Download 0718 - a novel temperature-sensitive immortalized human adult

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ABSTRACT NO. 1203
PAPER NO.
Corresponding Author:
Mary
First Name
Goldring
Last Name
Presenting Author:
Mary
First Name
Goldring
Last Name
0718
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Cartilage Biology
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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