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To understand the mechanisms regulating the in vitro maturation of hPSC-derived hepatocytes, we developed a 3D differentiation system and compared gene regulatory elements in human primary hepatocytes with those in hPSC-hepatocytes that were differentiated in 2D or 3D conditions by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Regulome comparisons showed a reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and active enhancers without a reduced transcription of THRB. The addition of thyroid hormone T3 increased the binding of THRB to the CYP3A4 proximal enhancer, restored the super-enhancer status and gene expression of NFIC, and reduced the expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status, and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the hPSC-hepatocytes resulted in the engraftment of human hepatocytes into the mouse liver without disrupting normal liver histology. This work implicates the environmental factor-nuclear receptor axis in regulating the maturation of hPSC-hepatocytes.

Organoid cultures are proving to be powerful in vitro models that closely mimic the cellular constituents of their native tissue. The organoids are typically expanded and cultured in a 3D environment using either naturally derived or synthetic extracellular matrices. Assessing the morphology and growth characteristics of these cultures has been difficult due to the many imaging artifacts that accompany the corresponding images. Unlike single cell cultures, there are no reliable segmentation techniques that allow for the localization and quantification of organoids in their 3D culture environment. Here we describe OrgaQuant, a deep convolutional neural network implementation that can locate and quantify the size distribution of intestinal organoids in brightfield images. OrgaQuant is an end-to-end trained neural network that requires no parameter tweaking, thus it can be fully automated to analyze thousands of images with no user intervention. To develop OrgaQuant we created a unique dataset of manually annotated intestinal organoid images and trained an object detection pipeline using TensorFlow. We have made the dataset, trained model and inference scripts publically available along with detailed usage instructions.

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties but are challenging to modify with large peptides or proteins. Here a facile mild enzymatic tethering approach is presented. Sortase mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG EGF) to pre formed PEG hydrogels or PEG macromers containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the post ligation of pre formed hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG EGF in the supernatant. Sortase tethered EGF was biologically active as demonstrated by stimulation of DNA synthesis in a 2D culture of primary human hepatocytes and endometrial epithelial cells. In addition results with the PEG macromers demonstrate that EGF integrates into 3D PEG hydrogels and that the EGF is released from crosslinked PEG EGF macromers upon sortase reaction on the gels. Furthermore the tethering of several EGF molecules to single PEG stars macromers allows a local clustering effect for growth factor presentation to cells. The simplicity specificity and reversibility of sortase mediated ligation and cleavage reactions make it an attrac tive approach for modification of hydrogel systems.

A major challenge in the in vitro organogenesis field is to create microscale tissues with intercalated vascular networks similar challenges exist in virtually every tissue type as blood and lymph networks must productively coexist while remaining physically separated by the intervening tissue cells. We address the important challenge of how to create a scaffold that provides both physical and chemical cues to organize liver derived cells to create such integrated vascular networks within a hepatic soft tissue. We use existing 3D microperfused liver models but dramatically expand the ability to create fine tissue architecture by exploiting a very recent scaffold microfabrication approach (projection microstereolithography) to fabricate complex three dimensional microstructures with very high resolution. Cells including hepatocytes in the liver undergo different interactions e.g. with integrins and the local extra cellular matric of the neighboring cells to generate a basal phenotype. Using bioconjugation techniques we functionalize our 3D printed scaffolds with integrin binding peptides and growth factors to render them biologically active. Our approach allows to create the kinds of biomaterials technologies i.e. integration of high resolution scaffold fabrication with practical functionalized materials that will enable entirely new kinds of in vitro tissues to be constructed.

© 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)

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed., National Institutes of Health (U.S.) (R01 CA96504)

Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases., National Institutes of Health (U.S.) (R01EB010246), National Institutes of Health (U.S.) (UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), John and Karinne Begg Fund, Begg New Horizon Fund for Undergraduate Research at MIT, Massachusetts Institute of Technology. Biophysical Instrumentation Facility, Manton Foundation, Ludwig Postdoctoral Fellowship for Cancer Research, Swiss National Science Foundation (Postdoctoral Fellowship)

Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function. This study's aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters. This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment. Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations. Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

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)

Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) (U24TR001951), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

With the rise of research utilizing microphysiological systems (MPSs), the need for tools that enable the physiological mimicking of the relevant cellular environment is vital. The limited ability to reproduce crucial features of the microenvironment, such as surrounding fluid flow and dynamic changes in biochemical stimuli, severely limits the types of experiments that can be carried out. Current equipment to achieve this, such as syringe and peristaltic pumps, is expensive, large, difficult to program and has limited potential for scalability. Here, we present a new pumping platform that is open-source, low-cost, modular, scalable, fully-programmable and easy to assemble that can be incorporated into cell culture systems to better recapitulate physiological environments. By controlling two commercially available piezoelectric pumps using a Raspberry Pi Zero microcontroller, the system is capable of producing arbitrary dynamic flow profiles with reliable flow rates ranging from 1 to 3000 µL/min as specified by an easily programmable Python-based script. We validated the accuracy of the flow rates, the use of time-varying profiles, and the practicality of the system by creating repeatable dynamic concentration profiles using a 3D-printed static micromixer., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)

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)

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/- and ADAM17-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

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)

Interest in “engineering liver” arises from multiple communities: therapeutic replacement; mechanistic models of human processes; and drug safety and efficacy studies. An explosion of micro- and nanofabrication, biomaterials, microfluidic, and other technologies potentially affords unprecedented opportunity to create microphysiological models of the human liver, but engineering design principles for how to deploy these tools effectively toward specific applications, including how to define the essential constraints of any given application (available sources of cells, acceptable cost, and user-friendliness), are still emerging. Arguably less appreciated is the parallel growth in computational systems biology approaches toward these same problems—particularly in parsing complex disease processes from clinical material, building models of response networks, and in how to interpret the growing compendium of data on drug efficacy and toxicology in patient populations. Here, we provide insight into how the complementary paths of engineering liver—experimental and computational—are beginning to interplay toward greater illumination of human disease states and technologies for drug development., National Institutes of Health (U.S.) (UH2TR000496), National Institutes of Health (U.S.) (R01-EB 010246), National Institutes of Health (U.S.) (R01-ES015241), National Institutes of Health (U.S.) (P30-ES002109)

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways., National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant U54-CA112967), National Institutes of Health (U.S.) (R01DE019523-13), Massachusetts Institute of Technology. Computational and Systems Biology Program. MIT-Merck Postdoctoral Fellowship, Swiss National Science Foundation (Postdoctoral Fellowship), National Institute of Environmental Health Sciences (Training Grant in Environmental Toxicology 5-T32-ES007020)

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)

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)

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

Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment-sensitive fluorophore or Förster resonance energy transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities., National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1 U54 CA112967), National Institutes of Health (U.S.) (R01 EB 010246)

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

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)