Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.

There are currently few treatment options for pulmonary fibrosis. Innovations may come from a better understanding of the cellular origin of the characteristic fibrotic lesions. We have analyzed normal and fibrotic mouse and human lungs by confocal microscopy to define stromal cell populations with respect to several commonly used markers. In both species, we observed unexpected heterogeneity of stromal cells. These include numerous cells with molecular and morphological characteristics of pericytes, implicated as a source of myofibroblasts in other fibrotic tissues. We used mouse genetic tools to follow the fates of specific cell types in the bleomcyin-induced model of pulmonary fibrosis. Using inducible transgenic alleles to lineage trace pericyte-like cells in the alveolar interstitium, we show that this population proliferates in fibrotic regions. However, neither these cells nor their descendants express high levels of the myofibroblast marker alpha smooth muscle actin (Acta2, aSMA). We then used a Surfactant protein C-CreER(T2) knock-in allele to follow the fate of Type II alveolar cells (AEC2) in vivo. We find no evidence at the cellular or molecular level for epithelial to mesenchymal transition of labeled cells into myofibroblasts. Rather, bleomycin accelerates the previously reported conversion of AEC2 into AEC1 cells. Similarly, epithelial cells labeled with our Scgb1a1-CreER allele do not give rise to fibroblasts but generate both AEC2 and AEC1 cells in response to bleomycin-induced lung injury. Taken together, our results show a previously unappreciated heterogeneity of cell types proliferating in fibrotic lesions and exclude pericytes and two epithelial cell populations as the origin of myofibroblasts.

SARS-CoV-2 papain-like protease activates nociceptors to drive sneeze and pain.

SARS-CoV-2, the virus responsible for COVID-19, triggers symptoms such as sneezing, aches and pain.1 These symptoms are mediated by a subset of sensory neurons, known as nociceptors, that detect noxious stimuli, densely innervate the airway epithelium, and interact with airway resident epithelial and immune cells.2-6 However, the mechanisms by which viral infection activates these neurons to trigger pain and airway reflexes are unknown. Here, we show that the coronavirus papain-like protease (PLpro) directly activates airway-innervating trigeminal and vagal nociceptors in mice and human iPSC-derived nociceptors. PLpro elicits sneezing and acute pain in mice and triggers the release of neuropeptide calcitonin gene-related peptide (CGRP) from airway afferents. We find that PLpro-induced sneeze and pain requires the host TRPA1 ion channel that has been previously demonstrated to mediate pain, cough, and airway inflammation.7-9 Our findings are the first demonstration of a viral product that directly activates sensory neurons to trigger pain and airway reflexes and highlight a new role for PLpro and nociceptors in COVID-19.

Autophagy restricts Mycobacterium tuberculosis during acute infection in mice.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

Quantitative prediction of human pharmacokinetic responses to drugs via fluidically coupled vascularized organ chips.

Analyses of drug pharmacokinetics (PKs) and pharmacodynamics (PDs) performed in animals are often not predictive of drug PKs and PDs in humans, and in vitro PK and PD modelling does not provide quantitative PK parameters. Here, we show that physiological PK modelling of first-pass drug absorption, metabolism and excretion in humans-using computationally scaled data from multiple fluidically linked two-channel organ chips-predicts PK parameters for orally administered nicotine (using gut, liver and kidney chips) and for intravenously injected cisplatin (using coupled bone marrow, liver and kidney chips). The chips are linked through sequential robotic liquid transfers of a common blood substitute by their endothelium-lined channels (as reported by Novak et al. in an associated Article) and share an arteriovenous fluid-mixing reservoir. We also show that predictions of cisplatin PDs match previously reported patient data. The quantitative in-vitro-to-in-vivo translation of PK and PD parameters and the prediction of drug absorption, distribution, metabolism, excretion and toxicity through fluidically coupled organ chips may improve the design of drug-administration regimens for phase-I clinical trials.

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

Non-invasive sensing of transepithelial barrier function and tissue differentiation in organs-on-chips using impedance spectroscopy.

Here, we describe methods for combining impedance spectroscopy measurements with electrical simulation to reveal transepithelial barrier function and tissue structure of human intestinal epithelium cultured inside an organ-on-chip microfluidic culture device. When performing impedance spectroscopy measurements, electrical simulation enabled normalization of cell layer resistance of epithelium cultured statically in a gut-on-a-chip, which enabled determination of transepithelial electrical resistance (TEER) values that can be compared across device platforms. During culture under dynamic flow, the formation of intestinal villi was accompanied by characteristic changes in impedance spectra both measured experimentally and verified with simulation, and we demonstrate that changes in cell layer capacitance may serve as measures of villi differentiation. This method for combining impedance spectroscopy with simulation can be adapted to better monitor cell layer characteristics within any organ-on-chip in vitro and to enable direct quantitative TEER comparisons between organ-on-chip platforms which should help to advance research on organ function.

A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.

The diverse bacterial populations that comprise the commensal microbiome of the human intestine play a central role in health and disease. A method that sustains complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro would thus enable the investigation of host-microbiome interactions. Here, we show the extended coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota, using a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients. When compared to aerobic coculture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human faeces. The intestine-on-a-chip may serve as a discovery tool for the development of microbiome-related therapeutics, probiotics and nutraceuticals.

Author Correction: A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.

In the version of this Article originally published, the authors mistakenly cited Fig. 5d in the sentence beginning 'Importantly, the microbiome cultured in these primary Intestine Chips...'; the correct citation is Supplementary Table 2. This has now been amended.

In vivo response to decellularized mesothelium scaffolds.

Biological surgical scaffolds are used in plastic and reconstructive surgery to support structural reinforcement and regeneration of soft tissue defects. Macrophage and fibroblast cell populations heavily regulate scaffold integration into host tissue following implantation. In the present study, the biological host response to a commercially available surgical scaffold (Meso BioMatrix Surgical Mesh (MBM)) was investigated for up to 9 weeks after subcutaneous implantation; this scaffold promoted superior cell migration and infiltration previously in in vitro studies relative to other commercially available scaffolds. Infiltrating macrophages and fibroblasts phenotypes were assessed for evidence of inflammation and remodeling. At week 1, macrophages were the dominant cell population, but fibroblasts were most abundant at subsequent time points. At week 4, the scaffold supported inflammation modulation as indicated by M1 to M2 macrophage polarization; the foreign body giant cell response resolved by week 9. Unexpectedly, a fibroblast subpopulation expressed macrophage phenotypic markers, following a similar trend in transitioning from a proinflammatory to anti-inflammatory phenotype. Also, α-smooth muscle actin-expressing myofibroblasts were abundant at weeks 4 and 9, mirroring collagen expression and remodeling activity. MBM supported physiologic responses observed during normal wound healing, including cellular infiltration, host tissue ingrowth, remodeling of matrix proteins, and immune modulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 716-725, 2018.

Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function.

Trans-epithelial electrical resistance (TEER) is broadly used as an experimental readout and a quality control assay for measuring the integrity of epithelial monolayers cultured under static conditions in vitro, however, there is no standard methodology for its application to microfluidic organ-on-a-chip (organ chip) cultures. Here, we describe a new microfluidic organ chip design that contains embedded electrodes, and we demonstrate its utility for assessing formation and disruption of barrier function both within a human lung airway chip lined by a fully differentiated mucociliary human airway epithelium and in a human gut chip lined by intestinal epithelial cells. These chips with integrated electrodes enable real-time, non-invasive monitoring of TEER and can be applied to measure barrier function in virtually any type of cultured cell.

Ear-Shaped Stable Auricular Cartilage Engineered from Extensively Expanded Chondrocytes in an Immunocompetent Experimental Animal Model.

Advancement of engineered ear in clinical practice is limited by several challenges. The complex, largely unsupported, three-dimensional auricular neocartilage structure is difficult to maintain. Neocartilage formation is challenging in an immunocompetent host due to active inflammatory and immunological responses. The large number of autologous chondrogenic cells required for engineering an adult human-sized ear presents an additional challenge because primary chondrocytes rapidly dedifferentiate during in vitro culture. The objective of this study was to engineer a stable, human ear-shaped cartilage in an immunocompetent animal model using expanded chondrocytes. The impact of basic fibroblast growth factor (bFGF) supplementation on achieving clinically relevant expansion of primary sheep chondrocytes by in vitro culture was determined. Chondrocytes expanded in standard medium were either combined with cryopreserved, primary passage 0 chondrocytes at the time of scaffold seeding or used alone as control. Disk and human ear-shaped scaffolds were made from porous collagen; ear scaffolds had an embedded, supporting titanium wire framework. Autologous chondrocyte-seeded scaffolds were implanted subcutaneously in sheep after 2 weeks of in vitro incubation. The quality of the resulting neocartilage and its stability and retention of the original ear size and shape were evaluated at 6, 12, and 20 weeks postimplantation. Neocartilage produced from chondrocytes that were expanded in the presence of bFGF was superior, and its quality improved with increased implantation time. In addition to characteristic morphological cartilage features, its glycosaminoglycan content was high and marked elastin fiber formation was present. The overall shape of engineered ears was preserved at 20 weeks postimplantation, and the dimensional changes did not exceed 10%. The wire frame within the engineered ear was able to withstand mechanical forces during wound healing and neocartilage maturation and prevented shrinkage and distortion. This is the first demonstration of a stable, ear-shaped elastic cartilage engineered from auricular chondrocytes that underwent clinical-scale expansion in an immunocompetent animal over an extended period of time.

Extensively Expanded Auricular Chondrocytes Form Neocartilage In Vivo.

OBJECTIVE: Our goal was to engineer cartilage in vivo using auricular chondrocytes that underwent clinically relevant expansion and using methodologies that could be easily translated into health care practice. DESIGN: Sheep and human chondrocytes were isolated from auricular cartilage biopsies and expanded in vitro. To reverse dedifferentiation, expanded cells were either mixed with cryopreserved P0 chondrocytes at the time of seeding onto porous collagen scaffolds or proliferated with basic fibroblast growth factor (bFGF). After 2-week in vitro incubation, seeded scaffolds were implanted subcutaneously in nude mice for 6 weeks. The neocartilage quality was evaluated histologically; DNA and glycosaminoglycans were quantified. Cell proliferation rates and collagen gene expression profiles were assessed. RESULTS: Clinically sufficient over 500-fold chondrocyte expansion was achieved at passage 3 (P3); cell dedifferentiation was confirmed by the simultaneous COL1A1/3A1 gene upregulation and COL2A1 downregulation. The chondrogenic phenotype of sheep but not human P3 cells was rescued by addition of cryopreserved P0 chondrocytes. With bFGF supplementation, chondrocytes achieved clinically sufficient expansion at P2; COL2A1 expression was not rescued but COL1A1/3A1genes were downregulated. Although bFGF failed to rescue COL2A1 expression during chondrocyte expansion in vitro, elastic neocartilage with obvious collagen II expression was observed on porous collagen scaffolds after implantation in mice for 6 weeks. CONCLUSIONS: Both animal and human auricular chondrocytes expanded with low-concentration bFGF supplementation formed high-quality elastic neocartilage on porous collagen scaffolds in vivo.

Type 2 alveolar cells are stem cells in adult lung.

Gas exchange in the lung occurs within alveoli, air-filled sacs composed of type 2 and type 1 epithelial cells (AEC2s and AEC1s), capillaries, and various resident mesenchymal cells. Here, we use a combination of in vivo clonal lineage analysis, different injury/repair systems, and in vitro culture of purified cell populations to obtain new information about the contribution of AEC2s to alveolar maintenance and repair. Genetic lineage-tracing experiments showed that surfactant protein C-positive (SFTPC-positive) AEC2s self renew and differentiate over about a year, consistent with the population containing long-term alveolar stem cells. Moreover, if many AEC2s were specifically ablated, high-resolution imaging of intact lungs showed that individual survivors undergo rapid clonal expansion and daughter cell dispersal. Individual lineage-labeled AEC2s placed into 3D culture gave rise to self-renewing "alveolospheres," which contained both AEC2s and cells expressing multiple AEC1 markers, including HOPX, a new marker for AEC1s. Growth and differentiation of the alveolospheres occurred most readily when cocultured with primary PDGFRα⁺ lung stromal cells. This population included lipofibroblasts that normally reside close to AEC2s and may therefore contribute to a stem cell niche in the murine lung. Results suggest that a similar dynamic exists between AEC2s and mesenchymal cells in the human lung.