Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET].

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Protective role of Gremlin-1 in myocardial function.

BACKGROUND: Gremlin-1 is a cystine knot protein and is expressed in organs developing fibrosis. Transient ischaemia leads to myocardial fibrosis, a major determinant of impaired myocardial function. MATERIALS AND METHODS: Expression of Gremlin-1 was investigated in infarcted myocardium by real-time PCR, Western blot analysis, histological and immunohistochemistry staining. We further elaborated the colocalization of Gremlin-1 and TGF-ß proteins by confocal microscopy and co-immunoprecipitation experiments. The interaction between Gremlin-1 and TGF-ß was analysed by photon correlation spectroscopy. Gremlin-1 modulation of the TGF-ß-dependent collagen I synthesis in fibroblasts was investigated using ELISA and immunohistochemistry experiments. The effect of prolonged administration of recombinant Gremlin-1 on myocardial function following ischaemia/reperfusion was accessed by echocardiography and immunohistochemistry. RESULTS: Gremlin-1 is expressed in myocardial tissue and infiltrating cells after transient myocardial ischaemia (P < .05). Gremlin-1 colocalizes with the pro-fibrotic cytokine transforming growth factor-ß (TGF-ß) expressed in fibroblasts and inflammatory cell infiltrates (P < .05). Gremlin-1 reduces TGF-ß-induced collagen production of myocardial fibroblasts by approximately 20% (P < .05). We found that Gremlin-1 binds with high affinity to TGF-ß (KD  = 54 nmol/L) as evidenced by photon correlation spectroscopy and co-immunoprecipitation. intravenous administration of m Gremlin-1-Fc, but not of equivalent amount of Fc control, significantly reduced infarct size by approximately 20%. In the m Gremlin-1-Fc group, infarct area was reduced by up to 30% in comparison with mice treated with Fc control (I/LV: 4.8 ± 1.2% vs 6.0 ± 1.2% P < .05; I/AaR: 15.2 ± 1.5% vs 21.1 ± 5%, P < .05). CONCLUSIONS: The present data disclose Gremlin-1 as an antagonist of TGF-ß and presume a role for Gremlin-1/TGF-ß interaction in myocardial remodelling following myocardial ischaemia.

Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations.

Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular "sensor" and "effector" of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.

Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling.

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

Critical Analysis of Non-Thermal Plasma-Driven Modulation of Immune Cells from Clinical Perspective.

The emerged field of non-thermal plasma (NTP) shows great potential in the alteration of cell redox status, which can be utilized as a promising therapeutic implication. In recent years, the NTP field considerably progresses in the modulation of immune cell function leading to promising in vivo results. In fact, understanding the underlying cellular mechanisms triggered by NTP remains incomplete. In order to boost the field closer to real-life clinical applications, there is a need for a critical overview of the current state-of-the-art. In this review, we conduct a critical analysis of the NTP-triggered modulation of immune cells. Importantly, we analyze pitfalls in the field and identify persisting challenges. We show that the identification of misconceptions opens a door to the development of a research strategy to overcome these limitations. Finally, we propose the idea that solving problems highlighted in this review will accelerate the clinical translation of NTP-based treatments.

Non-Thermal Plasma, as a New Physicochemical Source, to Induce Redox Imbalance and Subsequent Cell Death in Liver Cancer Cell Lines.

BACKGROUND/AIMS: Alteration of cancer cell redox status has been recognized as a promising therapeutic implication. In recent years, the emerged field of non-thermal plasma (NTP) has shown considerable promise in various biomedical applications, including cancer therapy. However, understanding the molecular mechanisms procuring cellular responses remains incomplete. Thus, the aim of this study was a rigorous biochemical analysis of interactions between NTP and liver cancer cells. METHODS: The concept was validated using three different cell lines. We provide several distinct lines of evidence to support our findings; we use various methods (epifluorescent and confocal microscopy, clonogenic and cytotoxicity assays, Western blotting, pharmacological inhibition studies, etc.). RESULTS: We assessed the influence of NTP on three human liver cancer cell lines (Huh7, Alexander and HepG2). NTP treatment resulted in higher anti-proliferative effect against Alexander and Huh7 relative to HepG2. Our data clearly showed that the NTP-mediated alternation of mitochondrial membrane potential and dynamics led to ROS-mediated apoptosis in Huh7 and Alexander cells. Interestingly, plasma treatment resulted in p53 down-regulation in Huh7 cells. High levels of Bcl-2 protein expression in HepG2 resulted in their resistance in response to oxidative stress- mediated by plasma. CONCLUSION: We show thoroughly time- and dose-dependent kinetics of ROS accumulation in HCC cells. Furthermore, we show nuclear compartmentalization of the superoxide anion triggered by NTP. NTP induced apoptotic death in Huh7 liver cancer cells via simultaneous downregulation of mutated p53, pSTAT1 and STAT1. Contrary, hydrogen peroxide treatment results in autophagic cell death. We disclosed detailed mechanisms of NTP-mediated alteration of redox signalling in liver cancer cells.

Antiviral vaccines license T cell responses by suppressing granzyme B levels in human plasmacytoid dendritic cells.

Human plasmacytoid dendritic cells (pDC) are important modulators of adaptive T cell responses during viral infections. Recently, we found that human pDC produce the serine protease granzyme B (GrB), thereby regulating T cell proliferation in a GrB-dependent manner. In this study, we demonstrate that intrinsic GrB production by pDC is significantly inhibited in vitro and in vivo by clinically used vaccines against viral infections such as tick-borne encephalitis. We show that pDC GrB levels inversely correlate with the proliferative response of coincubated T cells and that GrB suppression by a specific Ab or a GrB substrate inhibitor results in enhanced T cell proliferation, suggesting a predominant role of GrB in pDC-dependent T cell licensing. Functionally, we demonstrate that GrB(high) but not GrB(low) pDC transfer GrB to T cells and may degrade the ζ-chain of the TCR in a GrB-dependent fashion, thereby providing a possible explanation for the observed T cell suppression by GrB-expressing pDC. Modulation of pDC-derived GrB activity represents a previously unknown mechanism by which both antiviral and vaccine-induced T cell responses may be regulated in vivo. Our results provide novel insights into pDC biology during vaccinations and may contribute to an improvement of prophylactic and therapeutic vaccines.

WITHDRAWN: Static High-Gradient Magnetic Fields Activate Transient Receptor Potential Vanilloid 4 (TRPV4) Ion Channels Enabling Remote Control of Cell Function.

This manuscript has been withdrawn by the Author's request.

Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.

Impact of mechanical cues on key cell functions and cell-nanoparticle interactions.

In recent years, it has been recognized that mechanical forces play an important regulative role in living organisms and possess a direct impact on crucial cell functions, ranging from cell growth to maintenance of tissue homeostasis. Advancements in mechanobiology have revealed the profound impact of mechanical signals on diverse cellular responses that are cell type specific. Notably, numerous studies have elucidated the pivotal role of different mechanical cues as regulatory factors influencing various cellular processes, including cell spreading, locomotion, differentiation, and proliferation. Given these insights, it is unsurprising that the responses of cells regulated by physical forces are intricately linked to the modulation of nanoparticle uptake kinetics and processing. This complex interplay underscores the significance of understanding the mechanical microenvironment in shaping cellular behaviors and, consequently, influencing how cells interact with and process nanoparticles. Nevertheless, our knowledge on how localized physical forces affect the internalization and processing of nanoparticles by cells remains rather limited. A significant gap exists in the literature concerning a systematic analysis of how mechanical cues might bias the interactions between nanoparticles and cells. Hence, our aim in this review is to provide a comprehensive and critical analysis of the existing knowledge regarding the influence of mechanical cues on the complicated dynamics of cell-nanoparticle interactions. By addressing this gap, we would like to contribute to a detailed understanding of the role that mechanical forces play in shaping the complex interplay between cells and nanoparticles.

Mechanical regulation of mitochondrial morphodynamics in cancer cells by extracellular microenvironment.

Recently, it has been recognized that physical abnormalities (e.g. elevated solid stress, elevated interstitial fluid pressure, increased stiffness) are associated with tumor progression and development. Additionally, these mechanical forces originating from tumor cell environment through mechanotransduction pathways can affect metabolism. On the other hand, mitochondria are well-known as bioenergetic, biosynthetic, and signaling organelles crucial for sensing stress and facilitating cellular adaptation to the environment and physical stimuli. Disruptions in mitochondrial dynamics and function have been found to play a role in the initiation and advancement of cancer. Consequently, it is logical to hypothesize that mitochondria dynamics subjected to physical cues may play a pivotal role in mediating tumorigenesis. Recently mitochondrial biogenesis and turnover, fission and fusion dynamics was linked to mechanotransduction in cancer. However, how cancer cell mechanics and mitochondria functions are connected, still remain poorly understood. Here, we discuss recent studies that link mechanical stimuli exerted by the tumor cell environment and mitochondria dynamics and functions. This interplay between mechanics and mitochondria functions may shed light on how mitochondria regulate tumorigenesis.

A novel semisynthetic inhibitor of the FRB domain of mammalian target of rapamycin blocks proliferation and triggers apoptosis in chemoresistant prostate cancer cells.

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and its uncontrolled activation is a hallmark of cancer. Moreover, mTOR activation has been implicated in the resistance of cancer cells to many anticancer drugs, rendering this pathway a promising pharmacotherapeutic target. Here we explored the capability of a semisynthetic compound to intercept mTOR signaling. We synthesized and chemically characterized a novel, semisynthetic triterpenoid derivative, 3-cinnamoyl-11-keto-ß-boswellic acid (C-KßBA). Its pharmacodynamic effects on mTOR and several other signaling pathways were assessed in a number of prostate and breast cancer cell lines as well as in normal prostate epithelial cells. C-KßBA exhibits specific antiproliferative and proapoptotic effects in cancer cell lines in vitro as well as in PC-3 prostate cancer xenografts in vivo. Mechanistically, the compound significantly inhibits the cap-dependent transition machinery, decreases expression of eukaryotic translation initiation factor 4E and cyclin D1, and induces G(1) cell-cycle arrest. In contrast to conventional mTOR inhibitors, C-KßBA downregulates the phosphorylation of p70 ribosomal S6 kinase, the major downstream target of mTOR complex 1, without concomitant activation of mTOR complex 2/Akt and extracellular signal-regulated kinase pathways, and independently of protein phosphatase 2A, liver kinase B1/AMP-activated protein kinase/tuberous sclerosis complex, and F12-protein binding. At the molecular level, the compound binds to the FKBP12-rapamycin-binding domain of mTOR with high affinity, thereby competing with the endogenous mTOR activator phosphatidic acid. C-KßBA represents a new type of proapoptotic mTOR inhibitor that, due to its special mechanistic profile, might overcome the therapeutic drawbacks of conventional mTOR inhibitors.

Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis.

Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases.

The bispecific SDF1-GPVI fusion protein preserves myocardial function after transient ischemia in mice.

BACKGROUND: CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell-based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair. METHODS AND RESULTS: We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function. CONCLUSIONS: These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.

Thioredoxin-1 promotes anti-inflammatory macrophages of the M2 phenotype and antagonizes atherosclerosis.

OBJECTIVE: Oxidative stress is believed to play a key role in cardiovascular disorders. Thioredoxin (Trx) is an oxidative stress-limiting protein with anti-inflammatory and antiapoptotic properties. Here, we analyzed whether Trx-1 might exert atheroprotective effects by promoting macrophage differentiation into the M2 anti-inflammatory phenotype. METHODS AND RESULTS: Trx-1 at 1 µg/mL induced downregulation of p16(INK4a) and significantly promoted the polarization of anti-inflammatory M2 macrophages in macrophages exposed to interleukin (IL)-4 at 15 ng/mL or IL-4/IL-13 (10 ng/mL each) in vitro, as evidenced by the expression of the CD206 and IL-10 markers. In addition, Trx-1 induced downregulation of nuclear translocation of activator protein-1 and Ref-1, and significantly reduced the lipopolysaccharide-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1. Consistently, Trx-1 administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/30 g body weight challenged either with lipopolysaccharide at 30 µg/30 g body weight or with IL-4 at 500 ng/30 g body weight significantly induced the M2 phenotype while inhibiting differentiation of macrophages into the M1 phenotype in liver and thymus. ApoE2.Ki mice challenged once weekly with lipopolysaccharide for 5 weeks developed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. In contrast, however, daily injections of Trx-1 shifted the phenotype pattern of lesional macrophages in these animals to predominantly M2 over M1, and the aortic lesion area was significantly reduced (from 100%±18% to 62.8%±9.8%; n=8; P<0.01). Consistently, Trx-1 colocalized with M2 but not with M1 macrophage markers in human atherosclerotic vessel specimens. CONCLUSIONS: The ability of Trx-1 to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain its protective effects in cardiovascular diseases. These data provide novel insight into the link between oxidative stress and cardiovascular diseases.

Iron oxide nanoparticles trigger endoplasmic reticulum damage in steatotic hepatic cells.

Iron oxide nanoparticles (IONPs) are being actively researched in various biomedical applications, particularly as magnetic resonance imaging (MRI) contrast agents for diagnosing various liver pathologies like nonalcoholic fatty liver diseases, nonalcoholic steatohepatitis, and cirrhosis. Emerging evidence suggests that IONPs may exacerbate hepatic steatosis and liver injury in susceptible livers such as those with nonalcoholic fatty liver disease. However, our understanding of how IONPs may affect steatotic cells at the sub-cellular level is still fragmented. Generally, there is a lack of studies identifying the molecular mechanisms of potential toxic and/or adverse effects of IONPs on "non-heathy" in vitro models. In this study, we demonstrate that IONPs, at a dose that does not cause general toxicity in hepatic cells (Alexander and HepG2), induce significant toxicity in steatotic cells (cells loaded with non-toxic doses of palmitic acid). Mechanistically, co-treatment with PA and IONPs resulted in endoplasmic reticulum (ER) stress, accompanied by the release of cathepsin B from lysosomes to the cytosol. The release of cathepsin B, along with ER stress, led to the activation of apoptotic cell death. Our results suggest that it is necessary to consider the interaction between IONPs and the liver, especially in susceptible livers. This study provides important basic knowledge for the future optimization of IONPs as MRI contrast agents for various biomedical applications.

Lysosomal nanotoxicity: Impact of nanomedicines on lysosomal function.

Although several nanomedicines got clinical approval over the past two decades, the clinical translation rate is relatively small so far. There are many post-surveillance withdrawals of nanomedicines caused by various safety issues. For successful clinical advancement of nanotechnology, it is of unmet need to realize cellular and molecular foundation of nanotoxicity. Current data suggest that lysosomal dysfunction caused by nanoparticles is emerging as the most common intracellular trigger of nanotoxicity. This review analyzes prospect mechanisms of lysosomal dysfunction-mediated toxicity induced by nanoparticles. We summarized and critically assessed adverse drug reactions of current clinically approved nanomedicines. Importantly, we show that physicochemical properties have great impact on nanoparticles interaction with cells, excretion route and kinetics, and subsequently on toxicity. We analyzed literature on adverse reactions of current nanomedicines and hypothesized that adverse reactions might be linked with lysosomal dysfunction caused by nanomedicines. Finally, from our analysis it becomes clear that it is unjustifiable to generalize safety and toxicity of nanoparticles, since different particles possess distinct toxicological properties. We propose that the biological mechanism of the disease progression and treatment should be central in the optimization of nanoparticle design.

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment.

It has become evident that physical stimuli of the cellular microenvironment transmit mechanical cues regulating key cellular functions, such as proliferation, migration, and malignant transformation. Accumulating evidence suggests that tumor cells face variable mechanical stimuli that may induce metabolic rewiring of tumor cells. However, the knowledge of how tumor cells adapt metabolism to external mechanical cues is still limited. We therefore designed soft 3D collagen scaffolds mimicking a pathological mechanical environment to decipher how liver tumor cells would adapt their metabolic activity to physical stimuli of the cellular microenvironment. Here, we report that the soft 3D microenvironment upregulates the glycolysis of HepG2 and Alexander cells. Both cell lines adapt their mitochondrial activity and function under growth in the soft 3D microenvironment. Cells grown in the soft 3D microenvironment exhibit marked mitochondrial depolarization, downregulation of mitochondrially encoded cytochrome c oxidase I, and slow proliferation rate in comparison with stiff monolayer cultures. Our data reveal the coupling of liver tumor glycolysis to mechanical cues. It is proposed here that soft 3D collagen scaffolds can serve as a useful model for future studies of mechanically regulated cellular functions of various liver (potentially other tissues as well) tumor cells.

Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields.

Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.