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

药物和化学物质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（Food and Drug Administration）规定，在进行人体临床试验之前，必须先在动物身上进行药物测试；然而，动物实验是缓慢的，昂贵的，并不能总是预测药物/化学物质引起的人类肝脏毒性。因此，迫切需要体外（体外）人体肝脏模型，用于在活体人体试验之前筛选药物毒性。不幸的是，用于采集用于测试的人类肝细胞的供体器官严重短缺。另外，人类诱导多能干细胞（iPSC）衍生的人类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。该项目旨在解决这一关键挑战，通过开发由天然人类肝脏提取物制成的纳米结构3D支架，当ipsc衍生的肝细胞与肝脏的关键支持细胞类型共培养时，可以向其传递适当的生化和生物物理信号。该平台将以肝功能为特征，如代谢体内药物的能力。最终，这个人类肝脏培养平台可以降低开发更安全的人类药物/化学品的成本，并用于更好地了解人类肝脏疾病的影响。教育方面的努力将侧重于让高中教师和学生利用这个项目的发现和设备参与研究经验。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。生物制造研究生课程和本科毕业设计项目的新模块也将被开发。该项目的重点是解决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/化学物质对人类肝脏的毒性。使用原代人肝细胞（PHHs）的模型已经开发出来，可以完成这项任务，但是健康供体组织的稀缺性限制了PHHs在数千种化合物的常规筛选中的使用。虽然诱导多能干细胞（iPSCs）可以作为一种可持续的、丰富的细胞来源，但目前创造ipsc衍生的人肝细胞样细胞（iHeps）的方案无法使细胞完全成熟为成人PHH表型。因此，需要更好地了解iHep功能的微环境调节因子。该项目通过开发由脱细胞肝细胞外基质（ECM）制成的新型纳米结构3D支架，以及用于共培养分化为肝细胞表型和支持非实质细胞类型（npc）的iPSCs的新培养技术，解决了这一需求。静电纺丝纳米纤维将用于从合成和天然聚合物中制造纳米级3D支架材料。纳米纤维将进一步涂覆生长因子（GF）结合分子，如肝素，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。在存在或不存在肝脏npc的情况下，使用gf结合的ECM纳米纤维来控制iHeps在肝脏谱系中的分化将在两个目标下进行探索。第一个目标是开发从去细胞化的肝脏ECM中生成纳米纤维的工艺，同时使用胶原和Matrigel作为对照，并测试长期iHep功能+/-肝脏NPC刺激的效果。客观结果包括使用四种不同的ECM材料（人肝ECM、猪活ECM、Matrigel和大鼠尾胶原I型）构建了可控制纤维直径的基于ECM的新型纳米纤维支架，并深入评估了四种细胞类型（iHeps、原代人肝窦内皮细胞、肝星状细胞和kupffer细胞）及其在每种ECM纳米纤维支架上共培养的功能。第二个目标是确定关键gf和药物对iHep/NPC单培养物和共培养物的影响，这些培养物迭代地播种到第一个目标中开发的越来越复杂的ECM纳米纤维上。客观结果包括四种不同生长因子（VEGF， HGF， HB-EGF和OSM）的细胞因子递送控制，纳米纤维细胞因子递送对四种重要肝细胞类型的影响评估，GF递送和共培养模型的确定，该模型可在2-4周内最佳维持iHep功能成熟度和NPC表型标记物的保留。最后，这种方法的效用将被证明筛选化合物，以评估药物引起的肝损伤。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.