Co-cultures Of Hepatocytes Research Articles

Erratum to: Highlight report: co-cultures of hepatocytes and macrophages for hepatotoxicity testing.

Erratum to: Highlight report: co-cultures of hepatocytes and macrophages for hepatotoxicity testing.

Long-term hepatitis B infection in a scalable hepatic co-culture system

Hepatitis B virus causes chronic infections in 250 million people worldwide. Chronic hepatitis B virus carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. A prophylactic vaccine exists and currently available antivirals can suppress but rarely cure chronic infections. The study of hepatitis B virus and development of curative antivirals are hampered by a scarcity of models that mimic infection in a physiologically relevant, cellular context. Here, we show that cell-culture and patient-derived hepatitis B virus can establish persistent infection for over 30 days in a self-assembling, primary hepatocyte co-culture system. Importantly, infection can be established without antiviral immune suppression, and susceptibility is not donor dependent. The platform is scalable to microwell formats, and we provide proof-of-concept for its use in testing entry inhibitors and antiviral compounds.

3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells

In this study, a designed approach has been utilized for the development of a 3D liver system. This approach makes use of primary human sinusoidal endothelial cells, stellate cells and hepatocytes that are seeded sequentially on hollow fiber membranes (HF) in order to mimic the layers of cells found in vivo. To this purpose modified polyethersulfone (PES) HF membranes were used for the creation of a 3D human liver system in static and dynamic conditions. In order to verify the positive effect of non-parenchymal cells on the maintenance of hepatocyte viability and functions, homotypic cultures of hepatocytes alone on the HF membranes were further investigated. The membrane surface allowed the attachment and self-assembly of the cells, forming tissue-like structures around and between fibers. Sinusoidal cells formed tube-like structures that surrounded hepatocytes organized in cords within aggregates promoted by stellate cells. The co-culture of hepatocytes with sinusoidal endothelial and hepatic stellate cells preserved structural architecture of the construct and improved the liver-specific functions. Most importantly, cells co-cultured in a HF membrane bioreactor synthesized albumin and urea for 28 days. The liver membrane bioreactor also preserved the drug biotransformation activity with a continuous production of diazepam phase I metabolites for an extended period of time. Additionally, the cell oxygen uptake rates highlighted the maintenance of the actual oxygen concentration at a level compatible with their metabolic functions.

Metabolic Oscillations in Co-Cultures of Hepatocytes and Mesenchymal Stem Cells: Effects of Seeding Arrangement and Culture Mixing.

In vitro assembly of functional liver tissue is a prerequisite for the transplantation of tissue-engineered livers. There is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability. Recent reports have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) can provide a number of cues promoting hepatocyte growth and development. In this study, the effects of BM-MSCs co-culture on hepatocyte metabolism were evaluated as a function of scaffold seeding arrangement. BM-MSCs were co-cultured with hepatocytes in porous chitosan-heparin scaffolds using several seeding arrangements. The seeded scaffolds were subjected to orbital shaking to enhance mass transfer. BM-MSC-hepatocyte co-cultures exhibited higher rates of hepatocyte-specific functions, compared to hepatocyte-only cultures, regardless of the seeding arrangement. Cells formed smaller-compact spheroids in the heterotypic systems compared to mono-cultures of hepatocytes only. The spheroids exhibited reduction in size with time in all conditions except for the condition where BM-MSCs were seeded one day after seeding hepatocytes. In this condition, spheroids increased in size due to BM-MSC proliferation. Spheroid size reduction was hypothesized to be the result of cyclic shear stresses generated by the orbital shaking. Furthermore, results suggested that BM-MSC seeding onto preformed hepatocyte spheroids provide a degree of shear-protection and trophic stimuli. Overall, the results indicate that co-culturing hepatocytes with BM-MSCs enhanced their metabolic functions for the first week of culture. J. Cell. Biochem. 118: 3003-3015, 2017. © 2017 Wiley Periodicals, Inc.

Long-term liver-specific functions of hepatocytes in electrospun chitosan nanofiber scaffolds coated with fibronectin.

In this study, a new 3D liver model was developed using biomimetic nanofiber scaffolds and co-culture system consisting of hepatocytes and fibroblasts for the maintenance of long-term liver functions. The chitosan nanofiber scaffolds were fabricated by the electrospinning technique. To enhance cellular adhesion and spreading, the surfaces of the chitosan scaffolds were coated with fibronectin (FN) by adsorption and evaluated for various cell types. Cellular phenotype, protein expression, and liver-specific functions were extensively characterized by immunofluorescent and histochemical stainings, albumin enzyme-linked immunosorbent assay and Cytochrome p450 detoxification assays, and scanning electron microscopy. The electrospun chitosan scaffolds exhibited a highly porous and randomly oriented nanofibrous structure. The FN coating on the surface of the chitosan nanofibers significantly enhanced cell attachment and spreading, as expected, as surface modification with this cell adhesion molecule on the chitosan surface is important for focal adhesion formation and integrin binding. Comparison of hepatocyte mono-cultures and co-cultures in 3D culture systems indicated that the hepatocytes in co-cultures formed colonies and maintained their morphologies and functions for prolonged periods of time. The 3D liver tissue model developed in this study will provide useful tools toward the development of engineered liver tissues for drug screening and tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2119-2128, 2017.

Alteration of pancreatic carcinoma and promyeloblastic cell adhesion in liver microvasculature by co-culture of hepatocytes, hepatic stellate cells and endothelial cells in a physiologically-relevant model.

In vitro models of the liver microvasculature, especially with respect to cancer cell extravasation, should include not only endothelial and cancer cells but also surrounding cells to mimic the physiological situation. To this end, in the present study, we established a physiologically-relevant hierarchical co-culture model by stacking layers of primary rat hepatocytes (Hep), hepatic stellate cells embedded in collagen gel (LX-2) and endothelial cells (HUVECs) on a specially designed oxygen-permeable polydimethylsiloxane PDMS bottom plate. The model was used to investigate the role and contribution of each of the three cell types in pancreatic cancer and promyeloblast cell adhesion. In particular, we showed an increase in albumin production by the primary hepatocytes and in the consumption of the produced vascular endothelial growth factors (VEGFs). Furthermore, in co-culture, the HUVECs exhibited a mature vascular endothelial and non-inflamed phenotype, as evidenced by Stabilin-1, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), intercellular adhesion molecule (ICAM-1), and vascular adhesion protein-1 (VAP-1) expression. The HUVECs were also successfully activated with an inflammatory cytokine and their ICAM-1 response was found to be higher in monoculture compared to co-culture. Additionally, the adhesion of MiaPaCa-2 pancreatic cancer cells and HL60 promyeloblasts was tested in both cases (i.e.: activation or not by an inflammatory cytokine). It has been found that their adhesion was always reduced in the co-culture model. These results highlight the importance of integrating hepatic stellate cells in the design of biomimetic models of the hepatic endothelial barrier.

Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Coculture to Directly Determine the Degradation Rate Constant for CYP3A4.

The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug interactions (DDIs) that result from mechanism-based inactivation or induction of cytochrome P450 (P450). Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently extensive uncertainty exists in steady-state predictions for DDIs. In the current investigations, the stable isotope labeled amino acids in culture technique was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4 and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. The kdeg estimate was 0.026 ± 0.005 hour-1 This value was reproduced by cultures derived across four individual donors. For these cultures, the data indicated that CYP3A4 mRNA and total protein expression (i.e., labeled and unlabeled P450s), and activity were stable over the period where degradation had been determined. This kdeg value for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches but also employed micropatterned primary human hepatocytes as the in vitro model.

Rapid isolation of murine primary hepatocytes for chromosomal analysis

Primary hepatocyte culture is a crucial tool for investigations of liver function and for evaluating the toxic effects of drugs. In addition, chromosomal analysis of hepatocytes could also prove useful for understanding the mechanisms of hepatocarcinogenesis. However, cultivation of primary hepatocytes for chromosome analysis has been hampered by the specific equipment and skill required to perform the in situ perfusion step necessary for isolation of primary hepatocytes. In the present study, we aimed to establish a simple and efficient method of isolating hepatocytes suitable for chromosome analysis. We performed hepatocyte isolation without using collagenase perfusion, instead digesting liver tissues using collagenase in tubes. In addition, we examined hepatocyte and bone marrow cell (BMC) co-culture and cultivation of hepatocytes with medium containing BMC culture medium supernatants. We found that hepatocyte viability and attachment rate were significantly improved, both by co-culture with BMCs and medium containing BMC culture media supernatants, with the latter also significantly increasing the mitotic index. Using this simple method of isolation and cultivation, we could successfully perform chromosomal analysis of mouse primary hepatocytes. This method has the potential to help understand the mechanisms underlying chromosomal instability-mediated hepatocarcinogenesis.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

ABSTRACTEarly prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.

In Vitro Approaches to Model and Study Communication Between Adipose Tissue and the Liver.

Inter-organ communication is central to mammalian metabolism, allowing for the coordination of crucial homeostatic processes such as food intake, body weight, and blood glucose. Dissecting this process at a cellular and molecular level requires the establishment of a reductionist system in which cell types of interest can be investigated as the source and target of biologically relevant signals. Here, we describe a system to study the interaction between adipocytes and hepatocytes using either conditioned media from cultured adipocytes or co-culture of hepatocytes with adipose explants. These methods have the potential to identify novel polypeptides and metabolites involved in the adipose-liver metabolic axis.

Evaluation of the Interplay between Uptake Transport and CYP3A4 Induction in Micropatterned Cocultured Hepatocytes.

Previously we assessed the inductive response of prototypical inducers in hepatocyte monocultures and the long-term coculture model HepatoPac using cryopreserved hepatocytes from the same donors. We noted that the rifampicin EC50 generated using the HepatoPac model corresponded better to the EC50 based on clinical data compared with data generated in the monoculture system. We postulated that there may be differences in the functioning of uptake transporters between the two systems that may have led to the EC50 difference. In this study, we characterized the functional activity of multiple uptake transporters in the two systems using cryopreserved hepatocytes from the same donors. Our data suggest that uptake transporter activity is higher in HepatoPac compared with the monoculture system. As a follow up to this study, we measured the intracellular concentrations of rifampicin and bosentan, which are known substrates of uptake transporters; we observed significantly higher intracellular concentrations of both compounds in HepatoPac relative to the monoculture system. This finding equated to lower cytochrome P450 isoform 3A4 (CYP3A4) EC50 values in the HepatoPac system compared with the monoculture system for both mRNA and activity. In parallel, no significant EC50 shift was observed for carbamazepine and phenytoin, which are not known to be substrates of uptake transporters. Our data suggest that next generation liver models such as HepatoPac may be a useful in vitro tool to quantitatively predict drug-drug interactions when it is known that the perpetrator is also a substrate of drug transporters.

Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds.

Design of slowly metabolized compounds is an important goal in many drug discovery projects. Standard hepatocyte suspension intrinsic clearance (CLint) methods can only provide reliable CLint values above 2.5 μL/min/million cells. A method that permits extended incubation time with maintained performance and metabolic activity of the in vitro system is warranted to allow in vivo clearance predictions and metabolite identification of slowly metabolized drugs. The aim of this study was to evaluate the static HμREL coculture of human hepatocytes with stromal cells to be set up in-house as a standard method for in vivo clearance prediction and metabolite identification of slowly metabolized drugs. Fourteen low CLint compounds were incubated for 3 days, and seven intermediate to high CLint compounds and a cocktail of cytochrome P450 (P450) marker substrates were incubated for 3 h. In vivo clearance was predicted for 20 compounds applying the regression line approach, and HμREL coculture predicted the human intrinsic clearance for 45% of the drugs within 2-fold and 70% of the drugs within 3-fold of the clinical values. CLint values as low as 0.3 μL/min/million hepatocytes were robustly produced, giving 8-fold improved sensitivity of robust low CLint determination, over the cutoff in hepatocyte suspension CLint methods. The CLint values of intermediate to high CLint compounds were at similar levels both in HμREL coculture and in freshly thawed hepatocytes. In the HμREL coculture formation rates for five P450-isoform marker reactions, paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), and 1-OH-midazolam (3A4) were within the range of literature values for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation rate was lower. Further, both phase I and phase II metabolites were detected and an increased number of metabolites were observed in the HμREL coculture compared to hepatocyte suspension. In conclusion, HμREL coculture can be applied to accurately estimate intrinsic clearance of slowly metabolized drugs and is now utilized as a standard method for in vivo clearance prediction of such compounds in-house.

Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism

The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5days and maintained throughout 15days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism.

The Effect of Nitric Oxide on Ammonia Decomposition in Co-cultures of Hepatocytes and Hepatic Stellate Cells

Hepatic functions, such as albumin secretion and ammonia metabolism, are upregulated in response to hepatocyte growth factor (HGF) produced by hepatic stellate cells (HSC), as well as nitric oxide (NO) produced by endothelial cells under shear stress. However, the simultaneous effect of HSC and NO has not been previously investigated in a tri-co-culture model containing hepatocytes with HSC and endothelial cells under shear stress. We hypothesized that NO inhibits HGF production from HSC. To test this idea, we constructed a mono-culture model of hepatocytes and a co-culture model of hepatocytes and HSC and measured ammonia decomposition and HGF production in each model under NO load. Ammonia decomposition was significantly higher in the co-culture model under 0ppm NO load, but no significant increase was observed under NO load. In the co-culture model, HGF was produced at 1.0ng/mL under 0ppm NO load and 0.3ng/mL under NO load. Ammonia decomposition was increased by 1.0ng/mL HGF, but not by 0.3ng/mL HGF. These results indicated that NO inhibits HGF production from HSC; consequently, the effects of NO and co-culture with HSC cannot improve hepatic function simultaneously. Instead, the simultaneous effect of 1.0ng/mL HGF and NO may further enhance hepatic function in vitro.

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Induction of heme oxygenase-1 with dietary quercetin reduces obesity-induced hepatic inflammation through macrophage phenotype switching.

BACKGROUND/OBJECTIVESObesity-induced steatohepatitis accompanied by activated hepatic macrophages/Kupffer cells facilitates the progression of hepatic fibrinogenesis and exacerbates metabolic derangements such as insulin resistance. Heme oxyganase-1 (HO-1) modulates tissue macrophage phenotypes and thus is implicated in protection against inflammatory diseases. Here, we show that the flavonoid quercetin reduces obesity-induced hepatic inflammation by inducing HO-1, which promotes hepatic macrophage polarization in favor of the M2 phenotype.MATERIALS/METHODSMale C57BL/6 mice were fed a regular diet (RD), high-fat diet (HFD), or HFD supplemented with quercetin (HF+Que, 0.5g/kg diet) for nine weeks. Inflammatory cytokines and macrophage markers were measured by ELISA and RT-PCR, respectively. HO-1 protein was measured by Western blotting.RESULTSQuercetin supplementation decreased levels of inflammatory cytokines (TNFα, IL-6) and increased that of the anti-inflammatory cytokine (IL-10) in the livers of HFD-fed mice. This was accompanied by upregulation of M2 macrophage marker genes (Arg-1, Mrc1) and downregulation of M1 macrophage marker genes (TNFα, NOS2). In co-cultures of lipid-laden hepatocytes and macrophages, treatment with quercetin induced HO-1 in the macrophages, markedly suppressed expression of M1 macrophage marker genes, and reduced release of MCP-1. Moreover, these effects of quercetin were blunted by an HO-1 inhibitor and deficiency of nuclear factor E2-related factor 2 (Nrf2) in macrophages.CONCLUSIONSQuercetin reduces obesity-induced hepatic inflammation by promoting macrophage phenotype switching. The beneficial effect of quercetin is associated with Nrf2-mediated HO-1 induction. Quercetin may be a useful dietary factor for protecting against obesity-induced steatohepatitis.

Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes.

Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t1/2) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t1/2 for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t1/2 of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios.

Platelet GPIba Is Important for Thrombopoietin Production and Thrombopoietin-Induced Platelet Generation

Background: Platelets are generated from megakaryocytes in the bone marrow and play a key role in hemostasis. GPIba is the major subunit in the GPIb-IX complex expressed on platelets and megakaryocytes (MKs). Deficiency of GPIba results in Bernard Soulier Syndrome (BSS), a severe bleeding phenotype characterized by thrombocytopenia and abnormal platelet morphology.The generation of megakaryocytes and platelets is regulated by the hematopoietic growth factor, thrombopoietin (TPO). In patients with thrombocytopenia, the TPO receptor agonists and recombinant TPO (rTPO) have been used to stimulate thrombopoiesis. TPO production primarily occurs in liver parenchymal cells with minor amounts synthesized in other tissues. The major clearance mechanism of TPO from circulation is via interaction between TPO and its receptor, c-Mpl, following which the c-Mpl-TPO complex undergoes internalization and degradation. However, whether it is the production or clearance of TPO the main regulator of circulating TPO levels has been debated but remains unclear.The prevailing theory was that hepatic TPO is generated constitutively, and the levels of circulating TPO are fine-tuned by its uptake and metabolism in platelets and megakaryocytes. Recently, this concept was modified by studies in Ashwell-Morell receptor (AMR) deficient mice, which highlighted the important role played by platelets in the regulation of hepatic TPO mRNA expression. As the most glycosylated platelet surface protein, GPIba contributes to chilled platelets clearance viahepatocytes, as well as antibody-mediated thrombocytopenia. However, the relationship between GPIba and plasma TPO has not been exploredMethods and Results: We observed a significant decrease (approx. 3 fold) of TPO levels in both plasma and sera of GPIba-/- mice compared to wild type (WT) controls. Given the enlarged size of GPIba-/- platelets compared to WT, we investigated whether the low circulating TPO levels could be due to increased TPO clearance. Interestingly, we found similar levels of intracellular TPO in WT and GPIba-/- platelets, and the capacity to adsorb TPO in vitro was not enhanced. Further, we observed GPIba-/- platelets, surprisingly, have less c-Mpl expression compared to WT. Therefore, the decreased circulating TPO levels in GPIba-/- mice is unlikely due to enhanced TPO clearance.We next quantified TPO mRNA from hepatic liver cells as they are known as the major source of TPO. Consistent with the low circulatory TPO levels, we found reduced hepatic TPO mRNA in GPIba-/- mice, suggesting a deficiency in hepatic TPO generation. To determine whether exogenous platelet transfusions could influence steady-state hepatic TPO production, we transfused WT or GPIba-/- mice with WT or GPIba-/- platelets. Post-transfusion of WT, but not GPIba-/- platelets elevated TPO levels to around 150% in GPIba-/- mice. Additionally, our preliminary data showed that co-culture of hepatocytes with WT, but not GPIba-/- platelets in vitro increased hepatic TPO mRNA expression, further supporting a role for GPIba in TPO generation.To test whether TPO can equally induced platelet generation in GPIba-/- and WT mice. We injected rTPO into the two groups of mice, and found that in contrast to WT controls, rTPO did not significantly increase the platelet number of GPIba-/- mice in the circulation.Conclusion: Our data demonstrated that GPIba is important for TPO generation in liver and is necessary for TPO-induced platelet generation. These discoveries may have significant implications in TPO therapy in ITP. DisclosuresNo relevant conflicts of interest to declare.

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.