Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes.

Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.

Computational Construction of Toxicant Signaling Networks.

Humans and animals are regularly exposed to compounds that may have adverse effects on health. The Toxicity Forecaster (ToxCast) program was developed to use high throughput screening assays to quickly screen chemicals by measuring their effects on many biological end points. Many of these assays test for effects on cellular receptors and transcription factors (TFs), under the assumption that a toxicant may perturb normal signaling pathways in the cell. We hypothesized that we could reconstruct the intermediate proteins in these pathways that may be directly or indirectly affected by the toxicant, potentially revealing important physiological processes not yet tested for many chemicals. We integrate data from ToxCast with a human protein interactome to build toxicant signaling networks that contain physical and signaling protein interactions that may be affected as a result of toxicant exposure. To build these networks, we developed the EdgeLinker algorithm, which efficiently finds short paths in the interactome that connect the receptors to TFs for each toxicant. We performed multiple evaluations and found evidence suggesting that these signaling networks capture biologically relevant effects of toxicants. To aid in dissemination and interpretation, interactive visualizations of these networks are available at http://graphspace.org.

Investigating Trans-differentiation of Glioblastoma Cells in an In Vitro 3D Model of the Perivascular Niche.

Glioblastoma multiforme (GBM) is the deadliest form of brain cancer, responsible for over 50% of adult brain tumors. A specific region within the GBM environment is known as the perivascular niche (PVN). This area is defined as within approximately 100 µm of vasculature and plays an important role in the interactions between endothelial cells (ECs), astrocytes, GBM cells, and stem cells. We have designed a 3D in vitro model of the PVN comprising either collagen Type 1 or HyStem-C, human umbilical vein ECs (HUVECs), and LN229 (GBM) cells. HUVECs were encapsulated within the hydrogels to form vascular networks. After 7 days, LN229 cells were co-cultured to investigate changes in both cell types. Over a 14 day culture period, we measured alterations in HUVEC networks, the contraction of the hydrogels, trans-differentiation of LN229 cells, and the concentrations of two chemokines; CXCL12 and TGF-ß. Increased cellular proliferation ranging from 10- to 16-fold was exhibited in co-cultures from days 8 to 14. This was accompanied with a decrease in the height of hydrogels of up to 68%. These changes in the biomaterial scaffold indicate that LN229-HUVEC interactions promote changes to the matrix. TGF-ß and CXCL12 secretion increased approximately 2-2.6-fold each from day 8 to 14 in all co-cultures. The expression of CXCL12 correlated with cell colocalization, indicating a chemotactic role in enabling the migration of LN229 cells toward HUVECs in co-cultures. von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP) in up to 15% of LN229 cells after 24 h in co-culture. Additionally, when LN229 cells were co-cultured with human brain microvascular ECs, the percentages of GFAP+/vWF+ cells were up to 20% higher than that in co-cultures with HUVECs in collagen (2.2 mg/mL) and HyStem-C gels on day 14. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an EC phenotype. Designing in vitro models of trans-differentiation may provide additional insights into how vasculature and cellular phenotypes are altered in GBM.

Bovine Airway Models: Approaches for Investigating Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a multifactorial condition where different genera of bacteria, such as Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis, and viruses, like bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes virus-1, infect the lower respiratory tract of cattle. These pathogens can co-infect cells in the respiratory system, thereby making specific treatment very difficult. Currently, the most common models for studying BRD include a submerged tissue culture (STC), where monolayers of epithelial cells are typically covered either in cellular or spent biofilm culture medium. Another model is an air-liquid interface (ALI), where epithelial cells are exposed on their apical side and allowed to differentiate. However, limited work has been reported on the study of three-dimensional (3D) bovine models that incorporate multiple cell types to represent the architecture of the respiratory tract. The roles of different defense mechanisms in an infected bovine respiratory system, such as mucin production, tight junction barriers, and the production of antimicrobial peptides in in vitro cultures require further investigation in order to provide a comprehensive understanding of the disease pathogenesis. In this report, we describe the different aspects of BRD, including the most implicated pathogens and the respiratory tract, which are important to incorporate in disease models assembled in vitro. Although current advancements of bovine respiratory cultures have led to knowledge of the disease, 3D multicellular organoids that better recapitulate the in vivo environment exhibit potential for future investigations.

Interpretable network propagation with application to expanding the repertoire of human proteins that interact with SARS-CoV-2.

BACKGROUND: Network propagation has been widely used for nearly 20 years to predict gene functions and phenotypes. Despite the popularity of this approach, little attention has been paid to the question of provenance tracing in this context, e.g., determining how much any experimental observation in the input contributes to the score of every prediction. RESULTS: We design a network propagation framework with 2 novel components and apply it to predict human proteins that directly or indirectly interact with SARS-CoV-2 proteins. First, we trace the provenance of each prediction to its experimentally validated sources, which in our case are human proteins experimentally determined to interact with viral proteins. Second, we design a technique that helps to reduce the manual adjustment of parameters by users. We find that for every top-ranking prediction, the highest contribution to its score arises from a direct neighbor in a human protein-protein interaction network. We further analyze these results to develop functional insights on SARS-CoV-2 that expand on known biology such as the connection between endoplasmic reticulum stress, HSPA5, and anti-clotting agents. CONCLUSIONS: We examine how our provenance-tracing method can be generalized to a broad class of network-based algorithms. We provide a useful resource for the SARS-CoV-2 community that implicates many previously undocumented proteins with putative functional relationships to viral infection. This resource includes potential drugs that can be opportunistically repositioned to target these proteins. We also discuss how our overall framework can be extended to other, newly emerging viruses.

Environmental interplay: Stromal cells and biomaterial composition influence in the glioblastoma microenvironment.

Glioblastoma multiforme (GBM) is the most deadly form of brain cancer. Recurrence is common, and established therapies have not been able to significantly extend overall patient survival. One platform through which GBM research can progress is to design biomimetic systems for discovery and investigation into the mechanisms of invasion, cellular properties, as well as the efficacy of therapies. In this review, 2D and 3D GBM in vitro cultures will be discussed. We focus on the effects of biomaterial properties, interactions between stromal cells, and vascular influence on cancer cell survival and progression. This review will summarize critical findings in each of these areas and how they have led to a more comprehensive scientific understanding of GBM. STATEMENT OF SIGNIFICANCE: Glioblastoma multiforme (GBM) is the most deadly form of brain cancer. Recurrence is common, and established therapies have not been able to significantly extend overall patient survival. One platform through which GBM research can progress is to design biomimetic systems for discovery and investigation into the mechanisms of invasion, cellular properties, as well as the efficacy of therapies. In this review, 2D and 3D GBM in vitro cultures will be discussed. We focus on the effects of biomaterial properties, interactions between stromal cells and vascular influence on cancer cell survival and progression. This review will summarize critical findings in each of these areas and how they have lead to a more comprehensive scientific understanding of GBM.

The assembly of integrated rat intestinal-hepatocyte cultures.

The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin-covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4-fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine-liver cultures can be used as a platform for future investigations.

Advances in Human Induced Pluripotent Stem Cell-Derived Hepatocytes for Use in Toxicity Testing.

Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of hepatocyte-like cells (HLCs) from iPSCs has resulted in a new source for liver cells. Since healthy primary human hepatocytes and hepatic cells are difficult to obtain, HLCs are gaining attention. HLCs can be obtained from a continuous, stable source while maintaining their original donor genotype, which opens new avenues into patient-specific testing and therapeutics. Studies have utilized HLCs for toxicity testing to further understand their drug metabolizing capabilities. This review focuses on advances being made to achieve hepatic functions from HLCs, their current use in hepatotoxicity testing, and their potential for future liver-related toxicity evaluations.

Isolating Rat Intestinal Explants for In Vitro Cultures.

The small intestine is an important organ primarily involved in digestion of food and absorption of nutrients. In vitro intestinal models are being developed to study this organ in health and disease. Intestinal explants can be used in such investigations since they contain all the major intestinal cell types. A detailed procedure to isolate intestinal explants from the rat jejunum is described. A protocol for culturing them in vitro for up to 24 hr is also provided. © 2019 by John Wiley & Sons, Inc.

Fibroblasts stimulate macrophage migration in interconnected extracellular matrices through tunnel formation and fiber alignment.

In vivo, macrophages and fibroblasts navigate through and remodel the three-dimensional (3D) extra-cellular matrix (ECM). The orientation of fibers, the porosity, and degree of cross-linking can change the interconnectivity of the ECM and affect cell migration. In turn, migrating cells can alter their microenvironment. To study the relationships between ECM interconnectivity and migration of cells, we assembled collagen hydrogels with dense (DCN) or with loosely interconnected networks (LCN). We find that in DCNs, RAW 264.7 macrophages in monocultures were virtually stationary. In DCN co-cultures, Balb/c 3T3 fibroblasts created tunnels that provided conduits for macrophage migration. In LCNs, fibroblasts aligned fibers up to a distance of 100 µm, which provided tracks for macrophages. Intra-cellular and extra-cellular fluorescent fragments of internalized and degraded collagen were detected inside both cell types as well as around their cell peripheries. Macrophages expressed higher levels of urokinase-type plasminogen activator receptor associated protein (uPARAP)/mannose receptor 1 (CD206) compared to α2ß1 indicating that collagen internalization in these cells occurred primarily via integrin-independent mechanisms. Network remodeling indicated by higher Young's modulus was observed in fibroblast monocultures as a result of TGF-ß secretion. This work unveils new roles for fibroblasts in forming tunnels in networked ECM to modulate macrophage migration.

Multi-cellular transitional organotypic models to investigate liver fibrosis.

Hepatic fibrosis is the result of wound healing and inflammation resulting in organ dysfunction. Hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs) play critical roles in fibrogenesis. As the liver undergoes fibrosis, there are populations of cells that are healthy, fibrotic as well as those undergoing fibrosis. We investigated how a varying mechanical environment could induce changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment resulted in transitioning phenotypes in hepatic cells. We have designed detachable polyelectrolyte multilayers (PEMs) whose elastic moduli ranged from 21 to 43 kPa to serve as Space of Disse mimics. We assembled novel 3D organotypic liver models comprised of hepatocytes, LSECs, HSCs, KCs, and the Space of Disse mimic. We demonstrate how cells in contact with a mechanical gradient exhibit different properties compared to cells cultured using non-gradient PEMs. Significant differences were observed in HSC and KC proliferation between 3D cultures assembled with gradient and non-gradient PEMs. While HSCs on the stiffer regions of the gradient PEMs expressed both GFAP and α-SMA, cells in cultures assembled with homogeneous 43 kPa multilayers primarily expressed α-SMA. Over an 8-day culture, the elastic modulus in the 21 and 43 kPa regions of the gradient PEMs increased by 1.6 and 3.7-fold, respectively. This was accompanied by a 4-fold increase in hydroxyproline. Such in vitro tissues can be used to investigate the effects of liver fibrosis. STATEMENT OF SIGNIFICANCE: We have assembled a liver model assembled with four major primary hepatic cell types to investigate how a varying mechanical environment induces changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment results in transitioning phenotypes in hepatic cells. Our goal was to investigate the interplay between mechanical properties and a multi-cellular engineered liver tissue. In these models, Kupffer cell proliferation and hepatic stellate cell activation occurred due to mechanical cues and inter-cellular signaling across a distance of 2000 µm. These models are unique, in that, fibrosis was initiated purely through changes to the microenvironment. These models were not exposed to fibrogenic factors nor were the models assembled with cells from fibrotic rats. To the best of our knowledge, these are the first liver models that capture how a gradient microenvironment can result in transitioning cellular phenotypes.

Transcriptomic Analysis of Hepatic Cells in Multicellular Organotypic Liver Models.

Liver homeostasis requires the presence of both parenchymal and non-parenchymal cells (NPCs). However, systems biology studies of the liver have primarily focused on hepatocytes. Using an organotypic three-dimensional (3D) hepatic culture, we report the first transcriptomic study of liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) cultured with hepatocytes. Through computational pathway and interaction network analyses, we demonstrate that hepatocytes, LSECs and KCs have distinct expression profiles and functional characteristics. Our results show that LSECs in the presence of KCs exhibit decreased expression of focal adhesion kinase (FAK) signaling, a pathway linked to LSEC dedifferentiation. We report the novel result that peroxisome proliferator-activated receptor alpha (PPARα) is transcribed in LSECs. The expression of downstream processes corroborates active PPARα signaling in LSECs. We uncover transcriptional evidence in LSECs for a feedback mechanism between PPARα and farnesoid X-activated receptor (FXR) that maintains bile acid homeostasis; previously, this feedback was known occur only in HepG2 cells. We demonstrate that KCs in 3D liver models display expression patterns consistent with an anti-inflammatory phenotype when compared to monocultures. These results highlight the distinct roles of LSECs and KCs in maintaining liver function and emphasize the need for additional mechanistic studies of NPCs in addition to hepatocytes in liver-mimetic microenvironments.

High-throughput toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells.

High-throughput screening (HTS) of liver toxicants can bridge the gap in understanding adverse effects of chemicals on humans. Toxicity testing of mixtures is time consuming and expensive, since the number of possible combinations increases exponentially with the number of chemicals. The combination of organotypic culture models (OCMs) and HTS assays can lead to the rapidly evaluation of chemical toxicity in a cost and time-effective manner while prioritizing chemicals that warrant additional investigation. We describe the design, assembly and toxicant response of multi-cellular hepatic organotypic culture models comprised of primary human or rat cells assembled in 96-well plates (denoted as µOCMs). These models were assembled using automated procedures that did not affect hepatocyte function or viability, rendering them ideal for large-scale toxicity evaluations. Rat µOCMs were assembled to obtain insights into deviations from human toxicity. Four test chemicals (acetaminophen, ethanol, isoniazid, and perfluorooctanoic acid) were added to the µOCMs individually or in mixtures. HTS assays were utilized to measure cell death, apoptosis, glutathione depletion, mitochondrial membrane damage, and cytochrome P450 2E1 activity. The µOCMs exhibited increased toxicant sensitivity compared to hepatocyte sandwich cultures. Synergistic and non-synergistic interactions were observed when the toxicants were added as mixtures. Specifically, chemical interactions in the µOCMs were manifested by changes in apoptosis and decreased glutathione. The µOCMs accurately predicted hepatotoxicity for individual and mixtures of toxicants, demonstrating their potential for large-scale toxicity evaluations in the future.

Measuring Cytoplasmic Stiffness of Fibroblasts as a Function of Location and Substrate Rigidity Using Atomic Force Microscopy.

The cytoplasmic stiffness of cells plays a significant role during cell migration. As a cell migrates, differences in cytoplasmic properties occur that subsequently modulate migratory behavior. The properties of the substrate to which cells are adherent also play a role. To accurately measure the cytoplasmic stiffness of cells, we provide detailed instructions on how to assemble hydrogels that exhibit different elastic moduli, culturing cells on these substrates followed by a step-by-step process to measure and analyze the cytoplasmic properties of fibroblasts. In this study, we have measured the elastic moduli of cells at different locations to demonstrate how this property varies as a function of where the measurement is performed. The degree of anisotropy measured by the difference between cytoplasmic stiffness at the two edges of the cell also varied as a function of the elasticity of their underlying substrates. Larger differences in cytoplasmic stiffness between the leading and trailing edges were observed on substrates with a higher elastic modulus. The methods reported in this study can provide information on cellular properties, specifically, how the elastic modulus of cells can be probed and analyzed in vitro.

Extracellular matrix remodeling in 3D: implications in tissue homeostasis and disease progression.

The extracellular matrix (ECM) plays a critical role in regulating cell behavior during tissue homeostasis and in disease progression. Through a combination of adhesion, contraction, alignment of ECM proteins and subsequent degradation, cells change the chemical, mechanical, and physical properties of their surrounding matrix. Other contributing factors to matrix remodeling are the de novo synthesis of ECM proteins, post-translational modifications and receptor-mediated internalization. In this review, we highlight how each of these processes contributes to the maintenance of homeostasis and in disease conditions such as cancer and liver fibrosis. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Investigating acetaminophen hepatotoxicity in multi-cellular organotypic liver models.

In vivo studies clearly demonstrate the participation and subsequent death of non-parenchymal liver cells (NPCs) with corresponding hepatocyte effects. This results in a critical need to investigate how major liver cell types function cohesively during hepatotoxicity. However, virtually no studies replicate these phenomena in vitro. We report the design of multi-cellular three-dimensional (3D) organotypic liver models of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). LSECs and KCs were separated from hepatocytes by a detachable membrane that emulates the physical and chemical properties of the Space of Disse. Acetaminophen (APAP)-induced changes to cellular function and phenotype were investigated. LSECs exhibited approximately 40% cell death at 20mM APAP. KCs exhibited decreased interleukin-10 and increased tumor necrosis factor-alpha and interferon-gamma secretion. The secretion of these proteins altered hepatocyte function and signaling. Both LSECs and KCs maintained phenotypic markers. At 20mM APAP, the 3D models exhibited aspartate aminotransferase to alanine aminotransferase ratios from 2.1-2.5 and 45% glutathione depletion, corresponding to what is seen in vivo. At 10 and 20mM APAP, the 3D models exhibited cell death, primarily through necrosis. Therefore, the 3D cultures described in this report demonstrate significant potential as realistic models for hepatotoxicity studies.

In Vitro Intestinal and Liver Models for Toxicity Testing.

The human body is exposed to hundreds of chemicals every day. Many of these toxicants have unknown effects on the body that can be deleterious. Furthermore, chemicals can have a synergistic effect, resulting in toxic responses of cocktails at relatively low individual exposure levels. The gastrointestinal (GI) tract and the liver are the first organs to be exposed to ingested pharmaceuticals and environmental chemicals. As a result, these organs often experience extensive damage from xenobiotics and their metabolites. In vitro models offer a promising method for testing toxic effects. Many advanced in vitro models have been developed for GI and liver toxicity. These models strive to recapitulate the in vivo organ architecture to more accurately model chemical toxicity. In this review, we discuss many of these advances, in addition to recent efforts to integrate the GI and the liver in vitro for a more holistic toxicity model.

The design of antimicrobial LL37-modified collagen-hyaluronic acid detachable multilayers.

UNLABELLED: The design of antimicrobial membranes and thin films are critical for the design of biomaterials that can combat bacterial contamination. Since the long-term use of conventional antibiotics can result in bacterial resistance, there is a critical need to incorporate natural antimicrobial peptides (AMPs) that not only prevent a wide range of pathogens from causing infections but can also promote many beneficial outcomes in wounded tissues. We report the design and antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a naturally occurring human AMP. LL-37 was physically adsorbed and chemically immobilized on the surface of PEMs. The antimicrobial and cytotoxic properties of PEMs were tested with Gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. The ability to prevent bacterial adhesion and to neutralize an E. coli layer was investigated as a function of LL-37 concentration. An interesting trend was that even unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, bacterial adhesion was significantly lower on the surface of the films as well as in the surrounding broth. Immobilizing LL-37 resulted in less than 3% bacterial adhesion on the surface due to the presence of the peptide. LL-37 modified PEMs did not result in any cytotoxicity up to input concentrations of 16µM. More importantly, urea and albumin secretion by hepatocytes were unaffected even at high LL-37 concentrations. The COL/HA PEMs can serve as antimicrobial coatings, biological membranes and as in vitro platforms to investigate pathogen-tissue interactions. STATEMENT OF SIGNIFICANCE: Antimicrobial peptides (AMPs) are emerging as an alternative to conventional antibiotics. We report the antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a human AMP. The antimicrobial and cytotoxic properties were tested with gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. Unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, the sustained release of the active peptide killed planktonic bacteria. Immobilizing LL-37 resulted in less than 3% bacterial adhesion. LL-37 modified PEMs did not result in cytotoxicity up to input concentrations of 16µM. The COL/HA PEMs can serve as antimicrobial coatings and to investigate pathogen-cell interactions.

The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironment.

BACKGROUND: Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. METHODS: To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. RESULTS: All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based ß-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. CONCLUSIONS: Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.

Designing a fibrotic microenvironment to investigate changes in human liver sinusoidal endothelial cell function.

The deposition of extracellular matrix (ECM) proteins by hepatic cells during fibrosis leads to the stiffening of the organ and perturbed cellular functions. Changes in the elasticity of liver tissue are manifested by altered phenotype in hepatic cells. We have investigated changes in human liver sinusoidal endothelial cells (hLSECs) that occur as the elastic modulus of their matrix transitions from healthy (6kPa) to fibrotic (36kPa) conditions. We have also investigated the role played by Kupffer cells in the dedifferentiation of hLSECs. We report the complete loss of fenestrae and the expression of CD31 at the surface as a result of increasing elastic moduli. LSECs exhibited a greater number of actin stress fibers and vinculin focal adhesion on the stiffer substrate, as well. A novel finding is that these identical trends can be obtained on soft (6kPa) substrates by introducing an inflamed microenvironment through the addition of Kupffer cells. hLSEC monocultures on 6kPa gels exhibited fenestrae that were 140.7±52.6nm in diameter as well as a lack of surface CD31 expression. Co-culturing hLSECs with rat Kupffer cells (rKCs) on 6kPa substrates, resulted in the complete loss of fenestrae, an increase in CD31 expression and in a well-organized cytoskeleton. These results demonstrate that the increasing stiffness of liver matrices does not solely result in changes in hLSEC phenotype. Even on soft substrates, culturing hLSECs in an inflamed microenvironment can result in their dedifferentiation. Our findings demonstrate the interplay between matrix elasticity and inflammation in the progression of hepatic fibrosis.