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

University Of Illinois At Chicago, Chicago IL

Investigators

Abstract

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.