Metabolic characterization of directly reprogrammed renal tubular epithelial cells (iRECs)

Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future

Parameter-adaption for a vehicle dynamics model for the evaluation of powertrain concept designs

The powertrain design of multi-motor electric vehicles directly affects not only costs, consumption and acceleration, but also the handling of a vehicle. Therefore, a holistic powertrain design optimization needs to include a vehicle dynamics model in its objective function. While the parameters for the powertrain model result from the design variables that describe the powertrain, the parameters for the vehicle dynamics model must be adapted in a feasible way to ensure comparable results. Therefore, the authors present a method on how to adaptively parametrize a double-track vehicle dynamics model for the use in powertrain design optimization. Automated design calculations for all main chassis and suspension parts are used to determine the parameters for the model. A parameter variation proves the plausibility of the approach. The results show that an adaption of the suspension and chassis parameters due to changes in the powertrain make results more comparable but do not compensate for the effects on the vehicle handling. In particular, the steady state longitudinal load distribution still has major influences on the vehicle handling

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1$^{-/-}$ iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms, for diagnostic or anti-infective applications, but which can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerisation of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms which produced them. This ‘bacteria-instructed synthesis’ can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the ‘instructing’ cell types. We further expand on the bacterial redox chemistries to ‘click’ fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualisation of pathogens

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1(−/−) iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.We acknowledge funding from an EPSRC Platform grant which supported McMurray and a Wellcome Trust project grant which supported Wann and McMurray. Wann is now supported on an ARUK project grant. Thompson was funded by a BBSRC PhD studentshi

Deep learning is widely applicable to phenotyping embryonic development and disease

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. Statement of Significance The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation

A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

© 2019 The Authors. Published by Springer. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1038/s41467-019-13149-7The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.This work was funded by the Novo Nordisk Foundation (NNF17CC0027852) and Danish National Research Foundation (DNRF116). J.C. and D.G.W. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001217), the UK Medical Research Council (FC001217), and the Wellcome Trust (FC001217). S.C. was supported by an SNSF Early Postdoc Mobility fellowship (P2ZHP3_164840) and a Long Term EMBO Postdoc fellowship (ALTF 511-2016), and L.S. and J.B.A. by the Independent Research Fund Denmark (DFF; Sapere Aude2 4183-00118B).Published versio