Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells ...that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.
The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between ...the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3Kα
ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3Kα
-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.
Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part ...because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an
system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system
chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.
A proper understanding of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. Here we combine state-of-the-art data ...acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We apply PAMAF in an established in vitro model of TGFβ-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; -topological coupling between omics, -four distinct cell states during EMT, -omics-specific kinetic paths, -stage-specific multi-omics characteristics, -distinct regulatory classes of genes, -ligand-receptor mediated intercellular crosstalk by integrating scRNAseq and subcellular proteomics, and -combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, this study provides a resource on TGFβ signaling and EMT.
Highlights • Regenerative engineering has been recently proposed to regenerate complex tissue. • Therapeutic proteins have many limitations for their use in regenerating tissues. • ...Small-molecule-based therapeutics is emerging as an alternative strategy. • Small molecules may represent the next generation in musculoskeletal regenerative medicine.
The North Water Polynya is an area of high biological activity that supports large numbers of higher trophic-level organisms such as seabirds and marine mammals. An overall objective of the Upper ...Trophic-Level Group of the International North Water Polynya Study (NOW) was to evaluate carbon and contaminant flux through these high trophic-level (TL) consumers. Crucial to an evaluation of the role of such consumers, however, was the establishment of primary trophic linkages within the North Water food web. We used
δ
15N values of food web components from particulate organic matter (POM) through polar bears (
Ursus maritimus) to create a trophic-level model based on the assumptions that
Calanus hyperboreus occupies TL 2.0 and there is a 2.4‰ trophic enrichment in
15N between birds and their diets, and a 3.8‰ trophic enrichment for all other components. This model placed the planktivorous dovekie (
Alle alle) at TL 3.3, ringed seal (
Phoca hispida) at TL 4.5, and polar bear at TL 5.5. The copepods
C. hyperboreus,
Chiridius glacialis and
Euchaeta glacialis formed a trophic continuum (TL 2.0–3.0) from primary herbivore through omnivore to primary carnivore. Invertebrates were generally sorted according to planktonic, benthic and epibenthic feeding groups. Seabirds formed three trophic groups, with dovekie occupying the lowest, black-legged kittiwake (
Rissa tridactyla), northern fulmar (
Fulmarus glacialis), thick-billed murre (
Uria aalge), and ivory gull (
Pagophilia eburnea) intermediate (TL 3.9–4.0), and glaucous gull (
Larus hyperboreus) the highest (TL 4.6) trophic positions. Among marine mammals, walrus (
Odobenus rosmarus) occupied the lowest (TL 3.2) and bearded seal (
Erignathus barbatus), ringed seal, beluga whale (
Delphinapterus leucas), and narwhal (
Monodon monoceros) intermediate positions (TL 4.1–4.6). In addition to arctic cod (
Boreogadus saida), we suggest that lower trophic-level prey, in particular the amphipod
Themisto libellula, contribute fundamentally in transferring energy and carbon flux to higher trophic-level seabirds and marine mammals. We measured PCB 153 among selected organisms to investigate the behavior of bioaccumulating contaminants within the food web. Our isotopic model confirmed the trophic magnification of PCB 153 in this high-Arctic food web due to a strong correlation between contaminant concentration and organism
δ
15N values, demonstrating the utility of combining isotopic and contaminant approaches to food-web studies. Stable-carbon isotope analysis confirmed an enrichment in
13C between POM and ice algae (–22.3 vs. –17.7‰). Benthic organisms were generally enriched in
13C compared to pelagic species. We discuss individual species isotopic data and the general utility of our stable isotope model for defining carbon flux and contaminant flow through the North Water food web.
Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced ...pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRASG12D mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRASG12D isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular in vitro platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma.
A challenge for the design of scaffolds in tissue engineering is to determine a terminal sterilization method that will retain the structural and biochemical properties of the materials. Since ...commonly used heat and ionizing energy‐based sterilization methods have been shown to alter the material properties of protein‐based scaffolds, the effects of ethanol and ethylene oxide (EtO) sterilization on the cellular compatibility and the structural, chemical, and mechanical properties of uncrosslinked, UV crosslinked, or 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) crosslinked fibrin microthreads in neutral (EDCn) or acidic (EDCa) buffers are evaluated. EtO sterilization significantly reduces the tensile strength of uncrosslinked microthreads. Surface chemistry analyses show that EtO sterilization induces alkylation of EDCa microthreads leading to a significant reduction in myoblast attachment. The material properties of EDCn microthreads do not appear to be affected by the sterilization method. These results significantly enhance the understanding of how sterilization or crosslinking techniques affect the material properties of protein scaffolds.
The structural and chemical properties of fibrin‐based scaffolds are affected by crosslinking and sterilization protocols. Ethanol and ethylene oxide gas sterilization procedures are performed on uncrosslinked, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC), or ultraviolet light crosslinked fibrin microthreads to evaluate the mechanical, structural, and chemical properties of these materials. The data presented here inform the strategic design of fibrin‐based scaffolds.
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