Collaborative Research: Protein nanofiber growth factor delivery platforms for modulating phenotype of iPSC-derived human hepatocytes and liver non-parenchymal cells

合作研究：用于调节 iPSC 衍生的人肝细胞和肝脏非实质细胞表型的蛋白质纳米纤维生长因子递送平台

• 批准号: 1933540
• 负责人: Salman Khetani
• 金额: $ 30万
• 依托单位: University of Illinois at Chicago
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1933540&HistoricalAwards=false
• 关键词: Collaborative; Research; Protein; nanofiber; growth

Drug and chemical toxicity to the liver is a major cause of acute liver failures. The Food and Drug Administration mandates the testing of drugs in animals prior to human clinical trials; however, animal experiments are slow, costly, and cannot always predict drug/chemical-induced liver toxicities in humans. Therefore, there is an urgent need for in vitro (outside the body) models of the human liver that can be used to screen for drug toxicity prior to testing in live humans. Unfortunately, there is a severe shortage of donor organs for harvesting human liver cells for testing. Alternatively, human induced pluripotent stem cell (iPSC)-derived human liver cells could provide a nearly infinite and patient-specific source of cells, but current methods are not able to mature these cells to the same functional levels as in the native liver. This project seeks to address this critical challenge by developing nanostructured 3D scaffolds made from native human liver extracts that can deliver the appropriate biochemical and biophysical signals to iPSC-derived liver cells when they are co-cultured with key supportive cell types of the liver. This platform will be characterized for liver functions, such as the ability to metabolize drugs as in the body. Ultimately, this human liver culture platform could reduce the cost of developing safer drugs/chemicals for humans, and be used to better understand the effects of human liver diseases. Educational efforts will focus on engaging high school teachers and students in research experiences using the findings and devices of this project. Such efforts will introduce cutting-edge research concepts earlier in high school, thereby preparing students better for a rigorous engineering/bioengineering curriculum at the college level. New modules for a graduate program in biomanufacturing and undergraduate capstone design projects will also be developed. The focus of this project is on addressing the need for improved cell culture-based models of liver function that enable screening of drugs/chemicals for human liver toxicity. Models using primary human hepatocytes (PHHs) have been developed that can accomplish this task, but scarcity of healthy donor tissues limits the use of PHHs for routine screening of thousands of compounds. Though induced Pluripotent Stem Cells (iPSCs) can serve as a sustainable and abundant cell source, current protocols to create iPSC-derived human hepatocyte-like cells (iHeps) are unable to fully mature the cells towards the adult PHH phenotype. Thus a better understanding of microenvironmental regulators of iHep functions is needed. This project addresses this need by developing new nanostructured 3D scaffolds made from decellularized liver extracellular matrix (ECM) and new culture technologies for co-cultures of iPSCs differentiated into a hepatocyte phenotype and supporting non-parenchymal cell types (NPCs). Electrospinning nanofibers will be used to create nanoscale 3D scaffold materials from both synthetic and natural polymers. Nanofibers will be further coated with growth factor (GF)-binding molecules such as heparin towards mimicking the cell-ECM and cell-GF signaling that occurs in vivo. Scaffolds made from naturally-derived ECM allow cells to interact with many molecules present in vivo. The use of GF-binding ECM nanofibers for controlled differentiation of iHeps down the hepatic lineage in the presence or absence of liver NPCs will be explored under two objectives. The FIRST Objective is to develop processes for generating nanofibers from decellularized liver ECM while using collagen and Matrigel as controls and test effects on long-term iHep functions +/- liver NPC stimulation. Objective outcomes include new ECM-based nanofiber scaffolds with control over fiber diameter, using four different ECM materials (human liver ECM, porcine live ECM, Matrigel and rat tail collagen type I) and an in-depth evaluation of the functions of four cell types (iHeps, primary human liver sinusoidal endothelial cells, hepatic stellate cells and kupffer cells) and their co-cultures on each of these ECM nanofiber scaffolds. The SECOND Objective is to determine the effects of key GFs and drugs on iHep/NPC mono-cultures and co-cultures seeded iteratively onto ECM nanofibers of increasing complexities developed in the first objective. Objective outcomes include demonstrated controlled cytokine delivery from each of the elecrospun ECM nanofibers for four different growth factors (VEGF, HGF, HB-EGF, and OSM), evaluation of the effects of cytokine delivery from nanofibers on each of four important liver cell types, and determination of the GF delivery and co-culture model that results in the optimal maintenance of iHep functional maturity and retention of NPC phenotypic markers over 2-4 weeks. Finally, the utility of this approach will be demonstrated for screening compounds to evaluate drug induced liver injury.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

药物和化学品对肝脏的毒性是急性肝衰竭的主要原因。 美国食品和药物管理局要求在人体临床试验之前在动物中进行药物测试;然而，动物实验缓慢，昂贵，并且不能总是预测药物/化学品诱导的人类肝脏毒性。因此，迫切需要人肝脏的体外（体外）模型，其可用于在活体人体中测试之前筛选药物毒性。不幸的是，用于采集人类肝细胞进行测试的供体器官严重短缺。或者，人诱导多能干细胞（iPSC）衍生的人肝细胞可以提供几乎无限的和患者特异性的细胞来源，但目前的方法不能使这些细胞成熟到与天然肝脏相同的功能水平。该项目旨在通过开发由天然人类肝脏提取物制成的纳米结构3D支架来解决这一关键挑战，这些支架可以在与肝脏的关键支持细胞类型共培养时向iPSC衍生的肝细胞提供适当的生化和生物物理信号。该平台将表征肝功能，例如在体内代谢药物的能力。最终，这个人类肝脏培养平台可以降低为人类开发更安全的药物/化学品的成本，并用于更好地了解人类肝脏疾病的影响。教育工作的重点将是让高中教师和学生利用本项目的研究结果和设备进行研究。这些努力将在高中早期引入尖端的研究概念，从而使学生更好地为大学阶段严格的工程/生物工程课程做好准备。还将开发生物制造研究生课程和本科顶点设计项目的新模块。该项目的重点是解决对改进的基于细胞培养的肝功能模型的需求，从而能够筛选药物/化学品对人类肝脏的毒性。 使用原代人肝细胞（PHH）的模型已经开发出来，可以完成这一任务，但健康供体组织的稀缺性限制了PHH用于数千种化合物的常规筛选。 虽然诱导的多能干细胞（iPSC）可以作为一种可持续的和丰富的细胞来源，目前的方案，以创造iPSC衍生的人肝细胞样细胞（iHep）是无法完全成熟的细胞向成人PHH表型。因此，需要更好地了解iHep功能的微环境调节剂。 该项目通过开发由脱细胞肝脏细胞外基质（ECM）制成的新型纳米结构3D支架和用于分化为肝细胞表型和支持非实质细胞类型（NPC）的iPSC共培养的新培养技术来满足这一需求。 静电纺丝纳米纤维将用于从合成和天然聚合物中创建纳米级3D支架材料。纳米纤维将进一步用生长因子（GF）结合分子如肝素包被，以模拟体内发生的细胞-ECM和细胞-GF信号传导。由天然来源的ECM制成的支架允许细胞与体内存在的许多分子相互作用。将在两个目标下探索在存在或不存在肝NPC的情况下使用GF结合ECM纳米纤维用于iHep沿肝谱系的受控分化。第一个目的是开发从脱细胞肝ECM产生纳米纤维的方法，同时使用胶原蛋白和基质胶作为对照，并测试对长期iHep功能+/-肝NPC刺激的影响。 客观结果包括新的ECM为基础的纤维直径控制，使用四种不同的ECM材料（人肝ECM，猪肝ECM，Matrigel和大鼠尾胶原I型）和四种细胞类型（iHep，原代人肝窦内皮细胞，肝星状细胞和枯否细胞）的功能的深入评价和他们的共培养物上的每一个这些ECM纤维支架。 第二个目的是确定关键GF和药物对迭代接种到第一个目的中开发的复杂性增加的ECM纳米纤维上的iHep/NPC单培养物和共培养物的影响。 客观的结果包括证明了四种不同生长因子从每个电纺ECM纳米纤维的受控细胞因子递送（VEGF、HGF、HB-EGF和OSM），评价来自纳米纤维的细胞因子递送对四种重要肝细胞类型中的每一种的影响，和GF交付和共同确定，该培养模型导致iHep功能成熟的最佳维持和NPC表型标记的保留超过2-4周。最后，这种方法的实用性将被证明筛选化合物，以评估药物诱导的肝损伤。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。