Comparing in vitro human liver models to in vivo human liver using RNA-Seq.

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Network integration and modelling of dynamic drug responses at multi-omics levels.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.

A 3D Heterotypic Multicellular Tumor Spheroid Assay Platform to Discriminate Drug Effects on Stroma versus Cancer Cells.

Three-dimensional (3D) cell culture models are thought to mimic the physiological and pharmacological properties of tissues in vivo more accurately than two-dimensional cultures on plastic dishes. For the development of cancer therapies, 3D spheroid models are being created to reflect the complex histology and physiology of primary tumors with the hopes that drug responses will be more similar to and as predictive as those obtained in vivo. The effect of additional cell types in tumors, such as stromal cells, and the resulting heterotypic cell-cell crosstalk can be investigated in these heterotypic 3D cell cultures. Here, a high-throughput screening-compatible drug testing platform based on 3D multicellular spheroid models is described that enables the parallel assessment of toxicity on stromal cells and efficacy on cancer cells by drug candidates. These heterotypic microtissue tumor models incorporate NIH3T3 fibroblasts as stromal cells that are engineered with a reporter gene encoding secreted NanoLUC luciferase. By tracking the NanoLUC signal in the media over time, a time-related measurement of the cytotoxic effects of drugs on stromal cells over the cancer cells was possible, thus enabling the identification of a therapeutic window. An in vitro therapeutic index parameter is proposed to help distinguish and classify those compounds with broad cytotoxic effects versus those that are more selective at targeting cancer cells.

Synthetic Morphogenesis.

Throughout biology, function is intimately linked with form. Across scales ranging from subcellular to multiorganismal, the identity and organization of a biological structure's subunits dictate its properties. The field of molecular morphogenesis has traditionally been concerned with describing these links, decoding the molecular mechanisms that give rise to the shape and structure of cells, tissues, organs, and organisms. Recent advances in synthetic biology promise unprecedented control over these molecular mechanisms; this opens the path to not just probing morphogenesis but directing it. This review explores several frontiers in the nascent field of synthetic morphogenesis, including programmable tissues and organs, synthetic biomaterials and programmable matter, and engineering complex morphogenic systems de novo. We will discuss each frontier's objectives, current approaches, constraints and challenges, and future potential.

Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6.

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes.

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. We propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

A platform for rapid prototyping of synthetic gene networks in mammalian cells.

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.

Rapid neurogenesis through transcriptional activation in human stem cells.

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner.

Microbead-based biomimetic synthetic neighbors enhance survival and function of rat pancreatic ß-cells.

Diabetes is caused by the loss or dysfunction of insulin-secreting ß-cells in the pancreas. ß-cells reduce their mass and lose insulin-producing ability in vitro, likely due to insufficient cell-cell and cell-extracellular matrix (ECM) interactions as ß-cells lose their native microenvironment. Herein, we built an ex-vivo cell microenvironment by culturing primary ß-cells in direct contact with 'synthetic neighbors', cell-sized soft polymer microbeads that were modified with cell-cell signaling factors as well as components from pancreatic-tissue-specific ECMs. This biomimetic 3D microenvironment was able to promote native cell-cell and cell-ECM interactions. We obtained sustained maintenance of ß-cell function in vitro enhanced cell viability from the few days usually observed in 2D culture to periods exceeding three weeks, with enhanced ß-cell stability and insulin production. Our approach can be extended to create a general 3D culture platform for other cell types.

Rapid, modular and reliable construction of complex mammalian gene circuits.

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

Design and connection of robust genetic circuits.

Phenotypic robustness is a highly sought after goal for synthetic biology. There are many well-studied examples of robust systems in biology, and for the advancement of synthetic biology, particularly in performance-critical applications, fundamental understanding of how robustness is both achieved and maintained is very important. A synthetic circuit may fail to behave as expected for a multitude of reasons, and since many of these failures are difficult to predict a priori, a better understanding of a circuit's behavior as well as its possible failures are needed. In this chapter, we outline work that has been done in developing design principles for robust synthetic circuits, as well as sharing our experiences designing and constructing gene circuits.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA.

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional ß-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel ß-strand interactions between enzyme and target is suggested.

Host cell interactome of tyrosine-phosphorylated bacterial proteins.

Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use secretion systems to inject tyrosine kinase substrates into host cells. Upon phosphorylation, these effector proteins recruit cellular binding partners to manipulate host cell functions. So far, only a few interaction partners have been identified. Here we report the results of a proteomic screen to systematically identify binding partners of all known tyrosine-phosphorylated bacterial effectors by high-resolution mass spectrometry. We identified 39 host interactions, all mediated by SH2 domains, including four of the five already known interaction partners. Individual phosphorylation sites recruited a surprisingly high number of cellular interaction partners suggesting that individual phosphorylation sites can interfere with multiple cellular signaling pathways. Collectively, our results indicate that tyrosine-phosphorylation sites of bacterial effector proteins have evolved as versatile interaction modules that can recruit a rich repertoire of cellular SH2 domains.

A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome.

Bartonella henselae enters human endothelial cells (ECs) by two alternative routes: either by endocytosis, giving rise to Bartonella-containing vacuoles or by invasome-mediated internalization. Only the latter process depends on the type IV secretion system VirB/VirD4 and involves the formation of cell surface-associated bacterial aggregates, which get engulfed by EC membranes in an F-actin-dependent manner, eventually resulting in their complete internalization. Here, we report that among the VirB/VirD4-translocated effector proteins BepA-BepG only BepG is required for triggering invasome-mediated internalization. Expression of BepG in the Bep-deficient DeltabepA-G mutant restored invasome-mediated internalization. Likewise, ectopic expression of BepG in ECs also restored invasome-mediated internalization of the DeltabepA-G mutant, while no discernable cytoskeletal rearrangements were triggered in uninfected cells. Rather, BepG inhibited endocytic uptake of B. henselae into Bartonella-containing vacuoles and other endocytic processes, that is, invasin-mediated uptake of Yersinia enterocolitica and uptake of inert microspheres. BepG thus triggers invasome-mediated internalization primarily by inhibiting bacterial endocytosis. Bacteria accumulating on the cell surface then induce locally the F-actin rearrangements characteristic for the invasome. These cytoskeletal changes encompass both the rearrangement of pre-existing F-actin fibres and the de novo polymerization of cortical F-actin in the periphery of the invasome by Rac1/Scar1/WAVE- and Cdc42/WASP-dependent pathways that involve the recruitment of the Arp2/3 complex.

A translocated bacterial protein protects vascular endothelial cells from apoptosis.

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane-associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.