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Abstract: In vitro angiogenesis models are crucial to the study of blood vessel growth. In this study an assay-ready, pre-mixed co-culture of human telomerase-immortalized (hTERT) human aortic endothelial cells that express green fluorescent protein and hTERT mesenchymal stem cells produce a vascular network reminiscent of in vivo angiogenesis.

Characterization of hTERT-immortalized Prostate-derived Stromal and Epithelial Cells

An Authentic in vitro Model for Tumor Microenvironment Studies

4/1/2017 — 4/5/2017

Abstract: Tumor development begins with mutational changes to the genetic makeup of a cell; tumor progression is not solely determined by the mutated cell, but also by the tumor's microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalized by(human telomerase reverse transcriptase (hTERT) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT-immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.

Abstract: In this study we report the development of a “universal” CellMatrix Basement Membrane ECM that provides a suitable microenvironment to support a wide variety of cell biology applications, including: 3D organoids; spheroid formation; angiogenic vessel formation; “sandwich” culture of primary human hepatocytes; and the routine culture of iPSCs and neural progenitor cells.

Abstract: Growing concern over bacterial food contamination has led to increased examination of food testing protocols in today’s industry. Currently, the use of bacterial strains as positive controls in testing protocols is not widely practiced for fear of cross-contaminating samples. Due to ongoing scrutiny of food testing methodology and growing regulations under the Food and Drug Administration (FDA) Food Safety Modernization Act, it is imperative to have control strains with unique, easily detectable traits that distinguish positive control strains from actual food contaminants, diminishing the fear of cross-contamination and improving current practices. In this study, we developed GFP reporter-labeled Shiga toxin-producing Escherichia coli for use as controls in QC testing.

Abstract: Herpes simplex virus (HSV-1 and HSV-2) causes a wide range of clinical manifestations that result in lifelong infections. Quantitative PCR (qPCR) assays are routinely used for the detection of HSV-1 and HSV-2 infections in clinical samples. However, the accuracy of a qPCR assay is dependent upon the generation of a standard curve using a positive control with a known genome copy number. Moreover, an independent positive control is required to monitor variations in assay performance for molecular assays. ATCC has developed HSV-1 and HSV-2 quantitative molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Abstract: Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) is a major health concern and affects millions of people worldwide. Patients are routinely monitored by quantitative RT-PCR (qRT-PCR) for the presence of HCV RNA or by qPCR for HBV DNA in blood. Since these viruses are difficult to culture in vitro, obtaining control material for these molecular-based assays is a challenge. To address this problem, ATCC has developed HBV and HCV specific quantitative synthetic molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Abstract: Recent studies show that tumor cells derived from a subset of patients with non-small-cell lung cancer (NSCLC) harbor the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene; the result of a Paracentric chromosomal inversion on the short arm of chromosome 2. The EML4-ALK oncogene, like other ALK fusion oncogenes, is a druggable target that is responsive to ALK inhibitors. However, there is a lack of EML4-ALK in vitro models for drug screening. Here we set out to generate an isogenic EML4-ALK fusion non-small cell lung cancer model in the A549 lung cancer cell line, which harbors other naturally occurring genomic aberrations inherent in non-small cell lung cancer. This model could serve as a clinically relevant drug screening cell model. In this study, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genomic translocation event. By employing sgRNAs-Cas9 constructs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant genomic rearrangements that resulted in the expression of EML4–ALK fusions. Breakpoint junction analysis tested after sgRNA-CRISPR/Cas9 mediated genomic DNA cleavage in A549 cells showed the successful creation of the EML4-ALK fusion found in tumor cells from a subpopulation of NSCLC patients. Furthermore, single clonal isolation and functional screening demonstrated that the EML4-ALK isogenic cell line was sensitive to ALK inhibitors relative to the parental A549 cell line. This newly developed EML4-ALK isogenic lung cancer cell line could provide a very useful tool for oncology drug discovery and development.

Abstract: Human induced pluripotent stem cells (iPSCs) have the capacity to differentiate into all of the somatic cells types and hold great promise for both regenerative medicine and drug discovery. A need for better tools to address neurological disease modeling and neuro-toxicology screening exists. We have developed a scalable process that allows for the generation of large quantities of neural progenitor cells (NPCs) derived from normal and Parkinson’s disease iPSC lines, along with a serum-free defined NPC expansion medium and dopaminergic differentiation medium. To validate the process of NPC derivation, we generated NPCs derived from iPSCs that were reprogrammed with the Sendai virus from the following sources: human foreskin fibroblasts (HFF-1), human CD34+ cells, and human fibroblasts from a patient with Parkinson’s disease. Compared to Parkinson’s disease patient-derived NPCs, both normal NPC lines demonstrated greater proliferative capacity. Moreover, our CD34+ cell-derived NPCs possessed better tri-lineage differentiation efficiency than that of fibroblast-derived NPCs although all three types of NPCs were capable of differentiating into dopaminergic neurons, astrocytes, and oligodendrocytes.

Abstract: In this study we characterize the functionality of an hTERT immortalized adipose tissue-derived MSC cell line (hTERT-MSC) and its use in two co-culturing applications: wound healing and angiogenesis. We confirmed that primary keratinocytes and an hTERT-immortalized keratinocyte cell line are able to fully differentiate into skin equivalents in an air-liquid interface 3D culture model when co-cultured with hTERT-MSCs. We also established an in vitro angiogenesis co-culture model system using TeloHAEC-GFP and hTERT-MSCs. The model can form tubules in less than 7 days and responds effectively to VEGF stimulation and drug treatments. The co-culture models developed by using hTERT-MSCs in this report provide a more consistent and robust in vitro co-culture system for studying wound healing and vascular biology for drug screening and tissue engineering

Abstract: Noroviruses (NoV) are the most common cause of epidemic gastroenteritis, accounting for 95% of viral gastroenteritis outbreaks worldwide. NoV detection is difficult because they are genetically heterogeneous and cannot be grown in cell culture. The principle detection method utilized by diagnostic laboratories is quantitative RT-PCR (qRT-PCR). The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known genome copy number. To that end, we have developed quantitative synthetic standards that include conserved sequences from NoV GI and NoV GII for the detection and quantification of NoV from either clinical, food, or environmental samples. These quantitative synthetic molecular standards provide well-characterized reference materials for the detection and quantification of NoV by qRT-PCR. Further, they exhibit excellent compatibility with numerous published NoV assays and can be used as controls for assay development, verification, and validation.