Cultured Cell Populations Research Articles

Utilizing convolutional neural networks for discriminating cancer and stromal cells in three-dimensional cell culture images with nuclei counterstain.

Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures. We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy. In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels. Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image's cell distribution. This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.

Olfactory Mucosal Cells of Individuals with Alzheimer’s Disease Display Alterations Similar to the Alzheimer Brain

AbstractBackgroundA sense of smell is orchestrated in the first place by the cells of the olfactory mucosa (OM), located in the rooftop of the nasal cavity. Impairments in olfaction are implicated in aging but also in Alzheimer’s disease (AD). Here, we describe the observed alterations in cultures of OM cells derived from individuals with late‐onset AD or amnestic form of mild cognitive impairment (MCI) when compared to OM cells derived from their cognitively healthy control subjects.MethodPrimary cultures of OM cells were harvested from biopsies of OM derived from cognitively healthy individuals and individuals with late‐onset AD or amnestic MCI and cultured in vitro. First, the cell types present in cultures of OM cells and genes differentially expressed between the OM cells derived from control subjects and individuals AD or MCI OM were assessed by single cell RNA‐sequencing (scRNA‐seq). Next, a total RNA‐seq was applied to a larger cohort of samples. The results of total and scRNA‐seq were further validated with various functional in vitro assays including inductively coupled plasma mass spectrometry (ICP‐MS) and measurements of intracellular ATP levels.ResultThe scRNA‐seq unveiled a total of five distinct cell populations in OM cell cultures. A total of 240 previously AD‐related genes were found to be differentially expressed between the AD and control OM cell cultures. Similarly, the MCI OM cells harbored 224 differentially expressed genes when compared to OM cells derived from their control subjects. Common for both AD and MCI OM cells were the alterations in genes related to mitochondrial function when compared to controls. Additionally, the ICP‐MS revealed increased levels of zinc, calcium and sodium in the AD OM cells in comparison to the OM cells derived from cognitively healthy subjects.ConclusionThe results implicate the role of olfactory mucosa as a site displaying early alterations related to Alzheimer’s disease.

Cell population growth kinetics in the presence of stochastic heterogeneity of cell phenotype

Recent studies at individual cell resolution have revealed phenotypic heterogeneity in nominally clonal tumor cell populations. The heterogeneity affects cell growth behaviors, which can result in departure from the idealized uniform exponential growth of the cell population. Here we measured the stochastic time courses of growth of an ensemble of populations of HL60 leukemia cells in cultures, starting with distinct initial cell numbers to capture a departure from the uniform exponential growth model for the initial growth (“take-off”). Despite being derived from the same cell clone, we observed significant variations in the early growth patterns of individual cultures with statistically significant differences in growth dynamics, which could be explained by the presence of inter-converting subpopulations with different growth rates, and which could last for many generations. Based on the hypothesis of existence of multiple subpopulations, we developed a branching process model that was consistent with the experimental observations.

Single-cell mass distributions reveal simple rules for achieving steady-state growth.

Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

Globoside and the mucosal pH mediate parvovirus B19 entry through the epithelial barrier.

Parvovirus B19 (B19V) is transmitted primarily via the respiratory route, however, the mechanism involved remains unknown. B19V targets a restricted receptor expressed in erythroid progenitor cells in the bone marrow. However, B19V shifts the receptor under acidic conditions and targets the widely expressed globoside. The pH-dependent interaction with globoside may allow virus entry through the naturally acidic nasal mucosa. To test this hypothesis, MDCK II cells and well-differentiated human airway epithelial cell (hAEC) cultures were grown on porous membranes and used as models to study the interaction of B19V with the epithelial barrier. Globoside expression was detected in polarized MDCK II cells and the ciliated cell population of well-differentiated hAEC cultures. Under the acidic conditions of the nasal mucosa, virus attachment and transcytosis occurred without productive infection. Neither virus attachment nor transcytosis was observed under neutral pH conditions or in globoside knockout cells, demonstrating the concerted role of globoside and acidic pH in the transcellular transport of B19V. Globoside-dependent virus uptake involved VP2 and occurred by a clathrin-independent pathway that is cholesterol and dynamin-dependent. This study provides mechanistic insight into the transmission of B19V through the respiratory route and reveals novel vulnerability factors of the epithelial barrier to viruses.

Adaptive Laboratory Evolution of Microorganisms: Methodology and Application for Bioproduction.

Adaptive laboratory evolution (ALE) is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. It is expected that researchers can understand the evolutionary adaptation processes, and for industrial applications, researchers can create useful microorganisms that exhibit increased carbon source availability, stress tolerance, and production of target compounds based on omics analysis data. In this review article, the methodologies for ALE in microorganisms are introduced. Moreover, the application of ALE for the creation of useful microorganisms as cell factories has also been introduced.

Characterization of alternative mRNA splicing in cultured cell populations representing progressive stages of human fetal kidney development

Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme—a transient kidney-specific progenitor state—undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms’ tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms’ tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms’ tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.

Evaluating the Safety and Quality of Life of Colorectal Cancer Patients Treated by Autologous Immune Enhancement Therapy (AIET) in Vinmec International Hospitals.

(1) Colorectal cancer (CRC) is an increasingly prevalent disease with a high mortality rate in recent years. Immune cell-based therapies have received massive attention among scientists, as they have been proven effective as low-toxicity treatments. This study evaluated the safety and effectiveness of autologous immune enhancement therapy (AIET) for CRC. (2) An open-label, single-group study, including twelve patients diagnosed with stages III and IV CRC, was conducted from January 2016 to December 2021. Twelve CRC patients received one to seven infusions of natural killer (NK)-cell and cytotoxic T-lymphocyte (CTL). Multivariate modelling was used to identify factors associated with health-related quality-of-life (HRQoL) scores. (3) After 20–21 days of culture, the NK cells increased 3535-fold, accounting for 85% of the cultured cell population. Likewise, CTLs accounted for 62.4% of the cultured cell population, which was a 1220-fold increase. Furthermore, the QoL improved with increased EORTC QLQ-C30 scores, decreased symptom severity, and reduced impairment in daily living caused by these symptoms (MDASI-GI report). Finally, a 14.3 ± 14.1-month increase in mean survival time was observed at study completion. (4) AIET demonstrated safety and improved survival time and HRQoL for CRC patients in Vietnam.

2D and 3D liver models

2D and 3D liver models

SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo.

ABSTRACTCell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here, we introduce SyNPL: clonal pluripotent stem cell lines that employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered ‘sender’ and ‘receiver’ cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new adaptation of SynNotch technology that could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and that can be customised to generate synthetic patterning of cell fate decisions.

Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro

BackgroundMicroglia, the resident immune cells of the brain, play a critical role in numerous diseases, but are a minority cell type and difficult to genetically manipulate in vivo with viral vectors and other approaches. Primary cultures allow a more controlled setting to investigate these cells, but morphological and transcriptional changes upon removal from their normal brain environment raise many caveats from in vitro studies.MethodsTo investigate whether cultured microglia recapitulate in vivo microglial signatures, we used single-cell RNA sequencing (scRNAseq) to compare microglia freshly isolated from the brain to primary microglial cultures. We performed cell population discovery, differential expression analysis, and gene co-expression module analysis to compare signatures between in vitro and in vivo microglia. We constructed causal predictive network models of transcriptional regulators from the scRNAseq data and identified a set of potential key drivers of the cultured phenotype. To validate this network analysis, we knocked down two of these key drivers, C1qc and Prdx1, in primary cultured microglia and quantified changes in microglial activation markers.ResultsWe found that, although often assumed to be a relatively homogenous population of cells in culture, in vitro microglia are a highly heterogeneous population consisting of distinct subpopulations of cells with transcriptional profiles reminiscent of macrophages and monocytes, and are marked by transcriptional programs active in neurodegeneration and other disease states. We found that microglia in vitro presented transcriptional activation of a set of “culture shock genes” not found in freshly isolated microglia, characterized by strong upregulation of disease-associated genes including Apoe, Lyz2, and Spp1, and downregulation of homeostatic microglial markers, including Cx3cr1, P2ry12, and Tmem119. Finally, we found that cultured microglia prominently alter their transcriptional machinery modulated by key drivers from the homeostatic to activated phenotype. Knockdown of one of these drivers, C1qc, resulted in downregulation of microglial activation genes Lpl, Lyz2, and Ccl4.ConclusionsOverall, our data suggest that when removed from their in vivo home environment, microglia suffer a severe case of “culture shock”, drastically modulating their transcriptional regulatory network state from homeostatic to activated through upregulation of modules of culture-specific genes. Consequently, cultured microglia behave as a disparate cell type that does not recapitulate the homeostatic signatures of microglia in vivo. Finally, our predictive network model discovered potential key drivers that may convert activated microglia back to their homeostatic state, allowing for more accurate representation of in vivo states in culture. Knockdown of key driver C1qc partially attenuated microglial activation in vitro, despite C1qc being only weakly upregulated in culture. This suggests that even genes that are not strongly differentially expressed across treatments or preparations may drive downstream transcriptional changes in culture.

Skin Organoid Research Progress and Potential Applications

Abstract Skin diseases were characterized by various types and high incidence, which seriously affect people's health. At present, skin pathogenesis research and the therapeutic drug development for skin diseases are limited by the lack of reasonable research models that recapitulate the development of skin diseases. Organoids are three-dimensionally cultured cell populations derived from skin stem cells, which exhibits the ability of multicell assembly and the similar histological characteristics with the living tissues and organs. This article reviews the establishment of normal skin organoids and skin tumor organoids, and summarizes the application of skin organoids in the evaluation of drug sensitivity, pathological mechanism research, and individualized treatment. In addition, the advantages and limitations of organoids in skin disease research are also discussed, which provides a basis for revealing the pathogenesis of skin diseases and developing preventive and therapeutic drugs for skin diseases.

Radiation Resistance of C57Bl/6 Mouse Bone Marrow Stromal Cell Lines Induced By Apoptosis Inhibitor JP4-039 or Necroptosis Inhibitor Necrostatin-1, but Not By Simultaneous Administration of Both Mitigators

Introduction: Necroptosis is a TNFa activated, caspase-independent cell death pathway that involves active disintegration of mitochondrial, lysosomal and plasma membranes (Vandenabeele, et al, Nat Rev Mol Cell Biol., 2010 Oct; 11(10):700-14). We measured clonogenic radiation survival of C57/Bl6 mouse bone marrow stromal cell lines in the presence of a mitochondrial targeted inhibitor of apoptosis (JP4-039), an inhibitor of necroptosis (Necrostatin-1) or both drugs.Materials and Methods: C57BL/6 mouse long term bone marrow cultures (LTBMCs) were established from adult female C57Bl/6 mice. Bone marrow stromal cell lines were derived from the adherent layers of LTBMCs. Radiosensitivity of C57Bl/6 bone marrow stromal cell lines and fresh bone marrow CFU-GEMM (semisolid medium with added hemopoietic growth factors) was measured in clonogenic radiation survival curves. Stromal Cells were irradiated to doses of 0 to 8 Gy, plated in T-75 tissue culture flasks, incubated for 8 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted at day 7. CFU-GEMMs from fresh bone marrow were scored at day 14. Both cultured cell populations were tested with addition of radiation mitigators: JP4-039 (10µM), Necrostatin-1 (40µM), or both drugs added to culture medium 30 minutes post irradiation. C57BL/6 mouse LTBMCs generated hemopoietic cells for 20 weeks. Marrow stromal cell lines were derived from 20 week old hemopoieticly-inactive culture adherent layer cells. Statistical analysis was performed using Student's t-test, with a two-tail p-value < 0.05 considered significant.Results: Radiation survival curves showed that C57Bl/6 fresh bone marrow CFU-GEMM and stromal cell lines were comparable to those for other mouse strains (CFU-GEMM Do = 1.67 ± 0.02, stromal cells Do=1.43 ± 0.14). Both fresh marrow CFU-GEMM and stromal cells (ň = 5.26 ± 0.72) were radioresistant when grown in presence of JP4-039 (ň = 8.85 ± 1.23, p = 0.012) or necrostatin-1 (n=7.97 ± 1.12, p = 0.024). Addition of both JP4-039 and Necrostatin-1 (ň = 5.79 ± 1.44) at the same time after irradiation (30 min) did not improve clonogenic survival of either fresh marrow CFU-GEMM or stromal cell lines.Conclusions: Both freshly explanted CFU-GEMM marrow hematopoietic progenitor cells and LTBMC derived marrow stromal cell lines display radioresistance when treated with each of two radiation mitigators that target different cell death pathways. While each drug delivered independently induced radioresistance, combined delivery of JP4-039 and Necrostatin-1 at 30 min after irradiation did not. Thus, the timing of delivery of drugs that target different cell death pathways is likely critical in designing effective protocols for radiation mitigation. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia depletes contaminating CD45+ hematopoietic cells from murine bone marrow stromal cell (BMSC) cultures: Methods for BMSC culture purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSCs continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation is widely based on adherence to tissue culture plastic; however, high hematopoietic contamination is a significant impediment in murine models. Remarkably, when cultured at a physiological oxygen tension of 1% O2, a 10-fold reduction in CD45+ hematopoietic cells associated with a concomitant increase in PDGFRα+ stromal cells occur. This is due, in part, to a differential response of the two populations to hypoxia. In standard tissue culture conditions of 21% O2, CD45+ cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O2. In contrast, PDGFR α+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR α+ stromal cell population in murine BMSC cultures.

Strategies to Identify Mesenchymal Stromal Cells in Minimally Manipulated Human Bone Marrow Aspirate Concentrate Lack Consensus

Background: There is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC. Purpose: To establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies. Study Design: Descriptive laboratory study. Methods: Five published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile. Results: ISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells. Conclusion: Currently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences. Clinical Relevance: This study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Amnion-Derived Multipotent Progenitor Cells Suppress Experimental Optic Neuritis and Myelitis.

The human amnion has been used for decades in wound healing, particularly burns. Amnion epithelial cells (AECs) have been the focus of extensive research based on their possible pluripotent differentiation ability. A novel, cultured cell population derived from AECs, termed human amnion-derived multipotent progenitor (AMP) cells, secrete numerous cytokines and growth factors that enhance tissue regeneration and reduce inflammation. This AMP cell secretome, termed ST266, is a unique biological solution that accumulates in eyes and optic nerves following intranasal delivery, resulting in selective suppression of optic neuritis in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, but not myelitis at the administered dose. We tested the hypothesis that systemic AMP cell administration could suppress both optic neuritis and myelitis in EAE. Intravenous and intraperitoneal administration of AMP cells significantly reduced ascending paralysis and attenuated visual dysfunction in EAE mice. AMP cell treatment increased retinal ganglion cell (RGC) survival and decreased optic nerve inflammation, with variable improvement in optic nerve demyelination and spinal cord inflammation and demyelination. Results show systemic AMP cell administration inhibits RGC loss and visual dysfunction similar to previously demonstrated effects of intranasally delivered ST266. Importantly, AMP cells also promote neuroprotective effects in EAE spinal cords, marked by reduced paralysis. Protective effects of systemically administered AMP cells suggest they may serve as a potential novel treatment for multiple sclerosis.

Abstract B19: Role of cell-cell communication in endocrine therapy-resistant breast cancer

Abstract Endocrine therapy is the mainstay for the treatment of estrogen-receptor positive (ER+) breast cancer. However, frequent emergence of endocrine resistance is a major clinical challenge. Cell-cell cooperation in the tumor microenvironment plays a vital role in acquiring resistance, but its role is poorly understood. We studied the interactions among endocrine-resistant (R) and -sensitive (S) breast cancer cells in the presence and absence of ER-targeted endocrine therapy. We measured changes in admixed cell population remodeling by dual fluorescent labeling of LCC1 (S) with eGFP (green fluorescent protein) and their derived resistant variant (selected against the antiestrogen fulvestrant) LCC9 (R) with mCherry (red fluorescent protein), respectively. Thus, unlike most prior studies, these are genetically related cells, rather than ER+ and ER- cell lines obtained from different patients. Our results revealed that “S”cells (LCC1) acquired the phenotype of “R” cells when cultured in a mixed population. A ratio of 5:1 of S:R cells in mixed population was sufficient to confer resistance to fulvestrant to the S cells within a six-day period. Furthermore, we show that acquirement of this resistant phenotype for the S cells was partially blocked by inhibition of either gap junctions or exosome secretion, suggesting a role of cell-cell contact as well as extracellular vesicles. Global proteomic analysis of pure and admixed S and R cell populations showed that R and S can alter key molecular features of each other when present in mixed cell populations. We used a Bayesian approach to fit single culture cell populations to infer density-dependent growth parameters (growth rate, carrying capacity) and a generalized Lotka-Volterra model to understand how S and R coculture populations may be depressing or supporting growth of the other. Our results identify a net mutualistic interaction between the susceptible and resistant cancer strains, demonstrating that there are ecological dynamics to cancer resistance. In some tumors, ecological dynamics may be more mechanistically relevant than simple Darwinian-based clonal selection in driving tumor heterogeneity and determining response to treatment. Our study strongly indicates that different tumor cell types that may exist in tumors are able to influence each other, which leads to alteration of phenotype. Note: This abstract was not presented at the conference. Citation Format: Surojeet Sengupta, Rong Hu, Zachary Susswein, Adeola Fagunloye, Shweta Bansal, Robert Clarke. Role of cell-cell communication in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B19.

Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in mono or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.

Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells.

Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.