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

对肝脏的药物和化学毒性是急性肝功能衰竭的主要原因。食品和药物管理局要求在人类临床试验之前在动物身上进行药物测试；然而，动物实验速度慢、成本高，而且不能总是预测药物/化学物质对人类的肝脏毒性。因此，迫切需要体外(体外)人体肝脏模型，在活体人体试验之前，可以用来筛选药物毒性。不幸的是，用于采集人类肝细胞进行检测的捐献器官严重短缺。或者，人类诱导多能干细胞(IPSC)来源的人肝细胞可以提供几乎无限的、特定于患者的细胞来源，但目前的方法无法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肝提取物制成的纳米结构3D支架来应对这一关键挑战，当IPSC来源的肝细胞与肝脏的关键支持细胞类型共同培养时，该支架可以向IPSC来源的肝细胞传递适当的生化和生物物理信号。这个平台将以肝脏功能为特征，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，并被用来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参与到使用该项目的发现和装置的研究体验中。这些努力将在高中早期引入尖端研究概念，从而使学生更好地为大学水平的严格工程/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生顶石设计项目的新模块。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，该模型能够筛选对人类肝脏毒性的药物/化学品。使用原代人类肝细胞(PHHs)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限制了PHHs用于数千种化合物的常规筛选。虽然诱导多能干细胞(IPSCs)可以作为一种可持续和丰富的细胞来源，但目前建立IPSC来源的人肝细胞样细胞(IHEPs)的方案无法使细胞完全成熟为成年PHH表型。因此，需要更好地了解IHEP功能的微环境调节器。该项目通过开发由脱细胞的肝细胞外基质(ECM)制成的新的纳米结构3D支架和新的共培养技术来满足这一需求，该技术可使IPSCs分化为肝细胞表型并支持非实质细胞类型(NPC)。电纺纳米纤维将被用来从合成聚合物和天然聚合物中创建纳米级的3D支架材料。纳米纤维将进一步包裹生长因子(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。由天然提取的细胞外基质制成的支架允许细胞与体内存在的许多分子相互作用。在存在或不存在肝神经干细胞的情况下，使用与生长因子结合的细胞外基质纳米纤维控制向下分化iHEPs，将在两个目标下进行探索。第一个目标是开发从脱细胞的肝脏ECM产生纳米纤维的方法，同时使用胶原和Matrigel作为对照，并测试对长期IHEP功能/肝脏NPC刺激的影响。目标成果包括使用四种不同的ECM材料(人肝ECM、猪肝ECM、Matrigel和鼠尾胶原I型)构建可控制纤维直径的新型ECM纳米纤维支架，并深入评估四种细胞类型(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纳米纤维支架上的共培养功能。第二个目标是确定关键的GFS和药物对IHEP/NPC单细胞培养和共培养的影响，这些细胞外基质纳米纤维的复杂性在第一个目标中不断增加。目标结果包括：证实了电纺ECM纳米纤维对四种不同生长因子(VEGF、HGF、HB-EGF和OSM)的可控细胞因子输送，评估了纳米纤维输送细胞因子对四种重要肝细胞类型的影响，并确定了导致IHEP功能成熟和鼻咽癌表型标志物保持2-4周的最佳输送和共培养模式。最后，这种方法的实用性将被证明用于筛选化合物来评估药物引起的肝损伤。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.