The Role of Liver Zonation in Physiology, Regeneration, and Disease.

As blood flows from the portal triad to the central vein, cell-mediated depletion establishes gradients of soluble factors such as oxygen, nutrients, and hormones, which act through molecular pathways (e.g., Wnt/ß-catenin, hedgehog) to spatially regulate hepatocyte functions along the sinusoid. Such "zonation" can lead to the compartmentalized initiation of several liver diseases, including alcoholic/non-alcoholic fatty liver diseases, chemical/drug-induced toxicity, and hepatocellular carcinoma, and can also modulate liver regeneration. Transgenic rodent models provide valuable information on the key molecular regulators of zonation, while in vitro models allow for subjecting cells to precisely controlled factor gradients and elucidating species-specific differences in zonation. Here, we discuss the latest advances in both in vivo and in vitro models of liver zonation and pending questions to be addressed moving forward. Ultimately, obtaining a deeper understanding of zonation can lead to the development of more effective therapeutics for liver diseases, microphysiological systems, and scalable cell-based therapies.

Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity.

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants.

Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine.

Owing to species-specific differences in liver pathways, in vitro human liver models are utilized for elucidating mechanisms underlying disease pathogenesis, drug development, and regenerative medicine. To mitigate limitations with de-differentiated cultures, bioengineers have developed advanced techniques/platforms, including micropatterned cocultures, spheroids/organoids, bioprinting, and microfluidic devices, for perfusing cell cultures and liver slices. Such techniques improve mature functions and culture lifetime of primary and stem-cell human liver cells. Furthermore, bioengineered liver models display several features of liver diseases including infections with pathogens (e.g., malaria, hepatitis C/B viruses, Zika, dengue, yellow fever), alcoholic/nonalcoholic fatty liver disease, and cancer. Here, we discuss features of bioengineered human liver models, their uses for modeling aforementioned diseases, and how such models are being augmented/adapted for fabricating implantable human liver tissues for clinical therapy. Ultimately, continued advances in bioengineered human liver models have the potential to aid the development of novel, safe, and efficacious therapies for liver disease.

Meeting the Challenge of Predicting Hepatic Clearance of Compounds Slowly Metabolized by Cytochrome P450 Using a Novel Hepatocyte Model, HepatoPac.

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min/kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 11 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 82% (14 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 70% within 2-fold and 100% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

Advances in Engineered Human Liver Platforms for Drug Metabolism Studies.

Metabolism in the liver often determines the overall clearance rates of many pharmaceuticals. Furthermore, induction or inhibition of the liver drug metabolism enzymes by perpetrator drugs can influence the metabolism of victim drugs (drug-drug interactions). Therefore, determining liver-drug interactions is critical during preclinical drug development. Unfortunately, studies in animals are often of limited value because of significant differences in the metabolic pathways of the liver across different species. To mitigate such limitations, the pharmaceutical industry uses a continuum of human liver models, ranging from microsomes to transfected cell lines and cultures of primary human hepatocytes (PHHs). Of these models, PHHs provide a balance of high-throughput testing capabilities together with a physiologically relevant cell type that exhibits all the characteristic enzymes, cofactors, and transporters. However, PHH monocultures display a rapid decline in metabolic capacity. Consequently, bioengineers have developed several tools, such as cellular microarrays, micropatterned cocultures, self-assembled and bioprinted spheroids, and perfusion devices, to enhance and stabilize PHH functions for ≥2 weeks. Many of these platforms have been validated for drug studies, whereas some have been adapted to include liver nonparenchymal cells that can influence hepatic drug metabolism in health and disease. Here, we focus on the design features of such platforms and their representative drug metabolism validation datasets, while discussing emerging trends. Overall, the use of engineered human liver platforms in the pharmaceutical industry has been steadily rising over the last 10 years, and we anticipate that these platforms will become an integral part of drug development with continued commercialization and validation for routine screening use.

Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. METHODS: Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. RESULTS: HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. CONCLUSIONS: Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro.

UNLABELLED: Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e., personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECMs) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned coculture (iMPCC) platform in a multiwell format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for at least 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel). We assessed iHep maturity by global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal cytochrome P450 (CYP450) activities, phase II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. CONCLUSION: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multiorgan responses to compounds.

Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes.

Accurate prediction of in vivo hepatic drug clearance using in vitro assays is important to properly estimate clinical dosing regimens. Clearance of low-turnover compounds is especially difficult to predict using short-lived suspensions of unpooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been shown previously to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 enzymes in MPCCs created from unpooled cryopreserved PHH donors. MPCCs were then used to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 ml/min per kilogram). MPCCs predicted 73, 92, and 96% of drug clearance values for all tested drugs within 2-fold, 3-fold, and 4-fold of in vivo values, respectively. There was good correlation (R(2) = 0.94, slope = 1.05) of predictions between the two PHH donors. On the other hand, suspension hepatocytes and conventional monolayers created from the same donor had significantly reduced predictive capacity (i.e., 30-50% clearance values within 4-fold of in vivo), and were not able to metabolize several drugs. Finally, we modulated drug clearance in MPCCs by inducing or inhibiting P450s. Rifampin-mediated CYP3A4 induction increased midazolam clearance by 73%, while CYP3A4 inhibition with ritonavir decreased midazolam clearance by 79%. Similarly, quinidine-mediated CYP2D6 inhibition reduced clearance of dextromethorphan and desipramine by 71 and 22%, respectively. In conclusion, MPCCs created using cryopreserved unpooled PHHs can be used for drug clearance predictions and to model drug-drug interactions.

Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters.

Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1ß-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1ß exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1ß-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1ß interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1ß antibody. Unlike IL-1ß, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.

Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.

Meeting the challenge of predicting hepatic clearance of compounds slowly metabolized by cytochrome P450 using a novel hepatocyte model, HepatoPac.

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

Bioactivation and toxicity of acetaminophen in a rat hepatocyte micropatterned coculture system.

We have recently shown that primary rat hepatocytes organized in micropatterned cocultures with murine embryonic fibroblasts (HepatoPac™) maintain high levels of liver functions for at least 4 weeks. In this study, rat HepatoPac was assessed for its utility to study chemical bioactivation and associated hepatocellular toxicity. Treatment of HepatoPac cultures with acetaminophen (APAP) over a range of concentrations (0-15 mM) was initiated at 1, 2, 3, or 4 weeks followed by the assessment of morphological and functional endpoints. Consistent and reproducible concentration-dependent effects on hepatocyte structure, viability, and basic functions were observed over the 4-week period, and were exacerbated by depleting glutathione using buthionine sulfoximine or inducing CYP3A using dexamethasone, presumably due to increased reactive metabolite-induced stress and adduct formation. In conclusion, the results from this study demonstrate that rat HepatoPac represents a structurally and functionally stable hepatic model system to assess the long-term effects of bioactivated compounds.

Long-term stability of primary rat hepatocytes in micropatterned cocultures.

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac™). Here, we assess the long-term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96-well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel™ sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW-7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3-hydroxy-3-methylglutaryl-CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96-well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways.

Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.

Engineered Platforms for Maturing Pluripotent Stem Cell-Derived Liver Cells for Disease Modeling.

Several liver diseases (eg, hepatitis B/C viruses, alcoholic/nonalcoholic fatty liver, malaria, monogenic diseases, and drug-induced liver injury) significantly impact global mortality and morbidity. Species-specific differences in liver functions limit the use of animals to fully elucidate/predict human outcomes; therefore, in vitro human liver models are used for basic and translational research to complement animal studies. However, primary human liver cells are in short supply and display donor-to-donor variability in viability/quality. In contrast, human hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells and embryonic stem cells are a near infinite cell resource that retains the patient/donor's genetic background; however, conventional protocols yield immature phenotypes. HLC maturation can be significantly improved using advanced techniques, such as protein micropatterning to precisely control cell-cell interactions, controlled sized spheroids, organoids with multiple cell types and layers, 3-dimensional bioprinting to spatially control cell populations, microfluidic devices for automated nutrient exchange and to induce liver zonation via soluble factor gradients, and synthetic biology to genetically modify the HLCs to accelerate and enhance maturation. Here, we present design features and characterization for representative advanced HLC maturation platforms and then discuss HLC use for modeling various liver diseases. Lastly, we discuss desirable advances to move this field forward. We anticipate that with continued advances in this space, pluripotent stem cell-derived liver models will provide human-relevant data much earlier in preclinical drug development and reduce animal usage, help elucidate liver disease mechanisms for the discovery of efficacious and safe therapeutics, and be useful as cell-based therapies for patients suffering from end-stage liver failure.

Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro.

Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000 nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B.

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

Modulation of human iPSC-derived hepatocyte phenotype via extracellular matrix microarrays.

In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.

Engineered matrix microenvironments reveal the heterogeneity of liver sinusoidal endothelial cell phenotypic responses.

Fibrosis is one of the hallmarks of chronic liver disease and is associated with aberrant wound healing. Changes in the composition of the liver microenvironment during fibrosis result in a complex crosstalk of extracellular cues that promote altered behaviors in the cell types that comprise the liver sinusoid, particularly liver sinusoidal endothelial cells (LSECs). Recently, it has been observed that LSECs may sustain injury before other fibrogenesis-associated cells of the sinusoid, implicating LSECs as key actors in the fibrotic cascade. A high-throughput cellular microarray platform was used to deconstruct the collective influences of defined combinations of extracellular matrix (ECM) proteins, substrate stiffness, and soluble factors on primary human LSEC phenotype in vitro. We observed remarkable heterogeneity in LSEC phenotype as a function of stiffness, ECM, and soluble factor context. LYVE-1 and CD-31 expressions were highest on 1 kPa substrates, and the VE-cadherin junction localization was highest on 25 kPa substrates. Also, LSECs formed distinct spatial patterns of LYVE-1 expression, with LYVE-1+ cells observed in the center of multicellular domains, and pattern size regulated by microenvironmental context. ECM composition also influenced a substantial dynamic range of expression levels for all markers, and the collagen type IV was observed to promote elevated expressions of LYVE-1, VE-cadherin, and CD-31. These studies highlight key microenvironmental regulators of LSEC phenotype and reveal unique spatial patterning of the sinusoidal marker LYVE-1. Furthermore, these data provide insight into understanding more precisely how LSECs respond to fibrotic microenvironments, which will aid drug development and identification of targets to treat liver fibrosis.