Use Of Stable Cell Lines Research Articles

Stable human cartilage progenitor cell line stimulates healing of meniscal tears and attenuates post-traumatic osteoarthritis.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Abstract 2401: Oncogenic H-Ras is a key regulator of death receptor-induced apoptosis

Abstract Ras oncogenes, including H-Ras, K-Ras and N-Ras, are widely implicated in tumorigenesis. Oncogenic K-Ras mutations are found in about 30% of human tumors rendering constitutive activation of Ras-dependent pathways to promote cell proliferation, survival, and transformation. By contrast, H-Ras mutations are rarely detected; however, its expression levels are often elevated in cancer cells. This study aims to identify the molecular pathways by which H-Ras exerts its oncogenic activity. Our previous data drew a link between H-Ras and apoptosis via death receptors (DRs), particularly DR4 and DR5 that are known to induce apoptosis upon ligation of their cognate ligand namely Tumor Necrosis Factor-related apoptosis inducing ligand (TRAIL). We showed that wild-type H-Ras was overexpressed in several human cancer cell lines that were resistant to TRAIL (Chen et al, Oncotarget, 2014). In this study, we tested H-Ras expression and its relationship with cellular susceptibility to TRAIL in a large panel of human cancer cell lines and xenograft tumor models. This included the use of stable cell lines that were developed via transfection of doxycycline-inducible sh-RNA against H-Ras transcript. These cancer cell lines were used to detect surface expression of DR4 and DR5 as well as TRAIL-induced apoptosis. A xenograft mouse model was developed by injecting the TRAIL-resistant H-Ras mutant cells into athymic mice. The mice were fed with water containing doxycycline, which effectively induced sh-RNA mediated reduction of H-Ras protein in the xenografts, and were subjected to intratumor injection with TRAIL, and immunohistochemistry analysis. Our results show that wild-type H-Ras GTPase is upregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. The elevated H-Ras expression correlated with a loss of DR4 and DR5 receptor expression on the plasma membrane in TRAIL-resistant cells. Knockdown of H-Ras in TRAIL-resistant cells restored the surface expression of DR4 and DR5, and subsequently increased apoptosis in response to TRAIL treatment. The xenograft mice developed tumors upon injection of the H-Ras mutant cells. Knockdown of H-Ras and TRAIL injection showed an increase of apoptotic markers in the tumor. This study confirms our previous report that high expression levels of H-Ras, while is found in its wild-type state, protect cancer cells from TRAIL induced apoptosis. As a TNF-related cytokine, TRAIL has been implicated in tumor immune surveillance by inducing apoptosis in target cells. H-Ras appears to suppress surface expression of DR4 and DR5, thereby promoting cancer cell survival through evasion from the immune surveillance mechanisms. These data have potential implications in identifying novel therapeutic targets for the development of effective cancer therapy. Citation Format: Su-Ryun Kim, Ancy D. Nalli, Baolin Zhang. Oncogenic H-Ras is a key regulator of death receptor-induced apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2401.

Characterisation and Gene Expression Profiling of a Stable Cell Line Expressing a Cell Cycle GFP Sensor

The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has increased significantly in recent years. In this study we have useda range of complimentary analytical techniques to examine the characteristicsof a cell line stably expressing a EGFP cell cycle sensor relative to parentalU2OS cells. Analysis of cell cycle duration and cell cycle phase distribution bycell growth assays and flow cytometry revealed that the two cell lines hadidentical doubling times and cell cycle distributions. Measurement of EGFPfusion protein mRNA by quantitative RT-PCR indicated a EGFP sensorexpression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2).Microarray analysis showed a 0.9% (>2 fold at p

Oxygen Sensing by Human Recombinant K+ Channels: Assessment of the Use of Stable Cell Lines

This chapter assesses the use of stable cell lines for oxygen sensing by human recombinant K + channels. Central to the cardiorespiratory responses to reduced O 2 availability of all chemosensory tissue is the rapid inhibition of ion channels by hypoxia. Acute hypoxic modulation of K + channel activity is central to chemosensing in the carotid body, neuroepithelial body, and its immortalized cellular counterpart (H146cells). In this study, the impact that recombinant human K + channel expression systems have on the understanding of acute O 2 chemotransduction by native and model chemosensory tissues is discussed. Different aspects related to generating stable cell lines, including the choice of cell line, cell culture, choice of vector, ligation of cloned channels into pcDNA3.1-TOPO, transfection and selection, verification of expression by RT-PCR, verification of expression by immunocytochemistry, and functional screening using the patch-clamp technique are discussed. . Homomultimeric O 2 -sensitive human K + channels stably expressed in HEK293 cells and alternative approaches to the study of heteromultimeric O 2 -sensitive human K + channels are also described.

Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells.

Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1alpha (EF-1alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.