Sweetman, J. | bioRxiv Science

bioRxiv2024

Watch-breaker: establishment of a microwell array-based miniaturized thymic organoid model suitable for high throughput applications.

T-cell development depends critically on the thymic stroma, in particular the diverse array of functionally distinct thymic epithelial cell (TEC) types. However, a robust in vitro thymus model mimicking the native thymus and compatible with medium/high-throughput analyses is currently lacking. Here, we demonstrate a novel high-density microwell array-based miniaturized thymus organoid (mTO) model, that supports T-cell commitment and development, possesses key organizational characteristics of the native thymus and is compatible with live-imaging and medium/high-throughput applications. We establish the minimum cellular input required for functional mTO and show that mTO TEC phenotype and complexity closely mirrors the native thymus. Finally, we use mTO to probe the role of fetal thymic mesenchyme, revealing a requirement beyond maintenance of Foxn1 in differentiation/maintenance of mature TEC subpopulations. Collectively, mTO present a new in vitro model of the native thymus adaptable to medium/high-throughput applications and validated for exploration of thymus- and thymus organoid-biology.

immunology↗

bioRxiv2023

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis.

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC- fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells.

developmental biology↗

bioRxiv2022

How benthic sediment microbial communities respond to glyphosate and its metabolite: A microcosm experiment

Glyphosate is the most commonly used agricultural herbicide in the world. In aquatic ecosystems, glyphosate often adsorbs to benthic substrates or is metabolized and degraded by microorganisms. The effects of glyphosate on microbial communities varies widely as microorganisms respond differently to exposure. To help understand the impacts of glyphosate on the sediment microbiome we conducted a microcosm experiment examining the responses of benthic sediment microbial communities to herbicide treatments. Sediments from a prairie pothole wetland were collected and 16S rRNA gene sequencing was used to analyze community composition 2-hours and 14-days after a single treatment of low (0.07 ppm), medium (0.7 ppm), or high (7 ppm) glyphosate, aminomethylphosphonic acid (glyphosate metabolite), or a glyphosate-based commercial formula. We found no significant differences in microbial community composition between treatments, concentration levels, or time. These findings suggest that microbial species in the Prairie Pothole Region of North America may be tolerant to glyphosate exposure.

microbiology↗

bioRxiv2021

Deploying an in vitro gut model to assay the impact of a mannan-oligosaccharide prebiotic, Bio-Mos(R) on the Atlantic salmon (Salmo salar) gut microbiome.

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and the associated metabolism. Bio-Mos is a commercially available pre-biotic currently used in the agri-feed industry. To assess Bio-Mos for potential use as a prebiotic growth promotor in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. Inoculated from biological triplicates of adult farmed salmon pyloric caeca compartments, the microbial communities were stabilised in SalmoSim followed by a twenty-day exposure to the prebiotic and in turn followed by an eight day wash out period. Dietary inclusion of MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to MOS supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggests that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators. ImportanceIn this paper we report the results of the impact of prebiotic (MOS supplementation) on microbial communities within recently developed Atlantic salmon gut microbiome in vitro simulator. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production in the Atlantic salmon gut and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

bioengineering↗

bioRxiv2020

Deploying and in vitro gut model to assay the impact of a mannan-oligosaccharide prebiotic, Bio-MOS(R) on the Atlantic salmon (Salmo salar) gut microbiome.

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and associated metabolism. Bio-MOS is a commercially available pre-biotic currently used in the agri-feed industry. To assay Bio-MOS for potential use as a pre-biotic growth promotor in salmonid aquaculture, we modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on host microbial communities. In biological triplicate, microbial communities were stabilised in SalmoSim followed by twenty-day exposure to the pre-biotic and then an eight day wash out period. Exposure the MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several principally anaerobic microbial genera (Fusobacteria, Agarivorans, Pseudoalteromonas, Myroides). 16S rDNA sequencing confirmed a significant shift in microbial community composition in response to supplementation. In conjunction with previous in vivo studies linking enhanced VFA production alongside MOS supplementation to host growth and performance, our data suggest that Bio-MOS may be of value in salmonid production. Furthermore, our data highlight the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

microbiology↗

bioRxiv2020

Development of a three-compartment in vitro simulator of the Atlantic Salmon GI tract and associated microbial communities: SalmoSim

Atlantic salmon are a species of major economic importance. Intense innovation is underway to improve salmon feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the industry. Several gut models targeted at monogastric vertebrates are now in operation. Here we report progress in the development of an Atlantic salmon in vitro gut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum and mid gut) and associated microbial communities. The artificial gut model was established in a series of linked bioreactors seeded with biological material derived for adult marine phase salmon. In biological triplicate, the response of the in vitro system to two distinct dietary formulations (fish meal and fish meal free) was compared to a parallel in vivo trial over forty days. 16S rDNA sequencing, qPCR, ammoniacal nitrogen and volatile fatty acid measurements were undertaken to survey microbial community dynamics and function. SalmoSim communities were indistinguishable (p=0.230) from their founding inocula at 20 days and most abundant genera (e.g. Psycrobacter, Staphylococcus, Pseudomonas) proliferated the in vitro system. Real salmon and SalmoSim responded similarly to the introduction of the novel feed, with most taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa was affected non-identically across both systems. Consistent with a low impact of the novel feed on microbial community function, VFA profiles were not significantly different in SalmoSim pre and post the switch feed. This study represents an important first-step in the development of an in vitro gut system as a tool for the improvement of salmon nutrition.

microbiology↗