A Liver Extracellular Matrix Nanofiber Platform for the Functional Maturation of Induced Pluripotent Stem Cell-Derived Human Hepatocytes

Introduction:: Drug-induced liver injury (DILI) remains a leading cause of drug attrition and acute liver failures, partly due to the inadequacy of animal models to accurately predict human clinical outcomes, which necessitates the utilization of in vitro models of the human liver. However, primary human hepatocytes (PHHs) are in short supply for routine drug screening. In contrast, induced pluripotent stem cells (iPSCs)-derived hepatocyte-like cells (HLCs) are a nearly unlimited cell source but display a fetal-like (versus adult-like) phenotype when differentiated using conventional protocols on tissue culture plastic or glass adsorbed with 2D extracellular matrix (ECM) proteins. Electrospinning can produce porous nanoscale 3D fibers that have a large surface area and present a high density of receptor ligands to modulate cell phenotype [1]. However, the application of electrospinning to generate 3D liver-derived ECM substrates for HLC differentiation remains unexplored. Therefore, here we first developed methods to a) electrospin nanofibers of different porosities, diameters and stiffness using porcine liver ECM (PLECM) and chitosan with or without type I collagen or poly(vinyl alcohol) (PVA), and then utilized these fibers to determine functional modulation in iPSC-derived HLCs relative to conventional adsorbed ECM substrates.


Materials and Methods:: ECM and polymers dissolved in optimized solvents was electrospun from a high voltage DC power supply and collected onto oxidized glass cover slides. The nanofiber mechanical properties, such as Young’s modulus and stiffness, were assessed by using an atomic force microscope (AFM), and the samples were imaged when submersed in a phosphate-buffered saline (PBS, 1X) solution. iPSCs were differentiated into HLCs over 20 days using a published protocol that uses specific growth factor cocktails to differentiate iPSCs into definitive endoderm, specified hepatic endoderm, hepatoblasts, and finally immature HLCs.


Results, Conclusions, and Discussions:: Results and Discussion: The nanofiber diameters ranged from 98±28 (PVA) to 192±83 nm (collagen/PLECM), and the porosities were similar across conditions (Fig. 1A). The mechanical properties were evaluated in PBS to determine the stiffness that cells experience in culture. Chitosan and PVA nanofibers have higher stiffness when compared to the nanofibers with collagen (Fig. 1B). About 80% of HLCs express HNF4A (master liver transcription factor) on PLECM/collagen nanofibers, which is higher than HLCs on chitosan nanofibers (~60%) and on ECM adsorbed control (~70%) (Fig. 1C, D). HLCs on chitosan/collagen, PLECM/collagen, and chitosan nanofibers also secrete significantly more albumin than HLCs on the adsorbed ECM control (Fig. 1E).
Conclusion: We showed for the first time that introducing collagen and liver ECM to nanofibers decreases nanofiber stiffness, making the nanofibers softer than the 2D control. Including collagen and ECM in nanofibers also improves HLCs differentiation, resulting in higher HNF4A purity and albumin secretion than on adsorbed ECM control surfaces. Future efforts will focus on co-culturing HLCs with liver non-parenchymal cells on the nanofibers to study heterotypic cell-cell and cell-ECM interactions.


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References (Optional): : [1]. Jennifer S Liu , Liszt Y C Madruga , Yang Yuan , Matt J Kipper , Salman R Khetani, Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro, Adv Healthc Mater. 2023