Organ Culture Research Articles

Low Baseline Expression of Fibrotic Genes in an Ex Vivo Human Skin Model is a Potential Indicator of Excessive Skin Scarring.

An ex vivo model of skin fibrosis was used with abdominal and breast skin tissue from 45 patients undergoing breast reduction and/or abdominoplasty. Fibrosis was induced in skin explants in organ culture with transforming growth factor-β (TFGβ). Fibrotic gene response was assessed via quantitative real-time polymerase chain reaction and correlated with skin location, age, and baseline levels of fibrotic genes. The increase in TFGβ-induced fibronectin1 (FN1) gene expression in skin explants was significantly higher than for Collagen 1A1, alpha smooth muscle actin, and connective tissue growth factor. Also, FN1 expression positively correlated with donor age. Moreover, lower expression of the fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin correlated with a more pronounced fibrotic response, represented by higher induction levels of these genes. Skin sites exhibit different baseline levels of profibrotic genes. Further, low baseline expression levels of fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin, in donor skin may indicate a potential for excessive scarring of the skin.

Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing Methodologies.

Thymic epithelial cells (TECs) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Cortical (c)TECs, present in the cortex, are essential for early thymocyte development including selection of thymocytes expressing functional TCRs (positive selection). Medullary (m)TECs, located in the medulla, play a key role in late thymocyte development, including depletion of self-reactive T cells (negative selection) and selection of regulatory T cells. In recent years, transcriptomic analysis by single-cell (sc)RNA sequencing (Seq) has revealed TEC heterogeneity previously masked by population-level RNA-Seq or phenotypic studies. We summarize the discoveries made possible by scRNA-Seq, including the identification of novel mTEC subsets, advances in understanding mTEC promiscuous gene expression, and TEC alterations from embryonic to adult stages. Whereas pseudotime analyses of scRNA-Seq data can suggest relationships between TEC subsets, experimental methods such as lineage tracing and reaggregate thymic organ culture (RTOC) are required to test these hypotheses. Lineage tracing - namely, of β5t or Aire expressing cells - has exposed progenitor and parent-daughter cellular relationships within TEC.

Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

A Simplified Approach to Evaluating T Cell Development by Flow Cytometry.

T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multiparameter flow cytometry-based cell sorting are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with relatively simple flow cytometry screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a multi-laser flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.

Fetal Thymic Organ Culture and Negative Selection.

Negative selection removes potentially harmful T cell precursors from the conventional T cell pool. This process can involve the induction of apoptosis, anergy, receptor editing, or deviation into a regulatory T cell lineage. As such, this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate these various forms of negative selection are not clear. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes fetal thymic organ culture methods designed to analyze the signals that determine these unique cell fates.

Electron microscopy of desmosomal structures in the pemphigus human skin organ culture model.

Pemphigus is a chronic autoimmune skin blistering disease, characterized by acantholysis and by the production of autoantibodies directed against the structural desmosomal proteins desmoglein 1 (DSG1) and/or DSG3. Model systems allow the identification and testing of new therapeutic targets. Here, we evaluated ultrastructural desmosomal morphology in the human skin organ culture (HSOC) model injected with either anti-desmoglein (DSG) 1/3 single-chain variable fragment (scFv, termed Px4-3), Staphylococcus aureus exfoliative toxin (ETA) as a reference and positive control, and normal human IgG as a negative control. Each experimental condition was evaluated in abdominal skin biopsies from five different donors. After 24 h of incubation, we processed the samples for histological and ultrastructural electron microscopy analyses. We found that Px4-3 or ETA induced a loss of desmosomes and increased interdesmosomal widening, similar to patient skin biopsies and other pemphigus models. Thus, we propose the HSOC pemphigus model as an attractive tool to unravel novel therapeutic targets.

A critical bioenergetic switch is regulated by IGF2 during murine cartilage development

Long bone growth requires the precise control of chondrocyte maturation from proliferation to hypertrophy during endochondral ossification, but the bioenergetic program that ensures normal cartilage development is still largely elusive. We show that chondrocytes have unique glucose metabolism signatures in these stages, and they undergo bioenergetic reprogramming from glycolysis to oxidative phosphorylation during maturation, accompanied by an upregulation of the pentose phosphate pathway. Inhibition of either oxidative phosphorylation or the pentose phosphate pathway in murine chondrocytes and bone organ cultures impaired hypertrophic differentiation, suggesting that the appropriate balance of these pathways is required for cartilage development. Insulin-like growth factor 2 (IGF2) deficiency resulted in a profound increase in oxidative phosphorylation in hypertrophic chondrocytes, suggesting that IGF2 is required to prevent overactive glucose metabolism and maintain a proper balance of metabolic pathways. Our results thus provide critical evidence of preference for a bioenergetic pathway in different stages of chondrocytes and highlight its importance as a fundamental mechanism in skeletal development.

Ameliorating Fibrosis in Murine and Human Tissues with END55, an Endostatin-Derived Fusion Protein Made in Plants.

Organ fibrosis, particularly of the lungs, causes significant morbidity and mortality. Effective treatments are needed to reduce the health burden. A fragment of the carboxyl-terminal end of collagen XVIII/endostatin reduces skin and lung fibrosis. This fragment was modified to facilitate its production in plants, which resulted in the recombinant fusion protein, END55. We found that expression of END55 had significant anti-fibrotic effects on the treatment and prevention of skin and lung fibrosis in a bleomycin mouse model. We validated these effects in a second mouse model of pulmonary fibrosis involving inducible, lung-targeted expression of transforming growth factor β1. END55 also exerted anti-fibrotic effects in human lung and skin tissues maintained in organ culture in which fibrosis was experimentally induced. The anti-fibrotic effect of END55 was mediated by a decrease in the expression of extracellular matrix genes and an increase in the levels of matrix-degrading enzymes. Finally, END55 reduced fibrosis in the lungs of patients with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) who underwent lung transplantation due to the severity of their lung disease, displaying efficacy in human tissues directly relevant to human disease. These findings demonstrate that END55 is an effective anti-fibrotic therapy in different organs.

Plant-based expression platforms to produce high-value metabolites and proteins.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

Results of ultrathin Descemet stripping automated endothelial keratoplasty with donor corneas preserved in synthetic organ culture media.

To evaluate outcomes of ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) with donor corneas preserved at 31°C in Cornea Syn®, a medium formulated with recombinant human serum albumin (rHSA) to replace foetal calf serum, and deswelled-transported in the xeno-free medium Cornea Trans®. Prospective, multicentre, open-label study. We evaluated the endothelial cell loss (ECL) as the percentage variation of the endothelial cell density (ECD, cells/mm2) between 6 and 12 months after surgery, corneal transparency and thickness at 12 months, and adverse events within 12 months. Endothelial lenticules of mean 89 μm, ECD ≥ 2300 cells/mm2, minimum signs of cell mortality or morphology alterations, were dissected by microkeratome in the eye bank, and grafted in patients ≥ 18 years without corneal neovascularisation, conjunctivalization, or blinking impairment. Thirty-five patients underwent UT-DSAEK, 3 showed primary failure, 1 late failure, and 2 skipped the 6-month visit. We analysed data from 29 patients, 27 with Fuchs endothelial corneal dystrophy (FECD) and 2 with pseudophakic bullous keratopathy (PBK). The median ECL between 6 and 12 months was 2.6% (p = .054, CI 0 to 12.5) and the absolute mean (SD) was 158.4 (364.1) cells/mm2. After 12 months, 96.5% of corneas were clear, with mean pachymetry of 585.9 (50.4) µm. The ECL rate after UT-DSAEK match overall that observed in DSAEK or UT-DSAEK models of endothelial survival and the overall safety compared that reported for similar follow-up. Corneas maintained in Cornea Syn® and Cornea Trans® did not affect the ECD and functional outcomes of UT-DSAEK up to 12 months.

Cisplatin Induces Apoptosis in Mouse Neonatal Testes Organ Culture.

Chemotherapy is used for childhood cancer but may lead to infertility in patients. Spermatogonia stem cells are present in the testes of prepubertal boys, although they do not produce sperm at this age. Herein, we evaluated the toxicity of cisplatin, a known medicine for cancer treatment, in neonatal mouse testes using in vitro organ culture. Mouse testicular fragments (MTFs) derived from 5.5-d postpartum mouse testes were exposed to 1-10 μg/mL cisplatin. The results showed that cisplatin significantly downregulated the expression of germ cell marker genes, including differentiated and undifferentiated, in a dose-dependent manner. In particular, a high dose of cisplatin (10 μg/mL) led to germ cell depletion. In addition, the expression levels of the Sertoli cell marker gene, the number of SOX9+ Sertoli cells, and the levels of SOX9 protein were markedly decreased in cisplatin-treated MTFs compared to controls. The mRNA expression of steroidogenic enzyme-related genes significantly increased in cisplatin-treated MTFs, except for estrogen receptor 1 (Esr1). Consistently, 3β-hydroxysteroid dehydrogenase protein was also observed in the interstitial regions of cisplatin-treated MTFs. Altogether, our findings showed a significant impairment in germ cell development, Sertoli cell survival, and steroidogenesis in the MTFs of cisplatin-treated mice.

Semaphorin 4D induces articular cartilage destruction and inflammation in joints by transcriptionally reprogramming chondrocytes.

Proinflammatory cytokines play critical roles in the pathogenesis of joint diseases. Using a mass spectrometry-based cloning approach, we identified Semaphorin 4D (Sema4D) as an inflammatory cytokine that directly promoted cartilage destruction. Sema4d-deficient mice showed less cartilage destruction than wild-type mice in a model of rheumatoid arthritis. Sema4D induced a proinflammatory response in mouse articular chondrocytes characterized by the induction of proteolytic enzymes that degrade cartilage, such as matrix metalloproteinases (MMPs) and aggrecanases. The activation of Mmp13 and Mmp3 expression in articular chondrocytes by Sema4D did not depend on RhoA, a GTPase that mediates Sema4D-induced cytoskeletal rearrangements. Instead, it required NF-κB signaling and Ras-MEK-Erk1/2 signaling downstream of the receptors Plexin-B2 and c-Met and depended on the transcription factors IκBζ and C/EBPδ. Genetic and pharmacological blockade of these Sema4D signaling pathways inhibited MMP induction in chondrocytes and cartilage destruction in femoral head organ culture. Our results reveal a mechanism by which Sema4D signaling promotes cartilage destruction.

Self-Complementary Adeno-Associated Viral Gene Delivery in the Metanephric Organ Culture Identifies Pvt1 as Modulator of Cystogenesis

Self-Complementary Adeno-Associated Viral Gene Delivery in the Metanephric Organ Culture Identifies Pvt1 as Modulator of Cystogenesis

Diet-induced modifications to human microbiome reshape colonic homeostasis in irritable bowel syndrome.

Changes in microbiome composition are associated with a wide array of human diseases, turning the human microbiota into an attractive target for therapeutic intervention. Yet, clinical translation of these findings requires the establishment of causative connections between specific microbial taxa and their functional impact on host tissues. Here, we infuse gut organ cultures with longitudinal microbiota samples collected from therapy-naive patients with irritable bowel syndrome (IBS) under a low-fermentable oligo-, di-, mono-saccharides and polyols (FODMAP) diet. We show that post-diet microbiota regulates intestinal expression of inflammatory and neuro-muscular gene sets. Specifically, we identify Bifidobacterium adolescentis as a diet-sensitive pathobiont that alters tight junction integrity and disrupts gut barrier functions. Collectively, we present a pathway discovery platform for mechanistic dissection and identification of functional diet-host-microbiota modules. Our data support the hypothesis that the gut microbiota mediates the beneficial effects of a low-FODMAP diet and reinforce the potential feasibility of microbiome-based therapies in IBS.

A macrocyclic molecule with multiple antioxidative activities protects the lens from oxidative damage.

Growing evidence links oxidative stress to the development of a cataract and other diseases of the eye. Treatments for lens-derived diseases are still elusive outside of the standard surgical interventions, which still carry risks today. Therefore, a potential drug molecule OHPy2N2 was explored for the ability to target multiple components of oxidative stress in the lens to prevent cataract formation. Several pathways were identified. Here we show that the OHPy2N2 molecule activates innate catalytic mechanisms in primary lens epithelial cells to prevent damage induced by oxidative stress. This protection was linked to the upregulation of Nuclear factor erythroid-2-related factor 2 and downstream antioxidant enzyme for glutathione-dependent glutaredoxins, based on Western Blot methods. The anti-ferroptotic potential was established by showing that OHPy2N2 increases levels of glutathione peroxidase, decreases lipid peroxidation, and readily binds iron (II) and (III). The bioenergetics pathway, which has been shown to be negatively impacted in many diseases involving oxidative stress, was also enhanced as evidence by increased levels of Adenosine triphosphate product when the lens epithelial cells were co-incubated with OHPy2N2. Lastly, OHPy2N2 was also found to prevent oxidative stress-induced lens opacity in an ex vivo organ culture model. Overall, these results show that there are multiple pathways that the OHPy2N2 has the ability to impact to promote natural mechanisms within cells to protect against chronic oxidative stress in the eye.

A Deletion Encompassing the Furin Cleavage Site in the Spike Encoding Gene Does Not Alter SARS-CoV-2 Replication in Lung Tissues of Mink and Neutralization by Convalescent Human Serum Samples.

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results.

Perivascular cells support folliculogenesis in the developing ovary

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin+ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

In Vitro Mimicking of Obesity-Induced Biochemical Environment to Study Obesity Impacts on Cells and Tissues.

Obesity represents a heavy burden for modern healthcare. The main challenge facing obesity research progress is the unknown underlying pathways, which limits our understanding of the pathogenesis and developing therapies. Obesity induces specific biochemical environments that impact the different cells and tissues. In this piece of writing, we suggest mimicking obesity-induced in vivo biochemical environments including pH, lipids, hormones, cytokines, and glucose within an in vitro environment. The concept is to reproduce such biochemical environments and use them to treat the tissue cultures, explant cultures, and cell cultures of different biological organs. This will allow us to clarify how the obesity-induced biochemistry impacts such biological entities. It would also be important to try different environments, in terms of the compositions and concentrations of the constitutive elements, in order to establish links between the effects (impaired regeneration, cellular inflammation, etc.) and the factors constituting the environment (hormones, cytokines, etc.) as well as to reveal dose-dependent effects. We believe that such approaches will allow us to elucidate obesity mechanisms, optimize animal models, and develop therapies as well as novel tissue engineering applications.

Protein-imprinted particles for coronavirus capture from solution.

Molecular imprinting is a promising strategy to selectively adsorb viruses, but it requires discerning and validating epitopes that serve as effective imprinting templates. In this work, glycoprotein‐imprinted particles were synthesized for coronavirus capture. Adsorption was maximized at pH 6 (the glycoprotein isoelectric point) where the glycoprotein‐imprinted particles outperformed non‐imprinted particles, adsorbing 4.96 × 106 ± 3.33 × 103 versus 3.54 × 106 ± 1.39 × 106 median tissue culture infectious dose/mg of the target coronavirus, human coronavirus – organ culture 43, within the first 30 min (p = 0.012). During competitive adsorption, with pH adjustment (pH 6), the glycoprotein‐imprinted particles adsorbed more target virus than non‐target coronavirus (human coronavirus – Netherland 63) with 2.34 versus 1.94 log removal in 90 min (p < 0.01). In contrast, the non‐imprinted particles showed no significant difference in target versus non‐target virus removal. Electrostatic potential calculation shows that the human coronavirus – organ culture 43 glycoprotein has positively charged pockets at pH 6, which may facilitate adsorption at lower pH values. Therefore, tuning the target virus glycoprotein charge via pH adjustment enhanced adsorption by minimizing repulsive electrostatic interactions with the particles. Overall, these results highlight the effective use of glycoprotein‐imprinted particles for coronavirus capture and discern the merits and limitations of glycoprotein imprinting for the capture of enveloped viruses.

Дегидротермический кросслинкинг стромальных графтов роговицы свиньи: влияние температуры на основные трансплантологические свойства. экспериментальное исследование

Purpose. To evaluate the effect of DHT crosslinking of stromal corneal grafts at temperatures of 60, 100, 140, 180 and 220°C on their principal applicability in keratoplasty. Material and methods. A corneoscleral disc was excised from a porcine eye (&lt; 12 h post-mortem) with epithelium and Descemet's membrane removed, dried, and a 10 mm central corneal graft was cut out. Dry grafts (n = 36) were divided into 6 groups of 6 samples: in five groups they were kept under vacuum for 3 days at 60, 100, 140, 180, 220 °C; 6th group – untreated control; then the grafts were immersed in phosphate-buffered saline for 24 h. Parameters evaluated: gross appearance, image quality from 5 m, light transmission, water content, central thickness, suture retention load. A test for epithelialization was performed ex vivo after 3 days in fresh porcine cornea organ culture. Results. The 220 °C mode caused charring destruction of the material. The grafts after 180 °C (10 g) became significantly weaker than the 10–0 nylon thread (90 g). In the range of 60–140 °C suture retention load significantly lowered from 913 g (control) down to 137 g (140 °C). Grafts increased their light transmission from 30 % (control) up to 75 % (140 °C); the central thickness went from 3000 (control) down to 320 um (140 °C), water content decreased from 94 % (control) down to 44 % (140 °C). Subjective image quality of the grafts after treatment at 60, 100, and 140 °C increased and grafts allowed distinguishing optotypes 0,3, 1,0 and 1,0 decimal, respectively. In organ culture control grafts and grafts after 60 and 100 °C treatment were covered with epithelial cells. 140 °C samples showed smooth surface with no cells upon. Сonclusion. Grafts after dehydrothermal treatment at temperatures 220 and 180 °C are principally not suitable for surgery. Grafts after 140 °C treatment do not pass epithelialization test. Grafts after 100 °C and lower can be used for keratoplasty. Keywords: crosslinking, corneal surgery, grafts, epithelial cells