CAREER: Towards a stem cell-derived 3D human liver array for high-throughput screening

1351909 Khetani, Salman r .药物性肝损伤（DILI）是人类急性肝衰竭和肝移植的主要原因，可导致药物使用限制、药物黑框警告以及药物上市前和上市后的损耗。除了对患者福祉的影响外，DILI的经济影响也是显著的（将一种药物成功推向市场需要约10亿美元和12-15年）。美国食品和药物管理局（FDA）要求的动物试验并不能完全代表人类的情况，因为动物和人类在肝功能方面存在显著的进化差异。因此，使用人体细胞对化合物进行潜在毒性的临床前筛选现在构成了药物开发管道的关键部分。诱导多能干细胞衍生的人类肝细胞，称为肝细胞（iHeps），不仅可以为筛选数百万种化合物提供近乎无限的细胞来源，而且还可以实现个性化（患者特异性）药物筛选和未来的治疗应用。然而，迄今为止创造的iHeps在功能上不像成人原代肝细胞（从供体肝脏中分离出来）那样成熟，这是在药物在临床试验中对活体患者进行试验之前充分预测临床结果所必需的。为了解决这一技术差距，研究人员将利用半导体（微芯片）行业的微加工工具，利用iHeps和其他肝脏支持细胞类型，在体外（体外）显示功能成熟和稳定数周，来设计一个小型化的3D人体肝脏模拟物。这种肝脏模拟物将使用几个重要的标记物来表征其相对于成人原代肝细胞的功能成熟度。此外，研究人员还将探索肝脏模拟物是否有能力提供有关单药和多药治疗导致的DILI的有临床意义的信息，以尝试模拟临床药物给药方案。该研究员将把他的研究与教育项目结合起来，旨在为科罗拉多州立大学（CSU）和北科罗拉多地区新生的生物医学工程（BME）本科项目建立一个连续的指导和科学社区。特别是，研究者将设计和实施：a)一门新课程，让科罗拉多州立大学BME学院的学生在正式的研究实验室环境中获得指导的研究经验；b)一个循序渐进的项目，让高中生接触到BME的职业选择，并最终在科罗拉多州立大学BME实验室进行暑期实践研究经验；c)循环学习模块，让K-6年级的学生在课后项目中接触到科学和工程方面的进展。即将开发的基于ihep的3D人体肝脏模拟物最终可能导致在药物开发早期开发有效和安全的治疗方法，以增加临床成功的可能性，并限制患者接触不安全药物。该平台也可用于评估污染环境的工业化学品的伤害潜力，促进对肝脏疾病的更好的细胞/分子理解，以及帮助设计和实施个性化医疗战略。更广泛地说，这种工程装置最终可以与其他组织模型集成成一个单一的集成系统，提供多器官(例如：“全身”)对各种外源性侮辱的理解水平。上述教育努力将建立一个健全的机制，使不同年龄组的学生对科学和工程教育感兴趣，并使彼此参与到为年轻学生提供的教学和实施计划中来。由于该项目的跨学科性质，由CBET部门的生物技术、生化和生物质工程项目颁发的CAREER奖由生物基础设施部门的生物研究仪器开发项目共同资助。

1351909 Khetani, Salman R.Drug-induced liver injury (DILI) is a leading cause of acute liver failures and liver transplants in humans that can lead to restrictions on drug use, black-box warnings on drugs, and the pre-launch and post-market attrition of pharmaceuticals. In addition to its impact on the well-being of patients, the economic impact of DILI is significant (~$1B and 12-15 years to bring 1 successful drug to market). Animal testing that is required by the Food and Drug Administration (FDA) is not fully representative of the human condition due to significant and evolutionary differences between animals and humans in liver functions. Therefore, preclinical screening of compounds for potential toxicity using human cells now constitutes a critical part of the drug development pipeline. Induced pluripotent stem cell-derived human liver cells, called hepatocytes (iHeps), can not only provide a near unlimited source of cells for screening millions of compounds, but also enable personalized (patient-specific) drug screening and therapeutic applications in the future. However, iHeps created to date are not as functionally mature as adult primary hepatocytes (isolated from donor livers) which is required to adequately predict clinical outcomes before the drugs are tested on live patients in clinical trials. To address this technology gap, the investigator will utilize microfabrication tools adapted from the semiconductor (microchip) industry to engineer a miniaturized 3D human liver mimic using iHeps and other liver supportive cell types that displays functional maturity and stability for several weeks in vitro (outside the body). This liver mimic will be characterized using several important markers for its functional maturity relative to adult primary hepatocytes. Additionally, the liver mimic will be probed for its ability to provide clinically meaningful information on DILI due to single drug and multiple drug treatments in attempts to model clinical drug dosing regimens. The investigator will integrate his research with educational programs designed to build a continuum of mentorship and scientific community in the nascent biomedical engineering (BME) undergraduate program at Colorado State University (CSU) and in the Northern Colorado region. In particular, the investigator will design and implement: a) a new course that exposes CSU BME college students to mentored research experiences in a formal research laboratory setting; b) a step-wise program that exposes high school students to career options in BME and culminates in hands-on summer research experiences in CSU BME laboratories; and, c) recurrent learning modules that expose K-6 students in an after-school program to advances in science and engineering. The iHep-based 3D human liver mimic that will be developed could ultimately lead to the development of efficacious and safe therapeutics earlier in drug development towards increasing the likelihood of clinical success and limiting patient exposure to unsafe drugs. This platform may also be applicable for evaluating the injury potential of industrial chemicals that contaminate the environment, in promoting a better cellular/molecular understanding of diseases of the liver, and in helping to design and implement personalized medicine strategies. More broadly, the engineered device could ultimately be integrated with other tissue models into a single integrated system that provides a multi-organ (i.e. 'whole body') level of understanding of various exogenous insults. The aforementioned educational efforts will develop a robust mechanism to get students of different age groups interested in science and engineering education and to get each other involved in teaching and implementing programs for younger students.Due to the interdisciplinary nature of the project, this CAREER award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Instrument Development for Biological Research Program of the Division of Biological Infrastructure.