common cause of pediatric end-stage liver disease in the U.S. and is inevitably fatal within the first two years of life if untreated. Notably, BA is the most rapidly fibrosing liver disease in humans and is associated with significant morbidity and mortality in children. Compared to other liver diseases that gradually progress into cirrhosis over decades, BA infants characteristically develop fibrosis/cirrhosis within weeks to months after birth. Although there is a palliative surgical procedure, there is no known treatment to halt the progressive fibrosis; most infants born with the disease will need liver transplantation in order to survive. A main challenge in developing effective anti-fibrotic drugs has been the lack of a model of the human disease. The human induced pluripotent stem cell (iPSC) technology provides an alternative for generating functional, renewable and relevant cell sources for disease modeling using patient tissues. Based on our expertise on in vitro disease modeling, we have recently succeeded in developing BA patient- specific iPSCs and have demonstrated that these cells produce significantly more collagen and other fibrosis markers along with deficiency in biliary differentiation (key disease features of BA), compared to the iPSCs of healthy children. This new line of research on BA patient iPSCs makes it feasible to evaluate both efficacy and safety of potential drugs in a more human-relevant setting. Thus we believe this human cellular model of BA can serve as an ideal system to identify effective anti-fibrotic compounds in treating liver fibrosis in BA. In the current study, we propose to: 1) Develop a novel high throughput assay to assess anti-fibrotic effects of compounds on BA fibrosis using COL1A1 reporter BA-iPSC lines. We will determine the conditions for miniaturizing the assay and for robust assay automation. 2) Perform pilot screens to validate and optimize the assay using a clinical drug library in order to ensure automation reliability and assay reproducibility. 3) Develop independent secondary assays to prioritize hit selection by further verifying the anti-fibrotic effects of the hits and evaluating their protective/adverse effects on hepatobiliary tissues derived from patient iPSCs. At the conclusion of this study, we will have developed a robust, patient cell-based HTS assay capable of identifying new disease targets and leads for developing novel therapies for BA patients. Moreover, success of this project will be a step forward in translating basic iPSC discoveries to therapeutic applications, helping to fulfill their promise in developing regenerative medicine.