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MAC ABSTRACT NO. 1203 PAPER NO. Corresponding Author: Mary First Name Goldring Last Name Presenting Author: Mary First Name Goldring Last Name 0718 I prefer a Poster Presentation Please consider for New Investigator Recognition Awards Please consider for the American Geriatrics Society Award REQUIRED - Supply first keyword from one of these lists: Cartilage Biology Supply 4 remaining keywords from the list in the Instructions: None Articular Chondrocyte Collagen Type II Cytokines Proteoglycans A NOVEL TEMPERATURE-SENSITIVE IMMORTALIZED HUMAN ADULT ARTICULAR CHONDROCYTE LINE *+Goldring, M.B., *Tan, L., *Choy, B., *Robbins, J.R , *New England Baptist Bone & Joint Institute, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115: (617)667-0742, FAX (617)975-5299, [email protected] Introduction: The limited repair response by chondrocytes accounts for a major component of the loss of articular cartilage in osteoarthritis. While development of therapies for osteoarthritis primarily have addressed prevention of cartilage matrix degradation, recent work has focused on strategies for promoting cartilage repair. The validation of autologous chondrocyte transplantation for the repair of advanced cartilage lesions in older adults will require relevant and reproducible models for determining how to promote a fully regenerative chondrocyte phenotype in vitro that would be maintained after implantation in vivo. Since articular cartilage is the primary joint tissue requiring replacement or reconstruction after damage in arthritic conditions, we have developed an immortalized human adult articular chondrocyte line. Methods: Human adult articular chondrocytes at day 4 of primary culture were immortalized using a retrovirus packaged in the amphotropic PA317 cell line and encoding a temperature-sensitive mutant of SV40-large T antigen (TAg) and NeoR. After selection in G418 for 4 weeks and culture for >60 passages in DMEM/Ham’s F12 medium containing 10% FCS, the T/AC62 cell line was established with stable proliferative capacity in monolayer culture at the permissive temperature (32°C; doubling time ~2 days). Total RNA was extracted using Trizol (Gibco-BRL) and mRNAs were analyzed by RT-PCR. For analysis of proteoglycans, cultures were incubated with [35S]sodium sulfate in the presence of ascorbic acid during the final 18 h of culture. Proteoglycans in conditioned medium and cells were extracted separately, purified by DEAE cellulose chromatography, and separated by SDS-PAGE on 4 - 20% gradient gels. biglycan and decorin were also detected by Western blotting. Clonal lines of T/AC62 cells stably transfected with regulatory sequences of the type II collagen gene (COL2A1) in pGL3-luciferase reporter gene constructs have been established to correlate changes in expression with differentiated phenotype in monolayer and alginate cultures and to examine transcriptional responses to cytokines and growth factors. MONOLAYER T/AC62 32°C ±IL-1β β - + ALGINATE 37°C 39°C - - + + 32°C - + 39°C - + Collagen II -359 bp Aggrecan -350 bp Biglycan -356 bp Decorin -302 bp GAPDH -346 bp Fig. 1. Comparison of matrix protein mRNAs in monolayer and alginate cultures of T/AC62 cells at permissive and nonpermissive temperatures. Monolayer 32°C 39°C C M C M Alginate 32°C 39°C C M C M Aggrecan- Results and Discussion: Transfer to 39°C resulted in loss of TAg detected in cell extracts by Western blotting and complete inhibition of cell proliferation. After continuous monolayer culture at 32°C for 10 to 12 passages, passage through a 10-day alginate culture reestablished full phenotypic expression. In alginate culture, the T/AC62 cells were capable of depositing abundant alcian bluestainable matrix even at 32°C and clumps of cells appeared suggesting that they continued to proliferate in suspension. Transfer to 39°C immediately after encapsulation resulted in single cell suspensions with decreased DNA content and matrix gene expression compared to alginate cultures maintained at 32°C for several days prior to transfer to 39°C. Type II collagen and aggrecan mRNAs were expressed equally well in 10-day monolayer and 21-day alginate cultures at 32°C and 37°C, correlated with TGF-β and IGF-I mRNA expression, and were increased by BMP-2 or -4 and decreased by IL1β (Fig. 1) in 24 - 48 h incubations. IL-1β also increased expression of MMP-1, MMP-3, and MMP-13 mRNAs, but had no effect on the high levels of TIMP-1 mRNA. Analysis of proteoglycans demonstrated preferential retention of aggrecan in the cell-associated matrix in alginate cultures and an increased ratio of biglycan to decorin secreted into the culture medium in both monolayer and alginate cultures after switch to 39°C (Fig. 2). Collagen II, aggrecan, -206 kD Biglycan- -125 kD Decorin- Fig. 2. Characterization of proteo[35S)glycans. Availability of a cell line that reproduces the adult articular chondrocyte phenotype will permit identification of signals that enhance differentiation in vitro and elucidation of rational strategies for improving cartilage repair using chondrocyte transplantation approaches. Acknowledgements: Supported by Arthritis Foundation Biomedical Science Grant and NIH R01 AR45378. One or more of the authors have received something of value from a commercial or other party related directly or indirectly to the subject of my presentation. The authors have not received anything of value from a commercial or other party related directly or indirectly to the subject of my presentation. 45th Annual Meeting, Orthopaedic Research Society, February 1-4, 1999, Anaheim, California 718