Increased life span of human osteoarthritic chondrocytes by exogenous expression of telomerase

Abstract

Objective. To extend the life span of human osteoarthritic (OA) articular chondrocytes by introduction of the catalytic component of human telomerase while preserving the chondrocyte-specific phenotype.Methods. Human articular chondrocytes were isolated from the femoral head and tibial plateau of patients undergoing knee joint replacement for OA. The chondrocytes were cultured as monolayers and infected with a retroviral telomerase expression construct followed by selection with G418 for 10-14 days. Telomericrepeat amplification protocol assays and telomere terminal restriction fragment length assays were performed on pools of transduced cells in order to measure telomerase activity and telomere length. Growth kinetics and population doubling capacity were assessed by passaging the cells in monolayer culture. Redifferentiation of the monolayer chondrocyte cultures was induced by transfer to suspension culture on poly-(2-hydroxyethyl-methacrylate) (polyHEMA)-coated dishes. Induction of the chondrocyte-specific phenotype was monitored by analysis of gene expression utilizing reverse transcription-polymerase chain reaction.Results. OA chondrocytes isolated from 3 different donors (ages 41, 69, and 75 years) were transduced with a retroviral construct expressing telomerase. After selection, pooled populations of cells from all donors and a clonal cell line from 1 donor expressed telomerase activity and exhibited lengthening of telomeres. Chondrocytes expressing telomerase showed an increase of 5-9 population doublings over 234 days of culture in monolayer. The telomerase-transduced cells recovered a chondrocyte-specific gene expression pattern following culture on polyHEMA-coated dishes.Conclusion. The exogenous expression of telomerase may represent a way to expand human OA chondrocytes while allowing maintenance of the chondrocytespecific phenotype. These cells have the potential to be used for restoration of the articular cartilage defects occurring in this disease.

Gene-based approaches for the repair of articular cartilage

“…In this regard, IGF-1 gene transfer was also found to render chondrocyte cultures resistant to dedifferentiation in monolayer, enabling maintenance of the chondrocytic phenotype for at least 28 days in culture. 59 Similar results have been observed following transfer of human telomerase 60 and connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) into chondrocyte cultures. 61 From accumulating reports in the literature, it appears that the act of genetic modification either virally or nonvirally does not, in and of itself, adversely affect the physiology of the chondrocyte.…”

Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

“…Even though application of these stimuli was capable of containing cartilage degradation, it was not sufficient to compensate for the loss of matrix elements and cells and to reestablish an original cartilage surface. Growth, transcription and enzymatic factors are potent candidates to achieve these goals because of their anabolic or mitogenic properties such as fibroblast growth factor 2 (FGF-2) (20,21), bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4) (22,23), transforming growth factor β (TGF-β) (22,24,25), transcription factor sex-determining region Y-type high mobility group box 9 (SOX9) (20,26,27), glucuronosyltransferase-I (28), bcl-2 (29) and telomerase (30). A critical agent that may have the strongest value to readjust the disturbed homeostasis in OA cartilage is insulinlike growth factor (IGF)-I, since it has the ability to influence concomitantly metabolic and proliferative processes, affording protection against extracellular matrix degradation in horse and rabbit articular cartilage explant cultures experimentally treated with proinflammatory cytokines (13,14,22).…”

“…In contrast with vectors derived from adenoviruses (6,10,13,14,(16)(17)(18)(19)21,22,25) and retroviruses (7,11,12,23,26,30) or with nonviral compounds (8,15,28,29), systems based on the replication-defective, nonpathogenic human adeno-associated virus (AAV) may provide better tools for OA, since recombinant AAV (rAAV) can deliver genes in nondividing cells such as chondrocytes both in vitro and in situ in their dense extracellular matrix at high efficiencies and for extended periods of time (20,24,27,31). Also, removal of the viral protein coding sequences in rAAV make them less immunogenic than adenoviral vectors characterized by shortterm transgene expression levels (32).…”

Mesenchymal Stem Cell and Nucleus Pulposus Cell Coculture Modulates Cell Profile

“…MSCs exhibit a stable phenotype and telomerase activity in culture [27]. Proliferation activity and telomere length of chondrocytes reportedly reduce with donor [35] and ex vivo expansion [24,26].…”

“…Also, coculture of NPCs and notochordal cells increases proteoglycan synthesis in the cells [1]. More recently, MSCs were cultured from adult bone marrow and reportedly contributed to bone, cartilage, adipose, and ligament regeneration [27,32]. In other studies, MSCs injected into the IVD survived and proliferated in situ [10,30].…”