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© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systems, National Institutes of Health (U.S.) (Grant UH3TR000496), National Institutes of Health (U.S.) (Grant UH3TR000503)

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (Grant 4-UH3-TR000496-03)

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk., National Institutes of Health (U.S.) (grant UH3TR00069), United States. Defense Advanced Research Projects Agency (grant Microphysiological Systems Program (W911NF-12-2-00))

Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development., United States. Defense Advanced Research Projects Agency (Grant W911NF-12-2-0039), National Institutes of Health (U.S.) (Grant 4-UH3-TR000496-03)

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats., National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01), National Institutes of Health (U.S.) (3UH2 TR000506-02S1), National Institutes of Health (U.S.) (T32 HL007936-12), National Institutes of Health (U.S.) (RO1 HL093282), National Institutes of Health (U.S.) (R21 EB016381-01)

Distant metastasis is the major cause of breast cancer-related mortality, commonly emerging clinically after 5 or more years of seeming 'cure' of the primary tumor, indicating a quiescent dormancy. The lack of relevant accessible model systems for metastasis that recreate this latent stage has hindered our understanding of the molecular basis and the development of therapies against these lethal outgrowths. We previously reported on the development of an all-human 3D ex vivo hepatic microphysiological system that reproduces several features of liver physiology and enables spontaneous dormancy in a subpopulation of breast cancer cells. However, we observed that the dormant cells were localized primarily within the 3D tissue, while the proliferative cells were in contact with the polystyrene scaffold. As matrix stiffness is known to drive inflammatory and malignant behaviors, we explored the occurrence of spontaneous tumor dormancy and inflammatory phenotype. The microphysiological system was retrofitted with PEGDa-SynKRGD hydrogel scaffolding, which is softer and differs in the interface with the tissue. The microphysiological system incorporated donor-matched primary human hepatocytes and non-parenchymal cells (NPCs), with MDA-MB-231 breast cancer cells. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes, and was more responsive to inflammatory stimuli. The proportion of tumor cells entering dormancy was markedly increased in the hydrogel-supported tissue compared to polystyrene. Interestingly, an unexpected differential response of dormant cells to varying chemotherapeutic doses was identified, which if reflective of patient pathophysiology, has important implications for patient dosing regimens. These findings highlight the metastatic microphysiological system fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic breast cancer.

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL- 6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment., United States. Defense Advanced Research Projects Agency (W911NF-12-2-0039), National Institutes of Health (U.S.) (UH3TR000496)

Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

Multipotent stromal cells (MSCs) can be differentiated into osteoblasts and chondrocytes, making these cells candidates to regenerate cranio-facial injuries and lesions in long bones. A major problem with cell replacement therapy, however, is the loss of transplanted MSCs at the site of graft. Reactive oxygen species (ROS) and nonspecific inflammation generated at the ischemic site have been hypothesized to lead to MSCs loss; studies in vitro show MSCs dying both in the presence of ROS or cytokines like FasL. We questioned whether MSCs themselves may be the source of these death inducers, specifically whether MSCs produce ROS under cytokine challenge. On treating MSCs with FasL, we observed increased ROS production within 2 h, leading to apoptotic death after 6 h of exposure to the cytokine. N-acetyl cysteine, an antioxidant, is able to protect MSCs from FasL-induced ROS production and subsequent ROS-dependent apoptosis, though the MSCs eventually succumb to ROS-independent death signaling. Epidermal growth factor (EGF), a cell survival factor, is able to protect cells from FasL-induced ROS production initially; however, the protective effect wanes with continued FasL exposure. In parallel, FasL induces upregulation of the uncoupling protein UCP2, the main uncoupling protein in MSCs, which is not abrogated by EGF; however, the production of ROS is followed by a delayed apoptotic cell death despite moderation by UCP2. FasL-induced ROS activates the stress-induced MAPK pathways JNK and p38MAPK as well as ERK, along with the activation of Bad, a proapoptotic protein, and suppression of survivin, an antiapoptotic protein; the latter two key modulators of the mitochondrial death pathway. FasL by itself also activates its canonical extrinsic death pathway noted by a time-dependent degradation of c-FLIP and activation of caspase 8. These data suggest that MSCs participate in their own demise due to nonspecific inflammation, holding implications for replacement therapies., National Institute of General Medical Sciences (U.S.) (GM069668), National Institute of Dental and Craniofacial Research (U.S.) (DE019523)

Biomimetic scaffolds have been proposed as a means to facilitate tissue regeneration by multi-potent stromal cells (MSCs). Effective scaffold colonization requires a control of multiple MSC responses including survival, proliferation, differentiation, and migration. As MSC migration is relatively unstudied in this context, we present here a multi-level approach to its understanding and control, integratively tuning cell speed and directional persistence to achieve maximal mean free path (MFP) of migration. This approach employs data-driven computational modeling to ascertain small molecule drug treatments that can enhance MFP on a given materials substratum. Using poly(methyl methacrylate)-graft-poly(ethylene oxide) polymer surfaces tethered with epidermal growth factor (tEGF) and systematically adsorbed with fibronectin, vitronectin, or collagen-I to present hTERT-immortalized human MSCs with growth factor and extracellular matrix cues, we measured cell motility properties along with signaling activities of EGFR, ERK, Akt, and FAK on 19 different substrate conditions. Speed was consistent on collagen/tEGF substrates, but low associated directional persistence limited MFP. Decision tree modeling successfully predicted that ERK inhibition should enhance MFP on collagen/tEGF substrates by increasing persistence. Thus, we demonstrated a two-tiered approach to control MSC migration: materials-based “coarse-graining” complemented by small molecule “fine-tuning”., National Institutes of Health (U.S.) (NIH grant R01-DE019523), National Institutes of Health (U.S.) (NIH Cell Migration Consortium U54-GM064346), National Institutes of Health (U.S.) (NIH grant R01-GM018336)

The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues., United States. Army, Hertz Foundation (Graduate Fellowship), National Institute for Biomedical Imaging and Bioengineering (U.S.) (R01EB003805), National Institute of Dental and Craniofacial Research (U.S.) (R01DE019523), Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institute of Environmental Health Sciences P30ES002109), Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institute of Environmental Health Sciences R01ES015241), Armed Forces Institute of Regenerative Medicine

Bone marrow-derived multipotent stromal cells (MSCs) offer great promise for regenerating tissue. Although certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remains poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multivariate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterial surfaces presenting tethered epidermal growth factor, type I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of eight intracellular phosphoproteins: phospho-epidermal growth factor receptor, phospho-Akt, phospho-extracellular signal-related kinase 1/2, phospho-heat shock protein 27, phospho-c-Jun, phospho-glycogen synthase kinase 3α/β, phospho-p38, and phospho-signal transducer and activator of transcription 3. This combination provides the strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements; day-4 and day-14 signal measurements are also significantly predictive, indicating a broad time frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate. STEM CELLS 2009;27:2804–2814, National Institutes of Health (U.S.) (Grant NIH R01-GM059870-07), National Institutes of Health (U.S.) (Grant R01 DE019523- 10), United Negro College Fund ((UNCF)/Merck Postdoctoral Fellowship), Georgia Institute of Technology (Facilitating Academic Careers in Engineering and Sciences Fellowship)

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling., National Institutes of Health (U.S.) (Grant R01-CA096504), National Institutes of Health (U.S.) (Grant U54-CA112967)

Introduction Bone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation. Methods MSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers. Results We found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin. Conclusions Our findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis., National Institute of General Medical Sciences (U.S.) (Grant GM069668), National Institute of Dental and Craniofacial Research (U.S.) (Grant DE019523), National Institute on Aging (Grant AG026389)

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability., National Institutes of Health (U.S.) (Grant 1UH2TR000496-01), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)

Reliable in vitro human disease models that capture the complexity of in vivo tissue behaviors are crucial to gain mechanistic insights into human disease and enable the development of treatments that are effective across broad patient populations. The integration of stem cell technologies, tissue engineering, emerging biomaterials strategies and microfabrication processes, as well as computational and systems biology approaches, is enabling new tools to generate reliable in vitro systems to study the molecular basis of human disease and facilitate drug development. In this review, we discuss these recently developed tools and emphasize opportunities and challenges involved in combining these technologies toward regenerative science., National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant 5R01EB010246-02), National Center for Advancing Translational Sciences (U.S.) (Grant 1UH2TR000496), United States. Defense Advanced Research Projects Agency (Cooperative Agreement W911NF-12-2-0039)

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models., National Institutes of Health (U.S.) (R01 EB010246), National Institutes of Health (U.S.) (P50-GM068762-08), National Institutes of Health (U.S.) (R01-ES015241), National Institutes of Health (U.S.) (P30-ES002109), 5UH2TR000496-02, National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)

Clinical management of endometriosis is limited by the complex relationship between symptom severity, heterogeneous surgical presentation, and variability in clinical outcomes. As a complement to visual classification schemes, molecular profiles of disease activity may improve risk stratification to better inform treatment decisions and identify new approaches to targeted treatment. We use a network analysis of information flow within and between inflammatory cells to discern consensus behaviors characterizing patient subpopulations. Unsupervised multivariate analysis of cytokine profiles quantified by multiplex immunoassays identified a subset of patients with a shared “consensus signature” of 13 elevated cytokines that was associated with common clinical features of endometriosis, but was not observed among patient subpopulations defined by morphologic presentation alone. Enrichment analysis of consensus markers reinforced the primacy of peritoneal macrophage infiltration and activation, which was demonstrably elevated in ex vivo cultures. Although familiar targets of the nuclear factor κB family emerged among overrepresented transcriptional binding sites for consensus markers, our analysis provides evidence for an unexpected contribution from c-Jun, c-Fos, and AP-1 effectors of mitogen-associated kinase signaling. Their crucial involvement in propagation of macrophage-driven inflammatory networks was confirmed via targeted inhibition of upstream kinases. Collectively, these analyses suggest a clinically relevant inflammatory network that may serve as an objective measure for guiding treatment decisions for endometriosis management, and in the future may provide a mechanistic endpoint for assessing efficacy of new agents aimed at curtailing inflammatory mechanisms that drive disease progression., National Institutes of Health (U.S.) (R01EB10246), Singapore-MIT Alliance, United States. Army Research Office (Institute for Collaborative Biotechnologies W911NF-09-001), John D. and Catherine T. MacArthur Foundation

Background—Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1β is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1β (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor–like domain of neuregulin-1β (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1β. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. Methods and Results—NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). Conclusions—NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines., United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Breast Cancer Research Program (W81XWH-11-1-0035)), National Institutes of Health (U.S.) (grant HL112905), National Institutes of Health (U.S.) (grant AG032977), National Institutes of Health (U.S.) (grant DE019523), National Institutes of Health (U.S.) (grant DK090147), National Institutes of Health (U.S.) (grant U54-CA112967), National Institutes of Health (U.S.) (grant EB003805), United States. Dept. of Defense (Congressionally Directed Medical Research Programs, Orthopedic Research Program Career Development Award (W81XWH-11-1-0821)), Sarnoff Cardiovascular Research Foundation (fellowship), Hertz Foundation (Fellowship), Harvard Stem Cell Institute

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1, National Institutes of Health (U.S.) (Grant HL052212), National Institutes of Health (U.S.) (Grant HL066105), National Institutes of Health (U.S.) (Grant ES015241), National Institutes of Health (U.S.) (Grant GM068762)

Multipotential stromal cells or mesenchymal stem cells (MSCs) have been proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells postimplantation. This cell death in part is due to ubiquitous nonspecific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Furthermore, tethering EGF (tEGF) onto a two-dimensional surface altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived preosteoblasts showed increased Fas levels and became more susceptible to FasL-induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation, and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function., National Institute of General Medical Sciences (U.S.) (GM069668), National Institute of Dental and Craniofacial Research (U.S.) (DE019523)

Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48–72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling., National Science Foundation (U.S.) (Grant EFRI-0735997), National Institutes of Health (U.S.) (Grant R01 GM069668)

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24–204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nm and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease., National Institutes of Health (U.S.) (Grant R01EB010246), National Institutes of Health (U.S.) (Grant R01GM081336), Heptagon Fund (London, England), Cancer Research UK, Whitehead Foundation, Duke University. School of Medicine (Bridge Funding Program), Germany. Bundesministerium für Bildung und Forschung, China (National Fellowship from the Chinese Scholarship Council), Florida State University

Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.

Refer to Web version on PubMed Central for supplementary material., Idiosyncratic drug hepatotoxicity is a major problem in pharmaceutical development due to poor prediction capability of standard preclinical toxicity assessments and limited knowledge of its underlying mechanisms. Findings in animal models have shown that adverse effects of numerous drugs with idiosyncratic hepatotoxicity in humans can be reproduced in the presence of coincident inflammatory cytokine signaling. Following these observations, we have recently developed an in vitro drug/inflammatory cytokine co-treatment approach that can reproduce clinical drug hepatotoxicity signatures—particularly for idiosyncratic drugs—in cultured primary human hepatocytes. These observations have suggested that drug-induced stresses may interact with cytokine signaling to induce hepatic cytotoxicity, but the hepatocyte signaling mechanisms governing these interactions are poorly understood. Here, we collect high-throughput phosphoprotein signaling and cytotoxicity measurements in cultured hepatocytes, from multiple human donors, treated with combinations of hepatotoxic drugs (e.g. trovafloxacin, clarithromycin) and cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1α, and interleukin-6). We demonstrate, through orthogonal partial least-squares regression (OPLSR) modeling of these signal-response data, that drug/cytokine hepatic cytotoxicity is integratively controlled by four key signaling pathways: Akt, p70 S6 kinase, MEK–ERK, and p38–HSP27. This modeling predicted, and experimental studies confirmed, that the MEK–ERK and p38–HSP27 pathways contribute pro-death signaling influences in drug/cytokine hepatic cytotoxicity synergy. Further, our four-pathway OPLSR model produced successful prediction of drug/cytokine hepatic cytotoxicities across different human donors, even though signaling and cytotoxicity responses were both highly donor-specific. Our findings highlight the critical role of kinase signaling in drug/cytokine hepatic cytotoxicity synergies and reveal that hepatic cytotoxicity responses are governed by multi-pathway signaling network balance., Pfizer Inc., Institute for Collaborative Biotechnologies, MIT Center for Cell Decision Processes, National Institute of Mental Health (U.S.) (grant P50-GM68762), Massachusetts Institute of Technology. Biotechnology Process Engineering Center, Massachusetts Institute of Technology. Center for Environmental Health Sciences, National Institute of Mental Health (U.S.) (grant U19ES011399), Whitaker Foundation

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers., National Institute of Environmental Health Sciences (grant number 5P30ES002109-30), National Institutes of Health (U.S.) (NIH grant number 5R01ES015241), DuPont MIT Alliance, Pfizer Inc., National Science Foundation (U.S.) (NSF grant number EEC-9843342)

Author Manuscript 2011 April 29., Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF., National Institutes of Health (U.S.) (Grant R01-GM059870-07), National Institutes of Health (U.S.) (Grant R01-DE 019523-10), National Institutes of Health (U.S.) (Grant P40RR017447), United Negro College Fund (Merck Postdoctoral Fellowship), Georgia Institute of Technology (FACES Fellowship), National Center for Research Resources (U.S.) (Grant P40RR017447)

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IκB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic., National Institute of General Medical Sciences (U.S.). Cell Decision Processes Center, National Cancer Institute (U.S.). Integrative Cancer Biology Program, Massachusetts Institute of Technology. Presidential Fellowship, National Institutes of Health (U.S.)

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation., National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870), National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523)