Explant Culture System Research Articles

Limitations of an ex vivo breast cancer model for studying the mechanism of action of the anticancer drug paclitaxel

Paclitaxel is widely used in the treatment of breast, ovarian, lung, and other cancers. Its primary mechanism is to prevent microtubule depolymerization causing loss of dynamic instability crucial for normal microtubule function leading to mitotic arrest. Prolonged mitotic arrest results in cell death as a secondary response. The effects of paclitaxel are typically studied in cell lines which precludes assessment of the possible influence of tumor-associated cells. We therefore examined paclitaxel action ex vivo in fresh explant cultures of human breast tumors. Surprisingly, we found that paclitaxel failed to induce tumor cell death in explant culture, in contrast to several other cytotoxic agents including salinomycin and vincristine. The lack of effect was not due to defective drug uptake, and furthermore, analysis of H&E stained tumor slices indicated that paclitaxel treatment caused defective (granular) mitosis and chromosomal condensation in 5–10% of tumor cells after 72 h. These results suggest that while paclitaxel was able to penetrate into the tumor slice and disrupt mitosis in cycling tumor cells, any ensuing cell death likely occurred beyond the useful lifetime of the tumor slices. We conclude that explant culture systems may be inappropriate for the study of cytotoxic drugs where a delay exists between the drug's primary and secondary modes of action.

An in vitro ovarian explant culture system to examine sex change in a hermaphroditic fish.

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achieved in vivo through social manipulation, inhibition of aromatase activity, or steroid treatment. However, the induction of sex change in vitro has been largely unexplored. In this study, we established an in vitro culture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17β-estradiol (E2), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E2. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e., sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex change in vitro. The in vitro gonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.

Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1β (IL1-β), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Jmjd3 Plays Pivotal Roles in the Proper Development of Early-Born Retinal Lineages: Amacrine, Horizontal, and Retinal Ganglion Cells.

PurposeTrimethylation of histone H3 at lysine 27 (H3K27me3) is a critical mediator of transcriptional gene repression, and Jmjd3 and Utx are the demethylases specific to H3K27me3. Using an in vitro retinal explant culture system, we previously revealed the role of Jmjd3 in the development of rod bipolar cells; however, the roles of Jmjd3 in the development of early-born retinal cells are unknown due to limitations concerning the use of retinal explant culture systems. In this study, we investigated the roles of Jmjd3 in the development of early-born retinal cells.MethodsWe examined retina-specific conditional Jmjd3 knockout (Jmjd3-cKO) mice using immunohistochemistry and quantitative reverse transcription PCR and JMJD3 binding to a target locus by chromatin immunoprecipitation analysis.ResultsWe observed reductions in amacrine cells (ACs) and horizontal cells (HCs), as well as lowered expression levels of several transcription factors involved in the development of ACs and HCs in the Jmjd3-cKO mouse retina. JMJD3 bound the promoter regions of these transcription factors. Notably, an elevated number of retinal ganglion cells (RGCs) was observed at embryonic stages, whereas RGCs were moderately reduced at later postnatal stages in the Jmjd3-cKO retina. We also observed reduced expression of Eomes, which is required for the maintenance of RGCs, as well as lower H3K27me3 level and lower JMJD3 binding in the promoter region of Eomes in RGC-enriched cells.ConclusionsThe results indicated that Jmjd3 has critical roles in the development of early-born retinal subtypes, and suggested biphasic roles of Jmjd3 in RGC production and maintenance.

Ex vivo culture of head and neck cancer explants in cell sheet for testing chemotherapeutic sensitivity.

Tumor explant culture systems can mimic the in vivo tumor microenvironment, proposing as a substitute for preclinical studies for prediction of individual treatment response. Therefore, our study evaluated the potential usefulness of ex vivo tumor explants culture assembled into the cell sheets by anticancer drug screening in patients with head and neck squamous cell carcinoma (HNSCC). Our model included tumor explants incorporated into cell sheet composing of epithelium and subepithelial stroma using tumor and mucosal samples obtained from the HNSCC patients who underwent surgery. Cell growth, viability, and hypoxia were measured by cell counting kit-8, live/dead assay, propidium iodide, and LOX-1 staining, and were compared among the different treatment groups with vehicle, cisplatin or docetaxel. Tumor explants stably survived in the cell sheet over 10days after explantation, whereas most of the explants in non-matrix culture became nonviable within 5-8days with the significant daily decrease of viability. The live tissue areas of tumor explants in the cell sheet maintained over 30days without significant changes although hypoxic cell areas gradually increased up to 5days. Tissue viability and live cancer tissue areas significantly decreased after the treatment of cisplatin or docetaxel in the dose and time-dependent manners. Our cell sheet-based tumor explants model might be applied to the reliable ex vivo screening for anticancer chemotherapeutics for HNSCC.

2155-P: Peripheral Nervous System Innervation of the Developing Pancreas

Pancreatic organogenesis is a complex, multistep process whereby multiple cell types must migrate, proliferate, and differentiate in a strict spatiotemporal manner to ultimately form the complete organ. Although pancreatic epithelial organogenesis is well characterized, the peripheral nervous system (PNS) innervating the organ is poorly understood. Although the PNS has been shown to be critical in the formation of other budding exocrine organs, and therefore likely plays a role in the formation of the pancreas, there is a dearth of information on innervation of this developing organ. To this end, our lab has taken advantage of an explant culture system and single-cell RNA-sequencing (scRNAseq) to characterize the nerves supplying the developing pancreas. Imaging of whole pancreata reveals that peripheral nerves develop in parallel with the pancreas, with intrapancreatic ganglia being present from early stages. Using scRNAseq analysis we have begun to decipher the timing of pancreatic PNS progenitor cell differentiation, and the potential signaling pathways guiding axonal growth and morphogenesis. Continued investigation of the mechanisms guiding innervation of the pancreas, deciphering how nerve-epithelial interactions are established and defining signaling pathways activated by the nerves will allow us to discover the role of the PNS in pancreatic morphogenesis, as well as mis-regulation during disease. Disclosure S. de la O: None. L. Byrnes: None. D.M. Wong: None. S. Knox: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc.

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation.

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

Organotypic Culture of Adult Mouse Retina.

Retinal explant culture systems have the potential to mimic the functional dynamics of the organ beyond those of the dissociated cells, thus making this technique a very powerful intermediate model system between in vitro cell cultures and in vivo animal models. The different retinal layers made of highly specialized cell types remain intact, while glia cell reactions and/or intercellular interactions can be evaluated under well-defined conditions in the lab.In retinal disorders neurodegeneration of mature retinal cells takes place. Therefore, we investigated the adult murine neuroretina in organ culture to test its suitability for use in preclinical therapeutic applications. Here we describe a method for the organ culture of adult murine retina (>12weeks) used to establish survival, cellular changes and early degeneration patterns of neuronal and glial cells. After enucleation of the eyeball and careful dissection of the retina from the sclera and retinal pigment epithelium, the detached retina is cultured with photoreceptor facing down on a supporting track-etched polycarbonate membrane in a 6-well culture plate maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After 1, 2, 3, 4, 6, 8, or 10days retinal explants can be harvested and immediately processed for RNA isolation or fixed in paraformaldehyde for histological analysis.

Explant Type, Culture System, 6-Benzyladenine, Meta-Topolin and Encapsulation Affect Indirect Somatic Embryogenesis and Regeneration in Carica papaya L.

A protocol to propagate papaya hybrid plants through indirect somatic embryogenesis was developed considering the effect of explant type, culture system, particular cytokinins and encapsulation, in different stages of the process. Optimal 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations for non-embryogenic callus formation ranged between 9.0 and 27.1 μM in half-cut seeds, while higher concentrations were harmful. Non-embryogenic callus was also obtained with 22–158 μM 2,4-D from hypocotyl segments. Callus with embryogenic structures was only obtained in half-cut seeds cultured in the darkness on half-strength Murashige and Skoog culture medium supplemented with 2,4-D, while hypocotyl segments and isolated zygotic embryos failed to produce this type of callus regardless of the 2,4-D and sucrose (30 and 70 g l-1) concentrations tested in this study. Both, embryogenic callus development and quantity of somatic embryos formed per embryogenic callus, which ranged between 11 and 31 units after 14 months, required 2,4-D, but without any effect of the concentration. Histological studies confirmed the multicellular origin of the somatic embryos. In further steps, liquid medium induced over four times more somatic embryos than agar-gelled medium and showed significantly higher production of globular somatic embryos (85 vs. 57%). Both, 6-benzyladenine (BA) and meta-topolin (Mtop) stimulated sprouting (40–45%) of the somatic embryos (development of shoots only) in concentrations of up to 2.7 and 10 μM, respectively. Sprouting probability showed a 2nd order polynomial trend despite the range of concentration used for each cytokinin. This is the first report about the positive effect of Mtop on the apical shoot development of Carica papaya somatic embryos known to the authors. Radicle growth was observed in 5% or less of the cultivated embryos, regardless of the BA concentration. Finally, all encapsulation conditions tested (2.5, 3.5, and 4.5% sodium alginate, combined with 50 and 100 mM CaCl2) reduced sprouting of somatic embryos when compared to the non-encapsulated ones, whereas capsule hardness showed low correlation with embryo sprouting. Embryos were further cultivated until they became plantlets approximately 5 cm long. They were acclimatized and afterward planted in the field, where they flowered and produced fruit.

The role of Zhx2 transcription factor in bipolar cell differentiation during mouse retinal development

We found that the Zhx2 gene (whose product is known to act as a tumor suppressor in hepatocellular carcinoma) is expressed in embryonic retinal progenitors and in developing cone bipolar cells in the postnatal retina, as well as in Müller glia in the mature retina. To examine the functions of Zhx2 protein during retinal development, we performed loss- and gain-of-function analyses using a retinal explant culture system. We introduced a plasmid encoding Zhx2 shRNA into isolated mouse retinas at E17.5, and the retinas were cultured as explants. After 3 days of culture, proliferation was slightly enhanced, leading to retinas thicker than in the control, but this phenomenon was observed only transiently. After 14 days of the culture, the thickness and gross morphology of retinas expressing sh-Zhx2 were indistinguishable from those of the control. The numbers of rod cells, amacrine cells, and Müller glia were the same in both groups. However, although the total number of bipolar cells was the same, the experimental group saw an increased population of ON bipolar cells, and decreased numbers of a subset of OFF bipolar cells. We also examined the effects of overexpression of Zhx2. Although Zhx2 acts as a tumor suppressor, its overexpression in developing retinas did not lead to any discernible difference in retinal thickness, suggesting that proliferation activity was not affected. After 14 days of explant culture, the total number of bipolar cells decreased, and subset composition was altered. Taken together, these results suggest that Zhx2 plays roles in the regulation of bipolar cell subset fate determination during retinal development.

Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury

After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration—RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% downregulation 5 days after hydrogel injection. Our results show that local implantation of a AON-gapmer-loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.

Evaluation of Rasna panchaka (indigenous drug) as oxidative stress down-regulator using serum-free explant culture system.

CONTEXT:The importance of phytochemicals/natural products as potential therapeutic agents in the present context is gaining a lot of importance. India with a rich heritage of such preparations needs evaluation as potent drugs. Explant culture system is a method, which is sensitive, reliable, reproducible and is capable of mimicking the in situ conditions maintaining the tissue in sufficiently high level of integration.AIM:The current study aimed to test the antioxidant activity of test compounds, namely, traditional aqueous (4212) and aqueous-methanolic (4308) extracts of Rasna panchaka using liver explant cultures.MATERIALS AND METHODS:Dose-response optima of extracts (0.2–10 μg/mL) were determined using mouse liver explant culture system up to 48 h. The antioxidant property of extracts was assessed by primary oxidative defense parameters, namely, superoxide-dismutase (SOD), catalase, reduced glutathione (GSH), and malondialdehyde (MDA).RESULTS:The results indicated that the cellular architecture of the cultured tissue was well conserved in the first 6 h with a gradual display of specific changes in the next 24 h. There was a significant increase in MDA levels in experimental groups indicating the oxidative stress induction in explants. A dose of 2.0 μg/mL extracts have shown statistically significant (P < 0.05) protection against oxidative stress. MDA levels, a measure of lipid peroxidation, were significantly (P < 0.01) reduced by 50% in extract treated explants compared to control. This effect was accompanied by the increase in the first defense enzymes SOD (50%) and catalase (18%) with no change in reduced GSH levels.CONCLUSION:The study enforces the importance of “explant culture system,” as it not only reduces the use of nonclinical/animal model but also is rapid and sensitive. Further, results of the current study also suggest that aqueous-methanolic extract of Rasna panchaka is having superior antioxidant activity compared to traditional water extract.

Comparison of the Effects of Interleukin-1 on Equine Articular Cartilage Explants and Cocultures of Osteochondral and Synovial Explants.

Osteoarthritis (OA) is a ubiquitous disease affecting many horses. The disease causes chronic pain and decreased performance for patients and great cost to owners for diagnosis and treatment. The most common treatments include systemic non-steroidal anti-inflammatory drugs and intra-articular injection of corticosteroids. There is excellent support for the palliative pain relief these treatments provide; however, they do not arrest progression and may in some instances hasten advancement of disease. Orthobiologic treatments have been investigated as potential OA treatments that may not only ameliorate pain but also prevent or reverse pathologic articular tissue changes. Clinical protocols for intra-articular use of such treatments have not been optimized; the high cost of in vivo research and concerns over humane use of research animals may be preventing discovery. The objective of this study was to evaluate a novel in vitro articular coculture system for future use in OA treatment research. Concentrations and fold increases in various markers of inflammation (prostaglandin E2 and tumor necrosis factor-alpha), degradative enzyme activity [matrix metalloproteinase-13 (MMP-13)], cartilage and bone metabolism (bone alkaline phosphatase and dimethyl-methylene blue), and cell death (lactate dehydrogenase) were compared between IL-1-stimulated equine articular cartilage explant cultures and cocultures comprised of osteochondral and synovial explants (OCS). Results suggested that there are differences in responses of culture systems to inflammatory stimulation. In particular, the IL-1-induced fold changes in MMP-13 concentration were significantly different between OCS and cartilage explant culture systems after 96 h. These differences may be relevant to responses of joints to inflammation in vivo and could be important to the biological relevance of in vitro research findings.

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells.

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS)+ cells using novel small-molecule inducers. We screened small molecules that increased the induction rate of INS+ cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1)+ pancreatic progenitor cells. The differentiation protocol to generate INS+ cells from hiPSCs/hESCs was optimised using hit compounds, and INS+ cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS+ cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS+ cells increased from a mean±SD of 5.9±1.5% (n=3) to 16.5±2.1% (n=3). SCG induced neurogenin 3-positive cells at a mean±SD of 32.6±4.6% (n=3) compared with 14.2±3.6% (n=3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS+ cells from hiPSCs/hESCs. The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.

Extracellular Matrix Mediates BMP-2 in a Model of Temporomandibular Joint Osteoarthritis

Temporomandibular joint (TMJ) osteoarthritis (OA) is a complex disease that affects both cartilage and subchondral bone. It is accompanied by loss of extracellular matrix (ECM) and may be controlled by bone morphogenetic protein-2 (BMP-2). We analyzed the effect of BMP-2 in both cartilage and subchondral bone in a TMJ-OA animal model that is deficient in biglycan (Bgn) and fibromodulin (Fmod) (Bgn^-/-Fmod^-/-). Whole mandibles were dissected from 3-week-old wild-type (WT) and Bgn^-/-Fmod^-/- mice and incubated with and without 250 µg/mL BMP-2 for 2 days using an explant culture system. Condyle growth was measured by microCT and the expression levels of cartilage and bone-related genes were analyzed using RT-PCR or by immunohistochemistry from condyles that contained an intact cartilage/subchondral bone interface. Osteoclast activity was estimated by tartrate-resistant acid phosphatase (TRAP) staining and by TRAP, Rankl, and Adamts4 mRNA expression levels. Our results showed that most parameters examined were slightly up-regulated in WT samples treated with BMP-2, and this up-regulation was significantly enhanced in the Bgn^-/-Fmod^-/- mice. The up-regulation of both catabolic and anabolic agents did not appear to positively affect the overall growth of Bgn^-/-Fmod^-/- condyles compared to WT controls. In summary, the up-regulation of both anabolic and catabolic genes in the WT and Bgn^-/-Fmod^-/- TMJs treated with BMP-2 suggests that BMP increases matrix turnover in the condyle, and, further, that Bgn and Fmod could have protective roles in regulating this process.

Complimentary action: C1q increases ganglion cell survival in an in vitro model of retinal degeneration

Using a previously described retinal explant culture system as an acute injury model, we here explore the role of C1q, the initiator of the classical complement pathway, in neuronal cell survival and retinal homeostasis.Full-thickness adult rat retinal explants were divided into four groups, receiving the following supplementation: C1q (50nM), C1-inhibitor (C1-inh; Berinert; 500mg/l), C1q+C1-inh, and no supplementation (culture controls). Explants were kept for 12h or 2days after which they were examined morphologically and with a panel of immunohistochemical markers.C1q supplementation protects ganglion cells from degeneration within the explant in vitro system. This effect is correlated to an attenuated endogenous production of C1q, and a quiesced gliotic response.

Abstract 2608: Dietary polyunsaturated fatty acids modulate adipose inflammation and the adipokine secretome to influence mammary stem cell self-renewal

Abstract Obesity strongly increases risk for multiple malignancies. One potential contributor may be the chronic low-grade inflammation that is likely driven by dysfunctional adipose characterized by aberrant adipokine production and macrophage activation/polarization. In adipose tissue, free fatty acids and their lipid mediators serve as important regulators of adipokine gene transcription. The adipocyte fatty acid composition is influenced by dietary availability. Here we present data demonstrating that dietary fish oil supplementation in obese rats (achieving a 2.5:1 ω6:ω3 PUFA ratio, comparable to a Mediterranean diet) attenuates adipose inflammation characterized reduction in the M1/M2 macrophage polarization ratio compared to a typical Western diet with a 20:1 ω6:ω3 PUFA ratio. We developed a novel adipose explant culture system to directly assay the secretome, and demonstrate that quenching adipose inflammation in obese animals is associated with a favorably rebalanced adipokine milieu, most notably increased adiponectin concentration and decreased leptin secretion. We previously showed that these two adipokines differentially modulate mammary stem cell self-renewal; here, we studied the effect of adipose conditioned media from obese rats on mammary stem cells using the mammosphere formation assay. Adipokines from obese rats fed the high-ω6 diet increased mammary stem cell symmetric self-renewal by 86.7 ± 7.5% (± SEM), whereas adipose-derived factors from the obese animals fed a low-ω6 diet supplemented with fish oil reduced the number of secondary mammospheres by 30.3 ± 10.1% by promoting either apoptosis, quiescence, or symmetric division-differentiation of the primary stem cells. Using a neutralizing antibody and a soluble receptor, we demonstrate that leptin contributes only 19% of the adipose-derived effect on mammary stem cell self-renewal. Taken together, our data suggests that a simple dietary intervention to modify the dietary PUFA ratio is an effective method to quench adipose inflammation and rebalance the adipokine milieu, which may modulate the mammary stem cell pool. If normal stem cells can give rise to breast cancer as has been theorized, then dietary interventions to quench adipose inflammation and reduce the rate of symmetric stem cell self-renewal might protect against the increased incidence of breast cancer seen in obese post-menopausal women. Citation Format: Raymond M. Esper, Evan Hill, Yan Jiang, Nadeem Aslam, Becky Simon, Max Wicha, William Smith, Dean E. Brenner. Dietary polyunsaturated fatty acids modulate adipose inflammation and the adipokine secretome to influence mammary stem cell self-renewal. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2608.

607. Organotypic Culture of Adult Porcine Retina as an In Vitro Screening Model for AAV-Mediated Gene Therapy in Ophthalmology

Introduction: Gene therapy studies in vivo using animal models provide critical information regarding vector specificity, therapeutic efficacy, and safety. However, long durations to observe transgene expression, high degree of variability between animals, and reduced control of experimental conditions often leads to large sample sizes and expense. Organotypic cultures of the retina can provide an efficient in vitro model with a high degree of throughput and control that can be used for pharmacological screening. These explants preserve complex intercellular processes and communications among the neural retinal layers with minimal manipulation of tissue architecture and molecular pathways, making it a useful model in target-tissue validation of adeno-associated virus (AAV) vector variants. Porcine retina has similar anatomical and physiologic features to the human retina and is therefore a suitable surrogate for preclinical testing in ophthalmology. Methods: We have developed a porcine explant culture system from retinas isolated from adult pigs as a model to screen novel rAAV vectors for transduction efficiency and cellular tropism. Pig eyes were enucleated and the neural retina dissected away from the pigment epithelium within 1-2 hours postmortem. Tissue was cut into 4 mm pieces and placed photoreceptor layer down on polycarbonate transwells, and cultured in Neurobasal-A medium supplemented with B-27. Viability of the explant cultures was examined over time by gross morphology, quantification of glucose-6-phosphate dehydrogenase release from dying cells, and TUNEL assay. Transductions with AAV vectors were performed on retinal explants on the day the cultures were started, and transgene expression was analyzed by live cell imaging and immunofluorescence (IF) staining, or ELISA from tissue supernatants. Results: Retinal explants were viable for up to 4 weeks and IF staining of retinal protein markers including β-III-tubulin, Chx10, glutamine synthetase, rhodopsin and L/M opsin suggested that most retinal cell types were preserved in vitro, with tissue architecture closely resembling that of intact in vivo retina. Transduction with different rAAV serotypes encoding GFP displayed protein expression at 3 days, with gradually rising levels during the first 2 weeks following transduction. Furthermore, IF staining using retinal cell markers revealed differential cellular transgene expression from the AAV vectors encoding various cell-specific or ubiquitous promoters under investigation. Finally, high levels of secreted soluble VEGFR-1 protein expressed from rAAV were detected from explant culture media samples with expression increasing over a period of 3 weeks. Conclusions: Our results demonstrate viability of retinal explants for up to 4 weeks and efficient rAAV transduction with various serotypes. Our organotypic porcine retinal explant model facilitates the evaluation of efficacy, cellular tropism, and promoter selectivity of rAAV vectors in a fast, reproducible, and economical manner, contributing to a reduction in the use of animals for the development of therapies in ophthalmology.

N-terminal Dentin Sialoprotein fragment induces type I collagen production and upregulates dentinogenesis marker expression in osteoblasts

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly462-Asp463, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.

Characterisation of the lymphoid stress surveillance response in human intestine

BackgroundThe human intestine harbours an immense repertoire of T cells, many having proved difficult to isolate and characterise. The lymphoid stress surveillance response (LSSR), whereby unconventional T cells respond to epithelial dysregulation in synchrony with innate myeloid cells, was first described in mice. We investigated the potential for LSSR in human intestine, and whether this response could contribute to gut inflammation. MethodsWe obtained colonic biopsy samples at endoscopy from healthy donors. We applied a novel explant culture system (from one described in human skin) that permits outgrowth of large numbers of highly viable T cells, to complete flow-cytometric phenotyping and functional studies. To validate our method we compared T cell isolation methods. FindingsValidation of our method demonstrated that cell yield was twice as high as with conventionally isolated cells. γδ T cells comprised a substantial, underappreciated proportion of total intestinal lymphocytes (mean 9·5%, SD 9). Gut γδ T cells expressed a tissue-resident memory phenotype, including the activating receptor NKG2D. As previously demonstrated by us in human skin, specific cytokines licensed such cells to make purely innate-like cytotoxic responses, both to recombinant NKG2D ligands and to colonic tumour cell lines. Responses to colonic tumour cell lines were abrogated by antibodies that block NKG2D, demonstrating common functional potentials at distinct epithelial surfaces. InterpretationThe study shows that the human intestine harbours a large number of tissue-resident γδ T cells that can make rapid, innate-like responses to epithelial cell dysregulation, akin to those shown in our laboratory both in vivo for their mouse counterparts, and in skin. We can now investigate the contribution of LSSR to inflammatory gastrointestinal diseases characterised by tissue dysregulation and the upregulation of stress ligands. FundingFrancis-Crick Institute - National Institute for Health Research Biomedical Research Centre (Clinical Research Training Fellowship), Wellcome Trust.