Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

The cancer­stromal interaction has been demonstrated to promote tumor progression, and cancer-associated fibroblasts (CAFs), which are the main components of stromal cells, have attracted attention as novel treatment targets. Chitinase 3-like 1 (CHI3L1) is a chitinase-like protein, which affects cell proliferation and angiogenesis. However, the mechanisms through which cells secrete CHI3L1 and through which CHI3L1 mediates tumor progression in the cancer microenvironment are still unclear. Accordingly, the present study assessed the secretion of CHI3L1 in the microenvironment of colorectal cancer and evaluated how CHI3L1 affects tumor angiogenesis. CAFs and normal fibroblasts (NFs) established from colorectal cancer tissue, and human colon cancer cell lines were evaluated using immunostaining, cytokine antibody array, RNA interference, reverse transcription-quantitative PCR (RT-qPCR), ELISA, western blotting and angiogenesis assays. The expression and secretion of CHI3L1 in CAFs were stronger than those in NFs and colorectal cancer cell lines. In addition, interleukin-13 receptor α2 (IL-13Rα2), a receptor for CHI3L1, was not expressed in colorectal cancer cell lines, but was expressed in fibroblasts, particularly CAFs. Furthermore, the expression and secretion of IL-8 in CAFs was stronger than that in NFs and cancer cell lines, and recombinant CHI3L1 addition increased IL-8 expression in CAFs, whereas knockdown of CHI3L1 suppressed IL-8 expression. Furthermore, IL-13Rα2 knockdown suppressed the enhancement of IL-8 expression induced by CHI3L1 treatment in CAFs. For vascular endothelial growth factor-A (VEGFA), similar results to IL-8 were observed in an ELISA for comparison of secretion between CAFs and NFs and for changes in secretion after CHI3L1 treatment in CAFs; however, no significant differences were observed for changes in expression after CHI3L1 treatment or IL-13Rα2 knockdown in CAFs assessed using RT-qPCR assays. Angiogenesis assays revealed that tube formation in vascular endothelial cells was suppressed by conditioned medium from CAFs with the addition of human CHI3L1 neutralizing antibodies compared with control IgG, and also suppressed by conditioned medium from CAFs transfected with CHI3L1, IL-8 or VEGFA small interfering RNA compared with negative control small interfering RNA. Overall, the present findings indicated that CHI3L1 secreted from CAFs acted on CAFs to increase the secretion of IL-8, thereby affecting tumor angiogenesis in colorectal cancer.

New Stable Cell Lines Derived from the Proximal and Distal Intestine of Rainbow Trout (Oncorhynchus mykiss) Retain Several Properties Observed In Vivo.

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line.

Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing 'quick hit' experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results demonstrated that specific growth rates (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to medium exchange after 48h prior to reseeding at 72h (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (route A1); whereby SGR values were (0.021±0.004) and (0.019±0.004). Viability was higher in route B2 over 10 passages with average viability reported as (86.3%±8.1) compared to route A1 (83.3±8.8). The metabolite data demonstrated both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had consistent-specific metabolite rates (SMR) for glucose, but SMR values of route B1 was consistently lower than route B2 (0.00001 mmol, cell-1.d-1 and 0.000025).Results revealed interactions between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve culture for therapeutic use, biologics production, and reference lines.

Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers.

During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells.

Establishment and characterization of a cell line derived from fin of the endangered Yangtze sturgeon (Acipenser dabryanus).

Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.

Synthesis of a far-red emitting flavonoid-based lysosome marker for live cell imaging applications.

A bright far-red emitting flavonoid derivative (FuraET) was synthesized in good yields by inserting a π extension group (i.e., furan) into the flavonoid skeleton, via using the Suzuki-Miyaura cross-coupling reaction. FuaraET exhibited optical absorption at λab ≈ 450 nm and emission λem ≈ 660 nm by recognizing as the first far-red emitting flavonoid derivative reported. FuraET exhibited a large Stokes shift (Δλ > 150 nm) high fluorescent quantum yield (φfl ≈ 0.2-0.4), and good photostability indicating excellent characteristics for an imaging probe. Live cell fluorescent confocal microscopy imaging revealed the exceptional selectivity of the FuraET towards cellular lysosomes (Mander's overlap coefficients >0.9). The observed non-alkalinizing nature and high biocompatibility (LC50 > 50 µM) suggested that FuraET can a reliable lysosome marker for live cell imaging experiments. Our further study also indicated that FuraET may likely internalized into hydrophobic regions of the cellular lysosomes in contrast to acidic lysosomal lumen.

Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field.

Insect cell lines are used to study cellular interactions and gene functions in vitro in several research areas. However, suitable cell lines for experiments are not always available, especially in non-model species. Here, we established novel cell lines derived from fat bodies of six lepidopteran insects: Cydia kurokoi (named NARO-Cyku), Cephonodes hylas (NARO-Cehy), Haritalodes basipunctalis (NARO-Haba), Theretra oldenlandiae (NARO-Thol), Lymantria dispar (NARO-Lydi), and Hyphantria cunea (NARO-Hycu) collected in the field. The larval fat body was a promising tissue for the starting material when samples were limited due to field collection. It was critical that the medium volume was kept to a minimum for primary culture to maintain adherence of the fat body cells to the flask. The flask was coated with poly-L-lysine for effective induction of adherence and cell division. The identities of cell lines were confirmed using DNA barcoding with the mitochondrial cytochrome c oxidase I gene after cultures were passaged over 50 times. All lines except for NARO-Lydi and NARO-Hycu are adherent cells, and population doubling time of six cell lines ranged from 1.03 to 2.49. Induction of gene expression was practicable in the four adherent cell lines as revealed by transfection of expression vectors and found the immediate early 2 and the Bombyx actin 3 were effective gene promoters. The results suggest that these cell lines are capable of gene functional analysis. Thus, establishments of cell line using our methods for non-model lepidopterans could make a practical contribution to pest management and insect utilization.

Multiplexed clonality verification of cell lines for protein biologic production.

During the development of cell lines for therapeutic protein production, a vector harboring a product transgene is integrated into the genome. To ensure production stability and consistent product quality, single-cell cloning is then performed. Since cells derived from the same parental clone have the same transgene integration locus, the identity of the integration site can also be used to verify the clonality of a production cell line. In this study, we present a high-throughput pipeline for clonality verification through integration site analysis. Sequence capture of genomic fragments that contain both vector and host cell genome sequences was used followed by next-generation sequencing to sequence the relevant vector-genome junctions. A Python algorithm was then developed for integration site identification and validated using a cell line with known integration sites. Using this system, we identified the integration sites of the host vector for 31 clonal cell lines from five independent vector integration events while using one set of probes against common features of the host vector for transgene integration. Cell lines from the same lineage had common integration sites, and they were distinct from unrelated cell lines. The integration sites obtained for each clone as part of the analysis may also be used for clone selection, as the sites can have a profound effect on the transgene's transcript level and the stability of the resulting cell line. This method thus provides a rapid system for integration site identification and clonality verification.

Antioxidant Activities of an Exopolysaccharide (DeinoPol) Produced by the Extreme Radiation-Resistant Bacterium Deinococcus radiodurans.

Deinococcus radiodurans shows extreme resistance to a range of remarkable environmental stresses. Deinococcal exopolysaccharide (DeinoPol) is a component of the cell wall, but its role in stress resistance has not yet been well-described. In this study, we isolated and characterized DeinoPol from Deinococcus radiodurans R1 strain and investigated its application as an antioxidant agent. Bioinformatic analysis indicated that dra0033, encoding an ExoP-like protein, was involved in DeinoPol biosynthesis, and dra0033 mutation significantly decreased survival rates in response to stresses. Purified DeinoPol consists of different monosaccharides and has a molecular weight of approximately 80 to 100 kDa. DeinoPol also demonstrates highly protective effects on human keratinocytes in response to stress-induced apoptosis by effectively scavenging ROS. Taken together, these findings indicate that DeinoPol is the first reported deinococcal exopolysaccharide that might be used in cosmetics and pharmaceuticals as a safe and attractive radical scavenger.

1-O-alkyl glycerophosphate-induced CD36 expression drives oxidative stress in microglial cells.

Microglia, the tissue-resident macrophages in the central nervous system, are important for the initiation and perpetuation of neuroinflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-inducible transcription factor and plays an important role in fatty acid metabolism. Our previous study found that 1-O-alkyl glycerophosphate (AGP), a naturally occurring ether analog of lysophosphatidic acid, is a high-affinity, partial agonist of PPARγ. In this study, we investigated the role of AGP in microglial activation and illustrated the underlying molecular mechanism. We found that AGP treatment increased the production of intracellular reactive oxygen species and induced PPARγ activation in microglial cells. Interestingly, AGP also up-regulated the expression levels of the cluster of differentiation 36 (CD36) scavenger receptor, a high-affinity receptor for oxidized low-density lipoproteins. The findings suggest that AGP induces PPARγ activation, enhances CD36 expression and increases the production of intracellular reactive oxygen species (ROS) in microglial cells.

Generation of Rybp homozygous knockout murine ES cell line GIBHe001-A-1 by using CRISPR/Cas9 technology.

RYBP (Ring1 and YY1 Binding Protein) is critical for pluripotency and differentiation of embryonic stem cells (ESCs). RYBP depletion disturbs both neural and myocardial differentiation of ESCs. Moreover, low level of RYBP is correlated with diseases such as glioblastoma. To study the biological function of RYBP in neural differentiation of ESCs, here we generated Rybp homozygous knockout murine ESC line based on Sox1-GFP reporter using CRISPR/Cas9 genome editing technology. The last two exons of Rybp gene in which contain 115 amino acids have been replaced with PGK-Pruo by homologous recombination.

Generation of a PDX1-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-3, using the CRISPR/Cas9 system.

PDX1 plays a crucial role in the development and maintenance of ß-cells and directly regulates pancreatic ß-cell-specific transcription factors by binding to the insulin gene. Here, we introduced an EGFP reporter into the C-terminus of PDX1 in KSCBi005-A human induced pluripotent stem cells through homologous recombination using CRISPR/Cas9 nuclease. The cells had a normal karyotype, expressed several pluripotency markers, and maintained their differentiation potential. KSCBi005-A-3 cells can be used to monitor PDX1 expression in live cells during ß-cell differentiation; the cell line has been registered at the National Stem Cell Bank, Korea National Institute of Health.

Characterisation of Crandell-Rees Feline Kidney (CRFK) cells as mesenchymal in phenotype.

The Crandell-Rees Feline Kidney Cell (CRFK) is an immortalised cell line derived from the feline kidney that is utilised for the growth of certain vaccinal viruses. Confusion exists as to whether CRFK are epithelial or mesenchymal in phenotype. The aim of this study was to characterise CRFK cells via immunofluorescence, enzyme cytochemistry, western blotting, RT-qPCR for S100A4 and comparison to primary feline proximal tubular epithelial cells (FPTEC) and feline cortical fibroblasts (FCF). CRFK cells were of fusiform morphology and appeared similar to FCF. CRFK expressed the mesenchymal intermediate filament (IF) protein vimentin together with two cell adhesion molecules associated with feline fibroblasts (CD29 and CD44), and lacked expression of the epithelial IF cytokeratin, myogenic IF desmin and endothelial marker von Willebrand factor (vWF). In addition, CRFK did not demonstrate brush border enzyme activity typical of FPTEC. S100A4 gene expression, implicated in both neoplastic transformation and epithelial to mesenchymal transition, was highly upregulated in CRFK in comparison to the primary feline renal cells. CRFK appear phenotypically similar to fibroblasts, rather than tubular epithelial cells, and may have undergone neoplastic transformation or epithelial-to-mesenchymal transition after extensive passaging. This finding may have potential implications for future research utilising this cell line.

Generation of a WWTR1 mutation induced pluripotent stem cell line, MUSIi012-A-1, using CRISPR/Cas9.

WWTR1 or TAZ (WWTR1/TAZ) is a transcriptional coactivator that acts as a downstream regulatory target in the Hippo signaling pathway, which plays a pivotal role in regulating cell proliferation and anti-apoptosis. It has been shown in other cell types that WWTR1/TAZ plays a redundant role to its homolog YAP1. Using CRISPR/Cas9 gene editing, we established the WWTR1/TAZ-KO cell line, which features homozygous deletion of WWTR1 gene from human iPSCs. The established WWTR1/YAZ-KO cell line maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and normal karyotype.

Generation of a knock-in MAP2-tdTomato reporter human embryonic stem cell line with inducible expression of NEUROG2/1 (NYGCe001-A).

Overexpression of NEUROG2 and NEUROG1 (NEUROG2/1) in human embryonic stem cells (hESCs) rapidly produces functional networks of excitatory and inhibitory neurons. To facilitate the use of this efficient inducible human neuron model in neuroscience research, we generated hESCs with doxycycline-inducible NEUROG2/1 via lentivirus and a tdTomato fluorescent reporter knock-in at the MAP2 locus using the CRISPR nuclease Cas9. Upon doxycycline-driven induction of NEUROG2/1, these hESCs differentiate within days into cells that are uniformly MAP2 immunoreactive and tdTomato fluorescent.

Description and Characterization of a Novel Human Mast Cell Line for Scientific Study.

BACKGROUND: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. METHODS: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (ß-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. RESULTS: LADR cells were larger and more granulated as seen with Wright-Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. CONCLUSIONS: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.