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.

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.

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.

Background The selection of appropriate preclinical models comes always with the major question on how accurately and robustly they can represent the complexity of human disease. Patient-derived xenograft (PDX) models faithfully preserve the biological features and the genetic expression profile of human tumor specimens. However, one limiting aspect of patient-derived models is the replacement of the human host microenvironment by murine stroma within the tumor. Lack of cross-species compatibility compromises the induction of a broad range of signaling pathways that cannot be entirely recapitulated. With our in vitro 3D InSightTMTumor Microtissues derived from PDX lines, we provide a relevant physiological environment and a strategy to assess candidate drugs for novel therapeutic approaches. Aim Development of in vitro 3D InSightTM Tumor Microtissues from PDX lines aimed to retain the cellular heterogeneity found in the original human tumor tissue. Material & Methods and Results PDX cell suspensions of lung, breast and melanoma origin were successfully used to assess 3D aggregation in 96-well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each PDX sample, 3D PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). Furthermore, to provide a more physiological cancer niche, PDX cells were also co-cultured with tumor-matched cancer-associated fibroblasts (CAFs). 3D in vitro tumors were analyzed histologically and cancer phenotypic alterations were evaluated through the analysis of epithelial-to-mesenchymal transition (EMT) markers. The morphology, viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. To assess the distribution of various cell populations within the tumor, 3D PDX samples were screened for standard stromal vs. epithelial-tumor cells markers (e.g. FAP, pan-CK, E-Cadherin), and diagnostic cancer type-specific biomarkers. 3D PDX samples were also employed to investigate the efficacy of specific targeted therapies based on distinct molecular signatures of PDX tumor models. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Conclusion Further efforts will focus on employing this platform to establish more complex co-cultures with integration of additional relevant stromal and immune cells, to enable a reliable preclinical translational research of tumor/immune cell interactions. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment of immunomodulators and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Nicole Buschmann, Chloe' Pichon, Teresa Langova, Armin Maier, Julia Schueler, Patrick Guye. PDX-derived 3D InSightTM tumor microtissues as ex-vivo human experimental models for evaluating therapeutic responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 52.

Abstract Background A major issue for improving the overall success rate in drug development is the lack of accurate experimental human in-vitro models. While there is a significant and increasing need for such better ex-vivo cell-based models, the question on how closely these systems resemble and recapitulate the original tumors is of great importance. Patient-derived xenograft (PDX) models act as vehicle systems to propagate human tumor specimens, faithfully preserving the biological features and the genetic expression profile. The retainment of such criteria in in vitro 3D InSightTM Tumor Microtissues derived from PDX lines is crucial to provide a relevant physiological environment and strategy to assess candidate drugs for novel therapeutic approaches. Aim Development and characterization of in vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft models. Material & Methods and Results PDX cell suspensions of Lung, Breast and Melanoma origin were successfully used to assess 3D aggregation in 96 well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each in vitro 3D PDX sample, PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). The morphology, biomarker phenotype (IHC) as well as cell proliferation were assessed by histological analysis. In addition to screening for standard diagnostic marker such as proliferating vs. dead cells (e.g. Ki67, ClCasp3) and stromal vs. epithelial-tumor cells (e.g. FAP, pan-CK, E-Cadherin), we also assessed the expression of cancer type-specific biomarkers. Moreover, to monitor the dynamics of cancer phenotypic alterations, epithelial-to-mesenchymal transition (EMT) marker were evaluated and scored. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. The viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Further efforts will focus on employing this platform to investigate the efficacy of specific targeted therapies based on the distinct molecular signatures of PDX tumor models. Conclusion Development and characterization of In vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft (PDX) lines demonstrated that the morphological and molecular features of the parental tumors are well retained. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Armin Maier, Simon Messner, Julia Schueler, Patrick Guye. Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-034.

We sought to evaluate a new regional segmentation method for use with three-dimensional (3D) non-contrast abdominal CT images and to report the preliminary results.The proposed method was evaluated in ten cases. Manually segmented areas were used as the gold standard for evaluation. To compare the standard and the extracted liver regions, the degree of coincidence R% was redefined by transforming a volumetric overlap error. We also evaluated the influence of varying the density window size in terms of setting the starting points.We confirmed in ten cases that our method could segment the liver region more precisely than the conventional method. A size of window 15 voxels was optimal as the starting point in all cases.We demonstrated the accuracy of a 3D semiautomatic liver segmentation method for non-contrast CT. This method promises to offer radiologists a time-efficient segmentation aid.

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.

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.

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.

Des aspects de la presente invention concernent des procedes et des compositions pour la production d'un tissu heterogene a partir de cellules souches pluripotentes induites (hiPS).

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.

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.

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.

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.

Diabetes is caused by the loss or dysfunction of insulin-secreting β-cells in the pancreas. β-cells reduce their mass and lose insulin-producing ability in vitro, likely due to insufficient cell-cell and cell-extracellular matrix (ECM) interactions as β-cells lose their native microenvironment. Herein, we built an ex-vivo cell microenvironment by culturing primary β-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 β-cell function in vitro enhanced cell viability from the few days usually observed in 2D culture to periods exceeding three weeks, with enhanced β-cell stability and insulin production. Our approach can be extended to create a general 3D culture platform for other cell types.