Primary Culture System Research Articles

The Condensin II Subunit, NcapH2, Is Required for Proper Embryonic Erythroid Terminal Maturation and Embryonic Viability in a Primary Mouse Model

The process of nuclear condensation is an integral part of erythroid differentiation. We have previously shown that loss of the histone methyltransferase, Setd8, results in severe embryonic (primitive) erythroid anemia due to loss of chromatin condensation and proper erythroid terminal maturation (Malik 2017). Setd8 mediated H4K20 mono-methylation is an important mediator of higher order chromatin condensation and can recruit members of the Condensin II complex (Lau 2015). ondensin I and II complexes are five protein, large complexes responsible for DNA organization and packaging during mitosis. In eukaryotes, the complexes are composed of conserved SMC components, with each Condensin having a unique kleisin bridge protein and two HEAT subunits. Subunits of Condensin II are highly expressed in erythroid cells, suggesting an erythroid specific function. Overexpression of the Condensin II HEAT subunit, Cap-G2, accelerates erythroid differentiation in MEL cells (Xu 2006). To interrogate the role of Condensin II in a primary model, we mated endogenous EpoR driven cre expressing mice with mice containing a floxed allele around Condensin II kleisin subunit, NcapH2. Embryos homozygous for EpoR specific excision of NcapH2 suffered from severe primitive erythroid anemia with visible anemia beginning at embryonic day (E11.5) and lethality occurring at approximately E13.5. Peripheral blood analysis at E11.5 was significant for erythroblasts with enlarged cell size and nuclear size, as well erythroblasts with dysplastic appearing nuclei. Knockout embryos at E12.5 exhibited peripheral blood anemia, with an increase in the number of dysplastic appearing cells, and decreased fetal liver coloration suggesting a failure of both primitive and fetal definitive erythropoiesis. Failure of the embryonic erythroid lineage to expand was confirmed in a primary cell culture system in which loss of proliferation was limited to mid to late stage precursors, suggesting that Condensin II function is critical beyond the proerythroblast stage of maturation when nuclear condensation is occurring and mass spectrometry demonstrates that H4K20 mono-methylation accumulates. Characterization of primitive erythroblasts from knockout embryos showed increased cell size, increased scatter in the size of the nucleus, and a high frequency of cells with punctate nuclear staining. In addition, the subset of erythroid cells with punctate nuclei did not appear to have a nuclear envelope, suggesting a pre-mitotic error, likely during G2 of the cell cycle. Indeed, knockout cells showed a buildup of cells in G0-G1 of the cell cycle, suggesting arrest following a defective cell division. Together, our data suggests that Condensin II is required during erythroid maturation for proper cell cycle progression and chromatin organization, and that chromatin modifications, such H4K20me1, may be a required mechanism to control the higher-level organization of chromatin. This control is required in red cell precursors, as they undergo rapid expansion, maturation, and nuclear condensation simultaneously. Understanding these mechanisms will give valuable insight into the unique sensitivity of red cells to chromatin perturbations observed in bone marrow failure and myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

Using Primary SCG Neuron Cultures to Study Molecular Determinants of HSV-1 Latency and Reactivation.

We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.

Estrogen and EGFR Pathways Regulate Notch Signaling in Opposing Directions for Multi-Ciliogenesis in the Fallopian Tube.

The lumen of the fallopian tube (FT) is lined with columnar epithelium composed of secretory and ciliated cells, both of which are important for reproduction. However, the molecular mechanism regulating cell fate remains controversial. In this study, we established a primary culture system using porcine fallopian tube epithelial cells (FTECs) to study the differentiation mechanism. We found that estrogen promoted the differentiation of multi-ciliated cells (MCCs) through estrogen receptor β, following the reduction of DLL1, a ligand of Notch. Meanwhile, epidermal growth factor (EGF), a regulator of epithelial homeostasis and differentiation, suppressed ciliogenesis by the activation of Notch signaling. However, the estrogen pathway did not affect the activation of the EGF pathway. Taken together, the differentiation of MMCs in FT depends on the balance of EGF and estrogen signaling, either of which inhibits or stimulates the Notch signaling pathway respectively.

VIABILITY OF VERO E6 CELLS EXPOSED TO THE Bothrops leucurus SNAKE CRUDE VENOM

Primary and continuous cell culture systems are a good in vitro alternative for the quantitative assessment of snake venom toxicity. There are still few studies evaluating the cellular response against Bothrops leucurus venom (BlV). This study aimed to evaluate the cytotoxic effect of crude BlV on culture of VERO E6 cells. Neutral Red incorporation (NR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) tests were performed for viable cells, and Trypan Blue (TB) dye exclusion test was performed for non-viable cells. The differences between the groups were evaluated by ANOVA-One-Way and Dunnet through the program Graph Pad 5.0 with significance of 5%. The 50% cytotoxic concentration (CC50) of BlV was 7.86 μg/mL. There was a trend of dose-dependent reduction on cell viability in all assays evaluated. However, MTT and NR tests were more sensitive for the evaluation of BlV cytotoxicity on the VERO E6 cell line.

Pharmacology of Airway Goblet Cell Mucin Release

Airway mucus hypersecretion is one of the major clinical manifestations of patients suffering from various pulmonary diseases. However, no drugs are yet available to control airway mucus hypersecretion. For the past 15 years, a plethora of information has amassed with regard to the pharmacology of airway goblet cell mucin secretion using various primary cell culture systems. The recent discovery of various MUC genes has also greatly stimulated research in this field. Nevertheless, the heterogeneity of the oligosaccharide structure of mucin molecules makes it extremely difficult to assess the significance of the information derived from the pharmacological studies. Therefore, it seems crucially important to understand the roles of individual mucins in conjunction with normal airway physiology and pathology, which should be the future research directions necessary for studying airway goblet cell mucin pharmacology.

Soluble Epoxide Hydrolase Inhibition Attenuates Excitotoxicity Involving 14,15-Epoxyeicosatrienoic Acid-Mediated Astrocytic Survival and Plasticity to Preserve Glutamate Homeostasis.

Astrocytes play pivotal roles in regulating glutamate homeostasis at tripartite synapses. Inhibition of soluble epoxide hydrolase (sEHi) provides neuroprotection by blocking the degradation of 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid mediator whose synthesis can be activated downstream from group 1 metabotropic glutamate receptor (mGluR) signaling in astrocytes. However, it is unclear how sEHi regulates glutamate excitotoxicity. Here, we used three primary rat cortical culture systems, neuron-enriched (NE), astrocyte-enriched glia-neuron mix (GN), and purified astrocytes, to delineate the underlying mechanism by which sEHi and 14,15-EET attenuate excitotoxicity. We found that sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and 14,15-EET both attenuated N-methyl-D-aspartate (NMDA)-induced neurite damage and cell death in GN, not NE, cortical cultures. The anti-excitotoxic effects of 14,15-EET and AUDA were both blocked by the group 1 mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), as were their protective effects against NMDA-disrupted perineuronal astrocyte processes expressing glutamate transporter-1 (GLT-1) and subsequent glutamate uptake. Knockdown of sEH expression also attenuated NMDA neurotoxicity in mGluR5- and GLT-1-dependent manners. The 14,15-EET/AUDA-preserved astroglial integrity was confirmed in glutamate-stimulated primary astrocytes along with the reduction of the c-Jun N-terminal kinase 1 phosphorylation, in which the 14,15-EET effect is mGluR5-dependent. In vivo studies validated that sEHi and genetic deletion of sEH (Ephx2-KO) ameliorated excitotoxic kainic acid-induced seizure, memory impairment, and neuronal loss while preserving GLT-1-expressing perineuronal astrocytes in hippocampal CA3 subregions. These results suggest that 14,15-EET mediates mGluR5-dependent anti-excitotoxicity by protecting astrocytes to maintain glutamate homeostasis, which may account for the beneficial effect of sEH inhibition in excitotoxic brain injury and diseases.

Abstract 33: Ex vivo drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix

Abstract Small cell neuroendocrine carcinoma (SCNEC) of uterine cervix is a rare tumor accounting for approximately 1-2% of uterine cervical tumors. SCNEC of uterine cervix is highly invasive and has an extremely poorer prognosis than other histologic types for which no standard treatment is established. To establish a new management strategy against SCNEC of uterine cervix, we developed a drug sensitivity assay based on a 3D primary culture system. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method from patients’ tumors. To date, we established a panel of 12 SCNEC CTOS lines with the success rate of 100%. For each line, multiple CTOSs were placed in one well of a 96 well plate and dose response analysis was performed for seven anticancer drugs; paclitaxel, carboplatin, irinotecan, SN-38, cisplatin, etoposide and gemcitabine. After 7 days of culture, the CTOSs viability was evaluated by measuring intracellular ATP levels, corrected by the size of CTOS at day0. All drugs had substantial variations in sensitivity among the lines. Cerv54 was far more sensitive to irinotecan than other lines while was similarly sensitive to SN-38. We revealed that cancer cells of cerv54 had high levels of carboxylesterase(CES)1 expression, which converts irinotecan to SN38. Since cerv54 with CES1 knockdown by pGFP-C-shCES1 lentivirus had lower sensitivity than sh-scramble, CES1 expression is related to the irinotecan sensitivity in cerv54. CES1 expression in cancer cells may be useful for predicting the effect of irinotecan based chemotherapy. In addition, this study indicates that sensitivity test using CTOS might be applicable to precision medicine by selecting sensitive drugs for each patient. Citation Format: Mie Tanaka, Satoshi Kubota, Yu Ito, Yumiko Kiyohara, Hiroko Endo, Jumpei Kondo, Satoshi Nakagawa, Akiko Okazawa, Shinya Matsuzaki, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Shoji Kamiura, Tadashi Kimura, Masahiro Inoue. Ex vivo drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 33.

Dendropanax morbiferus leaf extract facilitates oligodendrocyte development.

Treatment of multiple sclerosis is effective when anti-inflammatory, neuroprotective and regenerative strategies are combined. Dendropanax morbiferus (DM) has anti-inflammatory, anti-oxidative properties, which may be beneficial for multiple sclerosis. However, there have been no reports on the effects of DM on myelination, which is critical for regenerative processes. To know whether DM benefits myelination, we checked differentiation and myelination of oligodendrocytes (OLs) in various primary culture systems treated with DM leaf EtOH extracts or control. DM extracts increased the OL membrane size in the mixed glial and pure OL precursor cell (OPC) cultures and changed OL-lineage gene expression patterns in the OPC cultures. Western blot analysis of DM-treated OPC cultures showed upregulation of MBP and phosphorylation of ERK1/2. In myelinating cocultures, DM extracts enhanced OL differentiation, followed by increased axonal contacts and myelin gene upregulations such as Myrf, CNP and PLP. Phytochemical analysis by LC-MS/MS identified multiple components from DM extracts, containing bioactive molecules such as quercetin, cannabidiol, etc. Our results suggest DM extracts enhance OL differentiation, followed by an increase in membrane size and axonal contacts, thereby indicating enhanced myelination. In addition, we found that DM extracts contain multiple bioactive components, warranting further studies in relation to finding effective components for enhancing myelination.

Copper Transport and Disease: What Can We Learn from Organoids?

Many metals have biological functions and play important roles in human health. Copper (Cu) is an essential metal that supports normal cellular physiology. Significant research efforts have focused on identifying the molecules and pathways involved in dietary Cu uptake in the digestive tract. The lack of an adequate in vitro model for assessing Cu transport processes in the gut has led to contradictory data and gaps in our understanding of the mechanisms involved in dietary Cu acquisition. The recent development of organoid technology has provided a tractable model system for assessing the detailed mechanistic processes involved in Cu utilization and transport in the context of nutrition. Enteroid (intestinal epithelial organoid)-based studies have identified new links between intestinal Cu metabolism and dietary fat processing. Evidence for a metabolic coupling between the dietary uptake of Cu and uptake of fat (which were previously thought to be independent) is a new and exciting finding that highlights the utility of these three-dimensional primary culture systems. This review has three goals: (a) to critically discuss the roles of key Cu transport enzymes in dietary Cu uptake; (b) to assess the use, utility, and limitations of organoid technology in research into nutritional Cu transport and Cu-based diseases; and (c) to highlight emerging connections between nutritional Cu homeostasis and fat metabolism.

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications.

Recent research has shown that the maintenance of relevant liver functions ex vivo requires models in which the cells exhibit an in vivo-like phenotype, often achieved by reconstitution of appropriate cellular interactions. Multiple different models have been presented that differ in the cells utilized, media, and culture conditions. Furthermore, several technologically different approaches have been presented including bioreactors, chips, and plate-based systems in fluidic or static media constituting of chemically diverse materials. Using such models, the ability to predict drug metabolism, drug toxicity, and liver functionality have increased tremendously as compared to conventional in vitro models in which cells are cultured as 2D monolayers. Here, the authors highlight important considerations for microphysiological systems for primary hepatocyte culture, review current culture paradigms, and discuss their opportunities for studies of drug metabolism, hepatotoxicity, liver biology, and disease.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on acetylcholinesterase during myogenic differentiation of contractile rat primary skeletal muscle cells

Emerging data indicate that prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could interfere with myogenic differentiation in vivo. Acetylcholinesterase (EC3.1.1.7; AChE), an enzyme critical for cholinergic neurotransmission, is abundantly expressed in neurons and mature myotubes, and we recently found that muscle AChE expression was suppressed in parallel with the inhibition of myogenic differentiation upon TCDD treatment in mouse C2C12 cells. This TCDD-induced suppression of muscle AChE was proposed to involve an aryl hydrocarbon receptor (AhR)-independent mechanism, but the precise underlying mechanism remains unclear. Considering the widely recognized role of muscular activity in AChE expression and its potential crosstalk with the AhR signaling pathway, we sought to investigate the effect of TCDD on muscle AChE expression in the presence of muscular activity. Therefore, we employed a highly contractile rat primary skeletal muscle culture system in which AChE activity and the expression of genes related to it (AChE T subunit and collagen Q (ColQ)) were increased during the myogenic differentiation process. Although TCDD treatment successfully induced the expression of genes regulated by AhR activation, the treatment exerted no notable effects on myogenic differentiation. Moreover, muscle AChE enzymatic activity and mRNA level remained unchanged following TCDD treatment, and only ColQ mRNA expression was slightly increased after 4-day treatment with TCDD (10−10 M). The compensatory role of muscle-contraction-related signaling pathways in this newly identified unresponsiveness of muscle AChE to TCDD warrants further investigation.

Primary cell culture of adult zebrafish spinal neurons for electrophysiological studies

BackgroundZebrafish (Danio rerio) are growing in popularity as a vertebrate model organism for the study of spinal neurocircuitry and locomotion. While many studies have used the zebrafish model system for electrophysiological analyses in embryonic and larval stages, there is a growing interest in studying spinal circuits and neurons from adult fish. New MethodTo expand upon the existing toolset available to the zebrafish research community, we have developed the first primary cell culture system of adult zebrafish spinal neurons. The intact spinal cord is dissected, and neurons are isolated through enzymatic digestion and mechanical dissociation. Identifiable neurons are viable for electrophysiological analyses after two days in culture. ResultsSpinal neurons in culture were confirmed by immunofluorescence labeling and found to exhibit distinct morphologies from other cell types, allowing neurons to be identified based on morphology alone. Neurons were suitable for calcium imaging and whole cell patch clamp recordings, which revealed excitable cells with voltage-gated whole cell currents, including tetrodotoxin-sensitive sodium currents. Comparison with Existing MethodsThis primary cell culture system is the only methodology available to isolate neurons from the adult zebrafish spinal cord. Other methods rely on keeping the spinal cord intact or the utilization of embryonic or larval stage fish. This method provides a robust platform for use in neurophysiological and pharmacological studies. ConclusionsThe novel primary cell culture system described here provides the first in vitro methodology available to isolate and culture neurons from the adult zebrafish spinal cord for use in electrophysiological analyses.

Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model

The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. We used a primary tissue culture system to generate a murine p53 null gastric tissue model containing both epithelium and mesenchymal stroma. To characterize the microenvironment and niche signaling, we used single cell RNA sequencing (scRNA-Seq) to determine the transcriptomes of 4,391 individual cells. Based on specific markers, we identified epithelial cells, fibroblasts and macrophages in initial tissue explants during organoid formation. The majority of macrophages were polarized towards wound healing and tumor promotion M2-type. During the course of time, the organoids maintained both epithelial and fibroblast lineages with the features of immature mouse gastric stomach. We detected a subset of cells in both lineages expressing Lgr5, one of the stem cell markers. We examined the lineage-specific Wnt signaling activation, and identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that this primary tissue culture system enables one to study gastric tissue niche signaling and immune response in vitro.

Secreting oviduct epithelial cells of Coturnix coturnix japonica (QOEC) and changes to their proteome after nonviral transfection.

The quail oviduct (Coturnix c. japonica) is a natural candidate avian bioreactor, while the secretive quail oviduct epithelial cells (QOECs) are potential in vitro producers of recombinant proteins and vaccines. In view of the need for highly performing and transformable cell lines, QOEC may potentially act as an alternative bioreactor platform to the existing ones, for example, to the Chinese hamster ovary. The aim of this work was to characterize QOECs and their response to nucleofection with a nonviral plasmid DNA carrying the human interferon-α 2a gene (hIFNλ2a), in vitro. Primary QOEC cultures from laying quails (10-15 weeks old) were characterized by their proliferation rate, doubling time, and multilineage differentiation. Electroporation to cell nuclei (nucleofection) was used to deliver nonviral plasmid DNA containing a reporter GFP and hIFN under the ovalbumin promoter. The posttransfection analysis included polymerase chain reaction, Western blot analysis, and liquid chromatography coupled to tandem mass spectrometry. QOEC showed a typical epithelial characteristic in a primary 2D monolayer culture system and retained secretive potential up to the first passage. QOEC showed differentiation into osteoblastic lineage after stimulation. The nucleofection mean efficiency was low (2.3%). Differences of up to 10% in the proteomic profiles between nontransfected and transfected QOEC were found, the most important of these were related to the absence of keratins and cell-adhesion proteins in the transfected QOEC. Concluding, with the practical information provided here, QOEC have the potential to serve as an avian secreting cellular platform. QOEC may be further transformed to cell lineage to meet the requirement for a stable, electrocompetent, and transfectable model. The first proteomic comparison of QOEC delivered in this study showed, in the majority, a stable proteome of the nontransfected vs transfected QOEC.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Methylation Changes of Primary Tumors, Monolayer, and Spheroid Tissue Culture Environments in Malignant Melanoma and Breast Carcinoma.

Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for PAX5, TMPRSS2, and SBDS. These findings will play an important role in developing in vitro model system to understand the effect of methylation inhibitors on primary tumor tissue. In a previous study PAX5, TMPRSS2, and SBDS genes that we are investigating were reported to be methylated more than 60% in breast cancer and malignant melanoma cell lines. However, these genes have never been studied in primary tumor tissues. Thus, primary tumor tissues of breast cancer and malignant melanoma were first grown in 2D and 3D cultures. Then these two types of tumor tissues and their 2D and 3D cultures were investigated for changes considering methylation levels in PAX5, TMPRSS2, and SBDS genes using real-time polymerase chain reaction. No differences were observed in the primary tissues and culture systems for both PAX5 and TMPRSS2 in malignant melanoma tissues. We found that PAX5 gene was an efficient marker to measure the effects of methylation inhibitors for in vitro systems for malignant melanoma tissue.

Establishment of primary human epithelial ovarian cancer cells and their application to cytotoxicity assessment

Background: Ovarian carcinomas are highly metastatic tumors and the most lethal gynecological malignancies in the world. Treatment of human epithelial ovarian cancer (hEOC) is based on the combination of surgery and chemotherapy. The acquisition of resistance is a major obstacle to the clinical use of platinum drugs for 75% of high-grade serous ovarian cancer treatment. Hence, the urgent strategy is to develop platform to predict the resistance to targeted therapy after surgery and to screen the potential monotherapy, including platinum, in clinical treatment. Materials and Methods: Our laboratory tried to take advantage of ascites from hEOC patients to understand the mechanism of platinum-based resistance and screen the potential monotherapy choice via the primary culture strategy. We first established the culture conditions for ascites of the ovarian cancer patients. Results: We showed that we could culture these primary cells up to 13th generation. We further treated these primary cells with various steroid hormones for the epithelial-to-mesenchymal transient and regular monotherapy, including cisplatin, docetaxel, and doxorubicin, for the cytotoxicity. Finally, we also demonstrated the responsiveness of this monotherapy with antibodies against cell cycle, autophagy, or other survival-related factors. Conclusion: Our screening platform will provide us to further examine the resistance mechanism of any monotherapy in a different individual patient. We hope that our primary culture system used for standard monotherapy screening might provide the alert to drug resistance and the better combinatory therapy for personalized therapy.

A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

Transcriptomic Analysis of Cholestatic Compounds In Vitro.

Drug-induced cholestasis is one of the most severe manifestations of drug-induced liver injury. Drug-induced cholestasis is characterized by an accumulation of endogenous metabolites normally excreted in the bile such as bile salts, cholesterol, bilirubin, or drug metabolites. The possibility to determine early in the drug development process whether a compound presents a risk of inducing drug-induced cholestasis is key information. Since preclinical repeated dose toxicity studies have limited predictive value, large efforts in identifying alternative in vitro models with improved prediction are being made. One of the best current models for in vitro human liver is primary human hepatocytes, and we recently reported that primary human hepatocytes can be kept as long-term cultures in 2D-sandwich configuration when regularly renewing the Matrigel overlay, thereby making the model useful for repeat exposure-related toxicities, as well as for the study of adaptive responses. This primary human hepatocyte culture system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased drug-induced cholestasis risk.This chapter describes the various steps for culturing and exposing primary human hepatocytes to drugs during long-term 2D-sandwich culture, performing RNA extraction, gene chip assay and selecting hepatotoxic signature using the IPA software and highlighting genes involved in bile acid homeostasis.

Suppression of parotid acinar cell dysfunction by the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one.

Salivary gland atrophy and consequent hyposalivation are serious problems in clinical dentistry, as saliva regulates the environment of the oral cavity. To clarify the mechanisms underlying salivary gland dysfunction, a system for primary culture of parotid acinar cells has been established. It has been reported previously that the process of cell isolation from parotid glands triggers stress signaling mediated by Src and p38 mitogen-activated protein (MAP) kinase (p38), leading to dedifferentiation of acinar cells, and that an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor suppresses this activation of Src and p38, suggesting that reactive oxygen species initiate the dedifferentiation signal. The present study examined the effect of a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (also termed MCI-186 or edaravone), on activation of the stress signal and the secretory function of parotid acinar cells. Activation of p38 during cell isolation was suppressed by addition of MCI-186. The retention of the activity of amylase, a major salivary protein, and the number of amylase-containing secretory granules were improved by isolation and culture in the presence of MCI-186. In addition, calcium elevation upon stimulation with a muscarinic agonist was higher in MCI-186-treated cells than in untreated cells. These results suggest that MCI-186 (edaravone) is a promising agent for prevention of salivary gland dysfunction.