ATCC Research

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Characterization of a Three-Dimensional (3D) Organotypic Skin Model using Keratinocytes and Mesenchymal Stem Cells Immortalized by hTERT

Poster presented at the ASCB Meeting, December 2014.

Abstract: In this study, we compared primary keratinocytes (ATCC® No. PCS-200-010) to hTERT immortalized keratinocytes (Ker-CT; ATCC® No. CRL-4048™), co-cultured with either primary fibroblasts (ATCC® No. PCS-201-010), primary adipose-derived mesenchymal stem cells (MSCs; ATCC® No. PCS-500-011), hTERT-immortalized fibroblasts (BJ-5ta; ATCC® No. CRL-4001™), or hTERT-immortalized MSCs (hTERT-MSCs; ATCC® No. SCRC-4000™). We confirmed that both primary keratinocytes and Ker-CT are able to fully differentiate into skin equivalents in a 3D culture model when co-cultured with primary fibroblasts, primary MSCs, BJ-5ta, or hTERT-MSCs. To confirm the functionality of the co-culture models, both the primary keratinocytes and the Ker-CT air-liquid interface (ALI) co-cultures were subjected to a scratch assay. Re-epithelialization occurred in both cell lines, and interleukin 8 (IL-8) showed an increase in expression from day 0 to day 1 and 3, corresponding to migration of cells into the wound. The continuous nature of the Ker-CT cell line makes it an invaluable model for the research of keratinocyte biology, as it eliminates the issue of short life span and donor variation seen with primary cells.

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Development and Characterization of an in vitro Co-culture Angiogenesis Assay System Using hTERT Immortalized Cells for High Throughput Drug Screening

Poster presented at the ASCB Meeting, December 2014.

Abstract: In this study, we established an in vitro co-culture model system using the aortic endothelial cell line TeloHAEC (hTERT immortalized human aortic endothelial cell line, ATCC® No. CRL-4052™) and the hTERT immortalized adipose-derived mesenchymal stem cell line hTERT-MSC (ATCC® No. SCRC-4000™). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts; TeloHAECs co-cultured with BJ primary fibroblasts (ATCC® No. CRL-2522™) for 14 days in optimized ATCC angiogenesis medium formed fine tubular structures as shown by staining with CD31 endothelial cell marker. The tubule length elongated with increasing doses of vascular endothelial growth factor (VEGF), and tubule formation can be completely blocked by suramin in a concentration-dependent manner. Next, we introduced GFP into the TeloHAEC cell line (TeloHAEC-GFP, ATCC® No. CRL-4054™), allowing for real time visualization of angiogenesis when co-cultured with BJ fibroblasts; another hTERT immortalized cell line, hTERT-MSC, replaced the BJ-primary fibroblast in the co-culturing system. It was observed that the new model forms the tubular structures in less than 7 days, and also responds effectively to VEGF and compounds such as suramin. Further, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by positive αSMA staining (a marker of smooth muscle cells), this indicates that the system has physiological relevance. Therefore, the co-culture models developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying vascular biology, drug screening and tissue engineering.

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Cell Line Genomic DNAs for the Molecular Diagnosis of Cancer 

Poster presented at the AMP Meeting, November 2014.

Abstract: Large-scale cancer genome programs have generated a rich data set comprising genetic abnormalities observed in thousands of clinical patient tumors, which provides a major opportunity for the molecular detection of cancer. However, the lack of controls for molecular tests has been a challenge. Because of the reproducible nature of the cell lines, genomic DNAs of fully characterized and authenticated cell lines provide a solution.

Genomic DNAs were extracted from over 70 commonly used human cancer cell lines derived from the breast, lung, colon, and pancreas, as well as hematopoietic and lymphoid tissue. Cancer gene mutations were identified by next-generation sequencing. Gene copy number changes were analyzed using the qBiomarker Copy Number PCR Assays kit (QIAGEN). Moreover, the selected cell lines were analyzed by quantitative polymerase chain reaction (qPCR), Western blot, and immunofluorescence (IF) staining to verify gene and protein expression mutation.

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Evaluation of NanoLuc and GFP Reporter-labeled Control Strains for Shiga toxin-producing Escherichia coli (STEC) Assays 

Poster presented at the IAFP Meeting, August 2014.

Abstract: Positive controls are essential for establishing assay performance and equipment efficacy. Yet, some food testing laboratories refrain from using bacterial strains as positive controls for fear of cross-contaminating their samples. Under the Food and Drug Administration (FDA) Food Safety Modernization Act, laboratories face an increasing number of regulations to expand testing for objectionable organisms. Control strains with unique, easily detectable traits which distinguish positive control strains from actual food contaminants can help differentiate true contamination from control strain cross-contamination.

In this study, we introduced shuttle vectors encoding either green fluorescent protein (GFP, Life Technologies) or NanoLuc® (Promega) into Escherichia coli strains, including Shiga toxin-producing O157 (stx1+, stx2+, eaeA+) and non-Shiga toxin-producing O157 (stx1-, stx2-, eaeA-), for use in food pathogen detection assays. Both reporters can be easily visualized without specialized detection equipment; GFP fluoresces when excited by UV light, while bacteria engineered with NanoLuc emit a strong light signal in the presence of a chemical substrate. Upon establishing the detectability of NanoLuc in the E. coli O157 strains, the reporter was transformed into the “Big Six” non-O157 E. coli strains (serogroups: O26, O45, O103, O111, O121, and O145) for use as reporter-labeled positive controls.

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Novel Fluorescent Reporters for Studying Pathogen-host Interactions

Poster presented at the asm2014 General Meeting, May 2014.

Abstract: Fluorescent proteins, such as green fluorescent protein (GFP), have diverse applications in the basic and applied sciences. While GFP has been frequently used in eukaryotic systems, its applications have been limited in microorganisms due to a lack of broad-range molecular tools. In this study, we have developed a vector to express GFP in pathogenic bacteria for use in bacterial pathogenesis and pathogen-host studies. A shuttle vector encoding the GFP variant mut31 (pUCP18-MCSgfpmut3) was generated and successfully transformed into various Gram-negative opportunistic pathogens from the ATCC collection, including: Escherichia coli (ATCC® 25922™), Salmonella enterica (ATCC® 14028™), Shigella flexneri (ATCC® 12022™), Pseudomonas aeruginosa (ATCC® 10145™), and the P. aeruginosa type strain PAO1 (ATCC® 15692™). P. aeruginosa was used as a model to test the characteristics of the vector and sensitivity of detection using a fluorescence plate reader, microscopy, flow cytometry, and in vivo imaging systems.

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Development and Verification of Synthetic RNA Controls for Determination of Influenza Virus Load

Poster presented at the asm2014 General Meeting, May 2014.

Abstract: Influenza is one of the most significant causes of acute respiratory infection worldwide. Rapid diagnostic tests for highly contagious pathogens, such as Influenza, are essential for decreasing the public health impact of emerging infectious diseases and bioterrorism agents. However, these tests require positive controls that are not always readily available. Consequently, if worldwide public health laboratories are unable to meet the costly regulations required for the import, transfer, and safe use of pathogens used as controls, then critical diagnostic, surveillance, and epidemiological information could be missed.

The use of in vitro synthesized viral RNA as a control would provide essential equivalency standards that would be accessible to any laboratory performing quantitative RT-PCR tests. Synthetic RNA controls are particularly useful for laboratories which lack appropriate biosafety containment facilities for propagating a particular pathogenic virus, or have difficulty gaining access to the organism in question due to international tightening of both import and export controls.

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Development of Synthetic Molecular Standards for Dengue Virus

Poster presented at the Clinical Virology Symposium (CSV) meeting, April 2014.

Abstract: Dengue fever is an acute illness caused by any one of four serotypes (1-4) of genetically related dengue viruses (DENV), with an estimated 390 million cases reported annually. Currently, quantitative RT-PCR (qRT-PCR) is the preferred method for the detection and quantification of DENV in clinical diagnostics and epidemiological surveillance. The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known viral genome concentration.

Native DENV RNA can be used as a standard for these assays; however, the full-length dengue viral RNA is on the Commerce Control List and requires a permit from the US Department of Commerce for international shipment. To make DENV RNA standards more accessible, ATCC has developed four synthetic molecular standards that represent DENV serotypes 1-4. Each standard contains short fragments from the capsid, membrane, and envelope genes of the DENV genome, as well as target regions encompassing the primer sequences from numerous published RT-PCR assays, including the DENV-1-4 Real-Time RT-PCR Assay developed by the CDC1. The synthetic RNA standards were quantified by Droplet Digital™ PCR (ddPCR™) in order to package precise copies of RNA. Moreover, given the inherent labile nature of RNA, a stabilization matrix was added to the quantitated RNA preparation. As compared to native RNA, these synthetic standards are easier to use as controls for qRT-PCR assays, exhibit less variability, have a longer shelf life, eliminate the need to culture viruses and can be used under BSL-1 conditions. Further, this synthetic quantitative RNA approach can be extended to other pathogenic viruses which are unculturable or need to be grown in a high-containment facility. 

In the following proof-of-concept study, we amplified the synthetic molecular standards with the published primers from the CDC assay1 and Waggoner et al2, and used the generated standard curves to quantify viral RNA extracted from various DENV strains.

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Whole Exome Sequencing Reveals 228 Conserved Mutations in Parental and Three LRRK2 Parkinson's Patient-derived iPSC Lines

Poster presented at the ISSCR Annual Meeting, June 2013.

Abstract: Patient-specific induced pluripotent stem cells (iPSCs) provide a unique tool for the study of human diseases such as Parkinson’s disease. To provide a better research tool for studying Parkinson’s disease, we generated three iPSC lines, from dermal fibroblasts of a 63 year old Caucasian male, diagnosed with Parkinson’s disease, by reprogramming with sendai viral, retroviral, or episomal expression of OCT3/4, SOX2, KLF4, and MYC genes. The Parkinson iPSC lines generated with different reprogramming methods all demonstrated similar cell morphology, pluripotent marker expression, and the ability to differentiate into three germ layers. Compared to an hiPSC line-derived from a healthy subject, these Parkinson’s iPSC lines showed similar efficiency of neural differentiation into neural progenitors from iPSC-derived embryoid bodies. To more effectively model Parkinson disease, we have sequenced all exons of the three Parkinson iPSC lines along with their parent fibroblast by exome sequencing with an Agilent’s SureSelect 51 Mb array. Compared to the hg19 human genome reference, each cell line has over 300 genes with missense mutations and there are 226 genes with missense mutations conserved among all four cell types. More importantly, there are three amino acid changes within the LRRK2 gene, the most common Parkinson’s disease-related gene, at positions 50 (R50H), 723 (I723V), and 2397 (M2397T), which have previously been reported in Parkinson’s patients. Via integrating and non-integrating reprogramming methods, we have created three fully characterized iPSC lines that carry LRRK2 mutations.

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Tumor Cell Panels: New Tools in Genomic Era

Poster presented at the American Association of Cancer Research Annual Meeting in Washington, DC, April 2013.

Abstract: Increased understanding of cancer genome is affecting every corner of cancer research. Although human tumor cell lines have been used as essential tools for decades, there are only a few cell line panels have been developed for the drug screening. There is a gap between the new knowledge of cancer genome and the cell line based platforms for both basic and translational research. Here, we show that new generation tumor cell panels are filling the gap. The panels were generated by selecting authenticated cell lines derived from variant cancer types, and annotated with genetic alteration information generated by large scale sequencing projects such as the Catalog of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE). To capture the genetic diversity of cancer, each panel includes cell lines with varying gene mutation complexity. To further facilitate targeted drug discovery, the molecular signature tumor cell line panels focus on individual driver genes, critical protein kinases, transcription factors and cell signaling pathways. Those panels have been analyzed to verify gene mutation, gene expression, protein expression and bio-functions.

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The Value of ATCC Biological and Molecular Standards in Assay Development

Presented at the AMP Annual Meeting, November 2014.

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ATCC Vendor Showcase Presentation at ASCB 2013

Presentation at The New Orleans Ernest N. Morial Convention Center, ASCB, December 2013.

hTERT Immortalized Cells — Unique tools for tissue-relevant research

ATCC Human telomerase (hTERT) immortalized cell lines combine the in vivo nature of primary cells and the long culture life of continuous cell lines. This section will provide an overview of the hTERT-immortalized cell line collection, and will examine the use of RPTEC/TERT1 (ATCC® No. CRL-4031™) and TIME (ATCC® No. CRL-4025™) cell lines to demonstrate how hTERT-immortalized cell lines can help you reach your research goals.

Induced Pluripotent and Mesenchymal Stem Cells — Cells with a lot of potential

ATCC has a wide selection of induced pluripotent and mesenchymal stem cells along with an array of associated culture media and reagents. This section will provide an overview of the ATCC stem cell collection and describe how these resources can be used in cell biology studies.

Transfex — for hard to transfect cells

ATCC offers a superior lipid-based transfection reagent (TransfeX) that can be used to transfect difficult cell types, like primary and stem cells, and uncomplicated continuous cell lines. In this section, we will show how TransfeX provides higher transfection efficiency and lower cytotoxicity than other commercially available transfection reagents. We will also describe the HEKPlus Expression System for protein expression.

ATCC Molecular Signature Panels — Powerful tools for the genomics age

ATCC molecular signature panels focus on key components of cell signaling pathways such as EGFR, AKT, PI3K, PTEN, or p53. This section will describe how we generated these panels using authenticated cell lines containing critical gene copy number changes and site mutations, as well as how each panel was experimentally validated for genetic alterations, protein expression, and cell functionality.

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Development of the PI3K Pathway Inhibitors: How to choose the right cell line

Presented at the Discovery on Target meeting, October 2012.

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iPS Cell Repository for Human Tissue and Disease Models presentation

Presented at the ISSCR meeting, June 2011.

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