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

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 3, March 2002, pp 683–693
DOI 10.1002/art.10116
© 2002, American College of Rheumatology
Increased Life Span of Human Osteoarthritic Chondrocytes by
Exogenous Expression of Telomerase
Sonsoles Piera-Velazquez, Sergio A. Jimenez, and David G. Stokes
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.
A number of current therapeutic protocols for
correction of focal cartilage defects and possible future
treatment of degenerative joint diseases such as osteoarthritis (OA) involve the expansion of autologous chondrocytes followed by reimplantation of these cells into
cartilage defects, their injection into affected joints, or
their utilization for engineering of replacement tissue ex
vivo (1,2). All of these protocols require an expansion
phase of chondrocytes in culture in order to obtain
sufficient cells to implement treatment. However, it is
well known that primary mammalian cells in culture
have a finite replicative life span and eventually enter a
state of senescence in which they remain metabolically
active but cease to proliferate (3). Furthermore, the
mitotic potential of primary cells in culture is dependent
on the age of the donor, with cells from older individuals
exhibiting a lower proliferative life span (3). Normal
adult chondrocytes also possess a limited mitotic potential and inevitably enter a state of replicative senescence
in which cellular proliferation ceases (4–6).
It has been demonstrated that chondrocytes from
various species show a relationship between the number
of population doublings achieved in vitro and the life
span of the organism (5). Recent work has shown that
both the proliferative activity and the telomere length of
human articular chondrocytes decrease with the age of
the donor (6). Indeed, chondrocytes from articular cartilage exhibit a number of age-related changes in their
Supported in part by a grant from Geron Corporation. Dr.
Stokes’ work was supported by NIH grant AG-19962-01. Dr. PieraVelazquez is the recipient of an Arthritis Foundation postdoctoral
fellowship.
Sonsoles Piera-Velazquez, PhD, Sergio A. Jimenez, MD,
David G. Stokes, PhD: Thomas Jefferson University, Philadelphia,
Pennsylvania.
Address correspondence and reprint requests to David G.
Stokes, PhD, Thomas Jefferson University, Division of Rheumatology,
233 S. 10th Street, Room 509 BLSB, Philadelphia, PA 19107-5541.
E-mail: David.G.Stokes@mail.tju.edu.
Submitted for publication August 7, 2001; accepted in revised
form October 12, 2001.
683
684
phenotype. Among these changes are decreased response to growth factors such as transforming growth
factor ␤ (TGF␤) and insulin-like growth factor 1, increased apoptosis, and decreased extracellular matrix
production (7,8). Compounding the latter events are the
phenotypic changes in chondrocytes that occur during
OA, a disease that has a high correlation with age (9,10).
These alterations, collectively, place a limit on the
usefulness of autologous chondrocytes isolated from
aged OA joints in the therapeutic strategies mentioned
above.
Numerous studies have shown that shortening of
telomere length, which occurs during each cell division,
is probably a signal for cellular senescence, since cells in
which telomere length has been shortened to a critical
level fail to undergo further mitotic events (3,11–13).
The enzyme telomerase is a reverse transcriptase that
solves the problem of DNA end-replication, and therefore affects telomere shortening due to the inability of
eukaryotic DNA polymerases to completely replicate
the ends of linear chromosomes (14,15). Telomerase
catalyzes the synthesis of telomeres which, in the case of
humans, are repeat sequences of ⬃1,000 copies of the
short guanine-rich sequence motif TTAGGG, located at
the ends of chromosomes (for review, see ref. 16).
Human telomerase is a ribonucleoprotein consisting of a
reverse transcriptase (hTERT or hTRT) protein subunit
and an RNA template subunit known as TER or hTR
(17,18). The hTR RNA subunit encodes ⬃1.5 telomeric
repeats, and thereby acts as a template for the hTERT
reverse transcriptase to add new telomeric sequences
onto the chromosome ends. However, most human
somatic cells no longer express telomerase and it is
believed that the absence of this activity is a major
contributing factor to their finite replicative life span
(19,20).
Recently, the life span of certain human somatic
cell types (skin fibroblasts, endothelial cells, and retinal
pigment epithelial cells) has been extended in culture by
the ectopic expression of the human telomerase reverse
transcriptase protein (21–25). The immortalized cells
maintained their differentiated phenotype and did not
acquire transformed or malignant characteristics
(23,24). Interestingly, the extension of the life span of
skin fibroblasts and retinal pigment epithelial cells by
introduction of telomerase did not require any of the
other components of the telomere-lengthening machinery such as telomere binding proteins or the RNA
template component (21,22,25). These results imply that
the life span of a wide variety of somatic cells can be
extended simply by the ectopic expression of telomerase,
PIERA-VELAZQUEZ ET AL
and support the telomere hypothesis of cellular aging
which proposes that most somatic cells become senescent because of progressive shortening of their telomeres with each cell division.
Although the effects of expression of exogenous
telomerase have been investigated in a number of human somatic cell types, there are no studies to date that
have examined these effects in human articular chondrocytes. Given the importance of extending the proliferative capacity and delaying the occurrence of senescence in articular chondrocytes, we undertook studies to
express the human telomerase protein subunit in OA
articular cartilage chondrocytes by utilizing a retroviral
complementary DNA (cDNA) expression construct.
Herein, we report the successful transfer of this cDNA
into human OA articular chondrocytes, and demonstrate
that expression of the enzyme in these cells results in an
extension of their telomere lengths and an increase in
their replicative capacity in culture, without altering
their chondrocyte-specific phenotype.
PATIENTS AND METHODS
Isolation and culture of chondrocytes. OA articular
cartilage was obtained from patients undergoing knee replacement surgery in the Department of Orthopedic Surgery at
Thomas Jefferson University Hospital. All tissues were procured following protocols reviewed and approved by the
Institutional Review Committee in accordance with the National Organ Transplant Act and the Pennsylvania Organ
Transplant Act. OA chondrocytes were obtained from 5 patients, a 60-year-old man (C203), a 57-year-old woman (C208),
a 75-year-old woman (C209), a 69-year-old man (C223), and a
41-year-old man (C222).
Articular cartilage was removed by careful dissection
and precautions were taken to avoid inclusion of the subchondral bone. Chondrocytes were isolated from these tissues with
the use of previously described procedures (26). Briefly, chondrocytes from OA human cartilage were obtained by excising
the remaining macroscopically healthy cartilage and then
mincing the tissue into small pieces with a razor blade. To
remove adherent fibrous tissues, the cartilage was incubated in
Dulbecco’s minimum essential medium (DMEM) containing 2
mg/ml of trypsin and bacterial collagenase (Worthington Biochemical, Lakewood, NJ) for 1 hour at 37°C. The medium was
discarded and the tissue fragments were digested overnight at
37°C in DMEM containing 10% fetal bovine serum (FBS) and
0.5 mg/ml of bacterial collagenase. The cells released by the
enzymatic digestion were filtered through a 70␮ nylon filter
and collected by centrifugation at 250g for 5 minutes, and then
resuspended and washed 4 times with collagenase-free medium.
Chondrocytes were then cultured in plastic tissueculture flasks to allow their expansion as dedifferentiated cells.
The cultures were maintained at 37°C in a 5% CO2 humidified
atmosphere in DMEM containing 10% FBS, 2 mM
EXOGENOUS EXPRESSION OF TELOMERASE FOR EXPANSION OF OA CHONDROCYTES
100 units/ml penicillin, 100 ␮g/ml streptomycin,
1% (volume/volume) vitamin supplement, 2.5 ␮g/ml Fungizone (Life Technologies, Carlsbad, CA), and 50 ␮g/ml ascorbic
acid. The medium was replaced every 3–4 days and the cells
were passaged at 7–10-day intervals. The monolayers were
subjected to enzymatic dissociation with trypsin at each passage and the cell numbers were determined by counting with a
hemocytometer. To allow redifferentiation, the monolayers
were dissociated as described above and aliquots of the cell
suspensions were cultured for 7 days at a density of 5–10 ⫻ 106
cells in 60-mm petri dishes that had been previously coated
with a 0.9% solution of poly-(2-hydroxyethyl-methacrylate)
(polyHEMA) prepared as previously described (26).
The experiments concerning the effect of basic fibroblast growth factor (bFGF) on chondrocyte proliferation were
performed using 3 monolayer cultures of the C203 cell line
cultured in the absence of bFGF or with either 5 ng/ml or 10
ng/ml bFGF (final concentrations). The medium was replaced
every 3–4 days and the cells were passaged at 7-day intervals.
Construction of a human telomerase retroviral vector
and infection and selection of chondrocytes. The human
telomerase cDNA was provided by Geron Corporation (Menlo
Park, CA). The telomerase retroviral expression plasmid was
constructed by inserting the human telomerase cDNA into the
pLNCX vector from Clontech (Palo Alto, CA). To obtain
retroviral vector particles containing the telomerase construct
or control vector (pLNCX alone), standard protocols were
used (27). Briefly, 15 ␮g of control vector plasmid or telomerase construct was transfected into the amphotropic PT67
packaging cell line (Clontech) using the Profection calcium
phosphate kit from Promega (Madison, WI). The cells were
then selected by culturing them for 10 days in the presence of
350 ␮g/ml of G418. Retrovirus was pooled from producing
cells derived from the stably transfected PT67 cells. The
supernatants were filtered through a 0.45-␮m filter and were
mixed with DMEM and 8 ␮g/ml polybrene (Sigma, St. Louis,
MO) to infect chondrocytes cultured as monolayers.
The chondrocytes were infected for 1 day, selected for
10 days with 350 ␮g/ml G418, and then expanded and assayed
for telomerase activity. In each case, chondrocytes were infected with pLNCX-telomerase retrovirus (referred to as
hTERT chondrocytes) or with pLNCX retrovirus as a control
(referred to as pLNCX chondrocytes). One hTERT- and 1
pLNCX-transduced clonal cell line was established from sample C222 by limiting dilution to 0.5–1 cells/well in 96-well plates
containing 200 ␮l medium/well. Medium was changed every
3–4 days until confluence, at which time the cell clones were
expanded.
Detection of telomerase activity by telomeric-repeat
amplification protocol (TRAP) assay. Telomerase activity was
detected with the use of the standard TRAPeze protocol
(Intergen, Purchase, NY) and the assay was performed according to the manufacturer’s instructions. Briefly, using aliquots
corresponding to 1 ⫻ 103 cells, polymerase chain reaction
(PCR) was carried out with specific primers for the
telomerase-extended product, using Taq polymerase. An aliquot equal to one-half volume (500 cells) of the PCR product
was electrophoresed on 10% polyacrylamide gels and the PCR
products were detected by staining with SYBR Green (BioWhittaker Molecular Applications, Rockland, ME) and a
FluorImager (Molecular Dynamics, Sunnyvale, CA). Relative
L-glutamine,
685
levels of activity were obtained by comparison with the activity
in control HeLa cells.
Assessment of telomere length by Southern analysis.
Genomic DNA was extracted from the cells transfected with
either the telomerase retroviral vector or with the empty vector
using the DNeasy tissue system (Qiagen, Valencia, CA).
Genomic DNA (3 ␮g) was digested with Hinf I and Rsa I and
electrophoresed on 0.6% or 0.7% agarose gels and transferred
onto a nylon membrane by capillary blotting. The membrane
was then hybridized with a 32P end-labeled telomere probe,
(CCCTAA)3, utilizing a standard protocol, and exposed to a
PhosphorImager screen (Molecular Dynamics) for 24 hours.
Images were scanned by using ImageQuant software (Molecular Dynamics) and mean telomere lengths were calculated
using the method of Harley et al (11).
Assessment of chondrocyte proliferation and morphology. Every 7–10 days, the cultures were subjected to enzymatic
dissociation with trypsin and the cell numbers were determined
by counting an aliquot of the resulting suspension in a hemocytometer. The population doublings were calculated with the
following equation: PD ⫽ Log10 (N/N0) ⫻ 3.33, where N is the
number of cells at the end and N0 is the number of cells at the
beginning of the experiment (28).
The morphology of pLNCX and hTERT chondrocytes
was examined by phase-contrast microscopy. Photomicrographs were obtained of cells that were cultured as monolayers
for various passages or following their redifferentiation by
culture on polyHEMA-coated dishes for 7 days.
Analysis of chondrocyte gene expression patterns. The
pattern of expression of genes encoding types I and II collagen
and aggrecan was examined by reverse transcription (RT)–
PCR analysis. Cells from either monolayer or polyHEMA
cultures were harvested and total RNA extracted using the
TRIzol reagent (Life Technologies). RT-PCR amplification of
target messenger RNA (mRNA) was performed with 1 ␮g of
RNA by using the Titan One-tube RT-PCR system (Roche,
Indianapolis, IN). PCR was performed using specific primers
for human GAPDH (5⬘-GCCAGTGGACTCCACGACGT-3⬘
and 5⬘-ATGGGGAAGGTGAAGGTCGG-3⬘), type I collagen
(5⬘-TAAAGGGTCACCGTGGCT-3⬘ and 5⬘-CGAACCACATTGGCATCA-3⬘), type II collagen (5⬘-AACAACCAGATTGAGAGC-3⬘ and 5⬘-TTACAAGAAGCAGACCGG-3⬘), and
aggrecan (5⬘-AGCATCCACCCAGGTCT-3⬘ and 5⬘-AGAGAAGATTCTGGGTC-3⬘). The products were electrophoresed
on 1% agarose gels stained with SYBR Green I. The reaction
products were visualized with a FluorImager (Molecular Dynamics). Images were quantitated by using ImageQuant software (Molecular Dynamics) and the signals were normalized
to those of GAPDH.
RESULTS
Establishment of culture conditions for optimal
proliferation of human adult OA chondrocytes. To
establish the optimal culture conditions for chondrocyte
growth that would permit an appropriate level of retroviral infection and allow for subsequent selection, freshly
isolated OA chondrocytes were cultured as monolayers
in tissue-culture flasks. However, since human OA chon-
686
Figure 1. Effect of basic fibroblast growth factor (bFGF) on osteoarthritic (OA) chondrocyte proliferation. Growth curves are shown for
OA chondrocytes (cell line C203 from a 60-year-old man) in monolayer that were left untreated (open squares) or treated with 5 ng/ml
bFGF (open circles) or 10 ng/ml bFGF (solid triangles).
drocytes do not readily proliferate, conditions that
would stimulate them to divide in culture were examined. It has been previously demonstrated that both
bFGF and TGF␤ are mitogenic for chondrocytes grown
as monolayers (29). Also, the expansion of dedifferentiated bovine chondrocytes in the presence of bFGF
enhances their ability to redifferentiate in 3-dimensional
culture systems (30).
Therefore, in order to determine whether bFGF
stimulates the cell division of human OA chondrocytes,
a requirement for retroviral infection, OA chondrocytes
isolated from a 60-year-old patient (C203) were treated
with varying doses of bFGF. The results, shown in
Figure 1, demonstrate that bFGF can stimulate human
OA chondrocytes to undergo increased cell division as
compared with the untreated cells. However, the difference in cell proliferation was observed only after weekly
subpassages conducted once per week for 4 weeks. The
cells treated with 5 ng/ml bFGF increased 181-fold (5
million to 1.8 billion), for a total of 8.52 population
doublings, and the 10 ng/ml bFGF–treated cells increased 758-fold (5 million to 7.58 billion), for a total of
10.5 population doublings. Since the effect of bFGF was
only evident at later time points, it was decided not to
use bFGF to stimulate growth of the chondrocytes
unless the starting cell numbers were limiting. With
respect to the 3 cell lines studied (C209, C223, and
C222), C209 required bFGF to stimulate cell growth
during the infection, selection, and early culture phases
PIERA-VELAZQUEZ ET AL
only. No bFGF was used to expand C223 or C222, the
other cell lines studied.
Generation of a human telomerase retroviral
expression vector and determination of telomerase expression in chondrocytes. The human telomerase cDNA
was provided by Geron Corporation. A telomerase
expression plasmid was constructed by inserting the
human telomerase cDNA into the pLNCX vector, which
places it under control of the cytomegalovirus promoter
(pLNCX-telomerase) (Figure 2). Packaging cell lines
were produced by transfecting amphotropic PT67 cells
(provided by Clontech) with either the pLNCX vector or
the pLNCX-telomerase construct. PT67 cells were transfected using the calcium phosphate method, and the
cells were selected by culturing them for 10 days in the
presence of 350 ␮g/ml of G418 (27).
The endogenous level of telomerase expression was
determined in uninfected OA chondrocytes, HeLa cells,
and in the transfected PT67 packaging cells by utilizing a
PCR-based TRAP assay (see Patients and Methods). Figure 3A shows that both the positive control HeLa cell
extract (lane 1) and the extract from the PT67 cells stably
transfected with the pLNCX-telomerase construct (lane 2)
displayed telomerase activity. However, no telomerase
activity was detected in OA chondrocytes (C208) that were
grown as monolayers in either the absence (lane 3) or
presence (lane 4) of bFGF. Furthermore, no telomerase
activity was found in the C209 OA chondrocytes cultured
either as monolayer (dedifferentiated) or on polyHEMAcoated dishes (differentiated) (lanes 5 and 6, respectively).
TRAP assays were then performed on uninfected
Figure 2. Schematic diagram of the human pLNCX-telomerase construct (pLNCXtelom). LTR ⫽ long terminal repeat; Amp(R) ⫽
ampicillin resistance gene; PSI ⫽ viral packaging signal; NEO(R) ⫽
neomycin resistance gene; CMV ⫽ cytomegalovirus.
EXOGENOUS EXPRESSION OF TELOMERASE FOR EXPANSION OF OA CHONDROCYTES
687
Figure 3. Telomeric-repeat amplification protocol (TRAP) assay performed on human osteoarthritic (OA) chondrocytes and various cell lines. A,
TRAP assays were performed on HeLa cells, PT67 retroviral packaging cells stably transfected with the pLNCX-hTERT construct, human OA
chondrocytes isolated from a 57-year-old woman (C208) and treated with 0 ng/ml basic fibroblast growth factor (bFGF) (lane 3) or 5 ng/ml bFGF
(lane 4) for 10 days, and OA chondrocytes isolated from a 75-year-old woman (C209) and grown as monolayer for 10 days (p1; lane 5) or cultured
on poly-(2-hydroxyethyl-methacrylate)–coated dishes for 10 days (pH; lane 6). B, TRAP assays were performed on OA chondrocytes isolated from
a 75-year-old woman (C209) and a 69-year-old man (C223) that were uninfected (U), infected with the pLNCX control retroviral vector (V), or
infected with the pLNCX-hTERT retroviral vector (T). C, TRAP assays were performed on OA chondrocytes isolated from a 41-year-old man
(C222) to serve as a control clonal line (C) and hTERT clonal line (T). IC ⫽ internal control; AS ⫽ after selection; P ⫽ passage number.
C209 and C223 OA chondrocytes or C209 and C223 OA
chondrocytes that had been transduced with the pLNCX
vector or the pLNCX-hTERT construct (Figure 3B). No
detectable telomerase activity was observed in either the
uninfected (lanes 1 and 6) or pLNCX-transduced (lanes
2 and 7) chondrocyte cell lines. In contrast, both
hTERT-transduced chondrocyte cell lines showed substantial levels of telomerase activity (lanes 3 and 8).
Furthermore, it was found that the telomerase activity in
these cells increased with subsequent passages (lanes 4,
5, and 9).
Since the pooled cell lines that have been characterized thus far were most likely composed of cells
with different expression levels of telomerase, clones of
hTERT and control chondrocyte cell lines were established. At passage 6, chondrocytes isolated from the
41-year-old OA patient that had been infected with
either the pLNCX-telomerase construct or the pLNCX
control vector were plated at a density of 0.5–1 cell/well
in 96-well plates. One hTERT and 1 control clonal cell
line was expanded. Figure 3C shows that the C222 clonal
hTERT cell line exhibited telomerase activity, whereas
the control line was negative.
Assessment of telomere lengths. Southern analyses were performed with digested genomic DNA from
the OA chondrocyte cell lines to determine the effects of
telomerase expression on telomere length. Analyses
were performed with genomic DNA isolated from uninfected, pLNCX-transduced, and hTERT-transduced
C209 and C223 chondrocytes immediately after selection with G418. The results, shown in Figure 4, indicate
that in the C209 cell line, telomerase expression caused
a detectable increase in telomere length. In contrast, the
same analysis performed on the initial pool of the
transduced C223 cell line failed to show any changes in
telomere length as a result of telomerase expression.
However, subsequent passaging of hTERT-transduced
C223 cells resulted in longer telomeres than that seen in
pLNCX-transduced cells (Table 1). The data shown in
Table 1 represent the average from at least 2 different
genomic DNA digestions analyzed by different hybridizations. Table 1 also shows the increase in telomere
length of the hTERT C222 clonal line as compared with
the control pLNCX C222 clonal cell line.
Effects of telomerase expression on OA chondrocyte proliferation and population doubling levels. The
effects of telomerase expression on chondrocyte proliferation and population doubling levels were examined in
the 2 cell lines and in a clonal cell line during culture
periods ranging from 80 days to 250 days. Cell line C209
688
PIERA-VELAZQUEZ ET AL
Figure 4. Terminal restriction fragment lengths. Mean telomere
lengths in the C209 and C223 cell lines were determined for uninfected
osteoarthritic (OA) chondrocytes (U), OA chondrocytes infected with
the pLNCX retroviral vector (V), or OA chondrocytes infected with
the pLNCX-hTERT retroviral vector (T). AS ⫽ after selection; P ⫽
passage number.
transduced with pLNCX alone reached senescence and
failed to increase in cell number after 40 days in culture
(Figure 5A). In contrast, C209 hTERT chondrocytes
displayed a 3-fold increase in cell number by day 20,
followed by a reduction in the cell number that reached
baseline levels at 60 days. However, a remarkable increase in cell number was observed in subsequent periods so that at 80 days, a nearly 4-fold increase was
observed. Cell growth continued so that by day 103, the
cell number had increased 20-fold and by 201 days, by
40-fold (Figure 5A). Similarly, the behavior of the
transduced C223 cell line showed that C223 pLNCXtransduced cells reached a state of senescence after 77
Table 1. Mean telomere lengths of osteoarthritic chondrocyte cell
lines
Mean telomere length, kb
Treatment
C209
C223
C222 clones
Uninfected
Early passage
pLNCX
hTERT
Later passage
pLNCX
hTERT
8.8
10.4
10.5
8.1
9.9
9.5
9.5
11.5
9.6
10.5
10
13.3
Figure 5. Growth curves of the osteoarthritic (OA) chondrocyte cell
lines A, C209, B, C223, and C, clonal C222, infected with pLNCX
vector or pLNCX-hTERT. OA chondrocytes infected with pLNCX
vector (open circles) or with pLNCX-hTERT (solid squares) were
grown in monolayer culture, trypsinized, counted, and replated at
lower density. At the end of the culture periods, the population
doublings (PD) were calculated using the equation, PD ⫽ Log10
(N/N0) ⫻ 3.33 (see Patients and Methods).
days in culture, while C223 hTERT-transduced cells still
continued growing after 80 days and 16 passages.
Population doubling levels were then calculated
for the 2 cell lines C209 and C223, and the clones
EXOGENOUS EXPRESSION OF TELOMERASE FOR EXPANSION OF OA CHONDROCYTES
689
Figure 6. Morphology of osteoarthritic (OA) chondrocyte cell lines cultured as monolayer or in poly-(2hydroxyethyl-methacrylate) (polyHEMA)–coated dishes. OA chondrocytes were infected with the pLNCX vector
(A and B) or the pLNCX-hTERT construct (C and D) and were cultured as monolayer or redifferentiated on
polyHEMA-coated dishes for 4 days (E and F).
derived from the C222 chondrocytes. In contrast to the
C209 pLNCX cells, which showed no detectable population doubling levels, the C209 hTERT cells exhibited 9
population doublings by the time the experiment was
terminated at 234 days. Similar results were observed
with the C223 hTERT cells, which exhibited 8 population doublings by 84 days of culture (Figure 5B), compared with C223 pLNCX cells, which exhibited only 3
population doublings. Interestingly, the C222 clonal cell
lines exhibited a higher population doubling level than
did the pooled cell lines. The C222 control clonal cells
senesced after ⬃20 population doublings, whereas the
hTERT clone continues to grow with ⬎28 population
doublings to date (Figure 5C).
Morphology and gene expression of telomeraseexpressing chondrocytes. Figure 6 shows the morphology of the C209 and C223 cell lines cultured either as
dedifferentiated chondrocytes in monolayer or as redifferentiated chondrocytes in polyHEMA-coated dishes.
The morphology of the hTERT chondrocytes was similar to that of the control cells (pLNCX chondrocytes)
under both culture conditions, and reacquisition of the
chondrocyte cell morphology was evident when the cells
were redifferentiated in polyHEMA-coated dishes for
up to 4 days (Figures 6E and F). In order to further
verify that the expression of hTERT had not altered the
phenotype of the chondrocytes, gene expression analysis
of control cells and of cells expressing telomerase cultured as either monolayer (dedifferentiated) or in
polyHEMA-coated dishes (redifferentiated) was performed. RT-PCR of total mRNA using human type I
collagen–specific primers showed that both C209
hTERT cells and C223 hTERT cells that had redifferentiated in polyHEMA culture for 7 days expressed less
type I collagen message (C209 at the ninth and nineteenth passage and C223 at the sixth and twelfth passage) than in plastic culture (Figure 7). We also detected
a higher level of aggrecan expression in both lines of
redifferentiated hTERT-infected chondrocytes; the difference observed by RT-PCR was 8.7-fold in C209 at
passage 9 and 3.7-fold in C223 at passage 6 which
increased to 5.2-fold in C223 at passage 12 (Figure 8).
The level of type II collagen mRNA, as measured
by RT-PCR, remained low in redifferentiated hTERTinfected chondrocytes (C223) after 6 or 12 passages on
plastic followed by 7 days on polyHEMA-coated dishes
(Figure 9). The low level of type II collagen mRNA
observed could be due to the short period of time in
which the cells were allowed to redifferentiate (7 days).
However, as shown in Table 2, the ratio of type II
690
Figure 7. Reverse transcription–polymerase chain reaction (RTPCR) analysis of RNA isolated from osteoarthritic (OA) chondrocyte
cell lines for COL1A1 mRNA expression. RT-PCR analysis was
performed on RNA isolated from the C209 (A) and C223 (B) OA
chondrocyte cell lines as described in Patients and Methods, with
primers specific for human COL1A1 and GAPDH mRNA. Upper
panels indicate the COL1A1 and GAPDH (as a control) RT-PCR
products, and lower panels show the corresponding quantitation.
Quantitation was performed with a FluorImager and the pixel volumes
(vol) of the COL1A1 bands were normalized to those of GADPH,
which were obtained in a separate RT-PCR reaction from the same
RNA samples. P ⫽ passage number; pl ⫽ pLNCX-hTERT–infected
chondrocytes as monolayer; pH ⫽ pLNCX-hTERT–infected chondrocytes as monolayer and then redifferentiated on poly-(2-hydroxyethylmethacrylate)–coated dishes for 7 days.
collagen to type I collagen increased ⬃2.5-fold when
C223 hTERT cells were transferred to polyHEMAcoated dishes for 7 days.
PIERA-VELAZQUEZ ET AL
Figure 9. RT-PCR analysis of RNA isolated from the C223 cell line
for COL2A1 mRNA expression. RT-PCR analysis was performed on
RNA isolated from the C223 OA chondrocyte cell line as described in
Patients and Methods, with primers specific for human COL2A1 and
GAPDH mRNA. Upper panels show the COL2A1 and GAPDH (as a
control) RT-PCR products, and lower panels show the corresponding
quantitation. Quantitation was performed with a FluorImager and the
pixel volumes of the RT-PCR bands for COL2A1 were normalized to
those of GADPH. pH (alone) ⫽ uninfected chondrocytes cultured on
polyHEMA; P ⫽ passage number; pl ⫽ pLNCX-hTERT–infected
chondrocytes as monolayer; pH (with passage number) ⫽ pLNCXhTERT–infected chondrocytes as monolayer and then redifferentiated
on polyHEMA-coated dishes for 7 days. See Figure 7 for other
definitions.
DISCUSSION
Figure 8. RT-PCR analysis of RNA isolated from OA chondrocyte
cell lines for aggrecan mRNA expression. RT-PCR analysis was
performed on RNA isolated from the C209 (A) and C223 (B) OA
chondrocyte cell lines as described in Patients and Methods, with
primers specific for human aggrecan and GAPDH mRNA. Upper
panels show the aggrecan and GAPDH (as a control) RT-PCR
products, and lower panels show the corresponding quantitation.
Quantitation was performed with a FluorImager and the pixel volumes
of the aggrecan bands were normalized to those of GAPDH, which
were obtained in a separate RT-PCR reaction from the same RNA
samples. pH (alone) ⫽ uninfected chondrocytes cultured on polyHEMA; P ⫽ passage number; pl ⫽ pLNCX-hTERT–infected chondrocytes as monolayer; pH (with passage number) ⫽ pLNCX-hTERT–
infected chondrocytes as monolayer and then redifferentiated on
polyHEMA-coated dishes for 7 days. See Figure 7 for other definitions.
One basis for cellular aging in vitro and in vivo is
the telomere length hypothesis. The telomere length
hypothesis proposes that as primary somatic cells divide,
their telomeric sequences shorten due to the lack of
telomerase activity (13,15,31,32). When telomeres become shortened to a certain threshold length, the cells
Table 2. Ratio of type II collagen (CII) to type I collagen (CI)
mRNA expression in transduced C223 cells cultured as monolayer in
plastic and following culture on polyHEMA for 7 days*
CII:CI
P6 monolayer
P6 f pH†
P12 monolayer
P12 f pH†
0.48
1.18
1.29
3.48
Fold increase
in CII:CI
2.5
2.7
* P ⫽ passage number.
† Redifferentiated 7 days in poly-(2-hydroxyethyl-methacrylate)
(polyHEMA)–coated dishes.
EXOGENOUS EXPRESSION OF TELOMERASE FOR EXPANSION OF OA CHONDROCYTES
respond by entering a metabolically active but nondividing state of senescence. Indeed, telomere length has
been shown to correlate with replicative life span in vitro
and somatic cells from older individuals possess a lower
replicative potential and shorter telomeres (12). In vivo
experiments also support the telomere length hypothesis. Mice deficient in telomerase activity (mTR⫺/⫺), due
to deletion of the RNA component, show decreased
viability after ⬃4–6 generations, depending on the genetic background (33). Additionally, mTR⫺/⫺ mice exhibit a reduced capacity to respond to stresses such as
wound healing and ablation of the hematopoietic system
(34). These results imply that loss of telomere length
contributes to the changes observed during aging and
age-related disease.
Chondrocytes, similar to other primary somatic
cell types, possess a limited replicative life span in vitro
and invariably enter into the nondividing, metabolically
active state of senescence (4,5,26). Studies have shown
that the population doubling capacity of normal human
articular chondrocytes is in the range of 35–40 and that
lapine articular chondrocytes exhibit a relationship between the age of the animal and the replicative life span
in vitro (4,5). During the aging process and during the
development and progression of OA, articular cartilage
loses both thickness and cellularity along with the development of changes in the cartilage matrix and metabolic
function of the chondrocytes (9,10). The loss of cellularity has been postulated to be due to a decreased ability
of the chondrocytes to proliferate or respond to mitogenic signals coupled with an increase in apoptosis (7,8).
Indeed, Martin and Buckwalter (6) have recently shown
that both mitotic potential and telomere length are
diminished in human articular chondrocytes isolated
from older individuals.
We report here, for the first time, the effect of
ectopic expression of the human telomerase reverse
transcriptase in human articular chondrocytes. Articular
chondrocytes isolated from patients undergoing knee
replacement surgery for OA were cultured and infected
with a human telomerase cDNA retroviral expression
construct. Uninfected chondrocytes or chondrocytes infected with the retroviral vector alone did not display
any detectable telomerase activity, whereas chondrocytes infected with the telomerase retroviral construct
exhibited telomerase activity comparable with that of
HeLa cells. Interestingly, the telomerase activity increased as the cells were passaged in culture, possibly
indicating a selective growth advantage. Indeed, articular chondrocytes from a 75-year-old patient (C209) that
were transduced with the hTERT expression vector were
691
able to grow for an additional 9 population doublings
and exhibited increased telomere lengths as compared
with control cells from the same donor. A second
hTERT chondrocyte cell line, C223 (from a 69-year-old
patient), grew for an additional 5 population doublings
as compared with control cells, and this line still continues to grow. We also established clonal hTERT and
control cell lines from the chondrocytes of a 41-year-old
OA patient. The clonal control cell line ceased dividing
after 20 population doublings, whereas the hTERT
clonal line has undergone 28 population doublings and
currently exhibits a population doubling rate of once
every 3 days.
The difference in population doubling levels
achieved by the pooled cell lines in comparison with the
C222 clonal lines is probably due to the heterogeneity of
telomerase expression levels in the pooled lines. Furthermore, the expression of telomerase in these chondrocytes did not impede the ability of the cells to
undergo the first stages of redifferentiation, as was
evidenced by a reduction in type I collagen gene expression, an increase in aggrecan gene expression, and
reappearance of a chondrocytic cell morphology after
transfer of the monolayer hTERT chondrocytes to
polyHEMA-coated dishes.
Recently, several laboratories have reported the
establishment of immortalized human primary cell lines
that retain their differentiated phenotype and normal
responses to external stimuli by engineering them to
express the hTERT protein. Bodnar et al (21) first
demonstrated that human foreskin fibroblasts and retinal pigment epithelial cells transfected with an hTERT
cDNA expression vector acquired an increased population doubling capacity in comparison with their hTERTnegative counterparts. Later it was shown that the
hTERT⫹ cell lines still retained their normal differentiated response to signals such as serum starvation,
contact growth inhibition, and anchorage-dependent
growth (23). Subsequently, normal human endothelial
cells were immortalized by ectopic telomerase expression also without acquiring malignant characteristics
(25). However, not all human primary cell types appear
to have the ability to be immortalized only by the
expression of telomerase. Human mammary epithelial
cells and human keratinocytes also required the downregulation or inactivation of the p16INK4a tumor suppressor gene (35,36). Also, expression of hTERT in
human epithelial cells resulted in cells that expressed
higher levels of the cMyc protooncogene (37).
Interestingly, telomerase expression has been
shown to provide a beneficial effect on age-related
692
PIERA-VELAZQUEZ ET AL
cellular processes such as apoptosis and skin regeneration. In a recent study, telomerase-expressing skin fibroblasts were compared with young or senescent fibroblasts in an in vivo model of skin growth by implantation
into SCID mice (38). The senescent fibroblasts gave rise
to skin that exhibited blistering and was more fragile
than the skin derived from either young or telomeraseexpressing fibroblasts. In other studies, the inhibition of
telomerase activity in pheochromocytoma cells and in
primary embryonic hippocampal neurons was found to
sensitize them to apoptosis-inducing insults such as
oxidative stress and the effect of the cytotoxic amyloid
␤-peptide associated with Alzheimer’s disease (39,40).
Finally, hTERT expression by human endothelial cells
also induced resistance to conditions that normally
result in apoptosis (25). These studies suggest that
expression of telomerase in aged, diseased, or senescent
cells may restore certain beneficial biologic functions.
However, the long-term effect of ectopic telomerase expression by somatic cells in vitro and in vivo is
unknown. Telomerase expression is associated with the
progression of cancer and the endogenous hTERT gene
has been shown to be up-regulated by the protooncogene cMyc (41). Therefore, aged or diseased somatic
cells engineered to continually express telomerase for
cell and gene therapy may have potential drawbacks,
such as the occurrence of malignant transformation.
Nevertheless, alternative strategies such as the transient
introduction of telomerase or the use of inducible or
tissue-specific promoters to drive expression of telomerase may allow for the controlled expansion of aged
somatic cells ex vivo which can then be used for therapeutic purposes and for tissue engineering. In conclusion, the engineering of aged human OA chondrocytes
to re-express telomerase may eventually represent a
novel way to expand these cells ex vivo for use in the
treatment of defects associated with the degeneration of
cartilage during OA.
ACKNOWLEDGMENT
We are grateful to David Hawkins for excellent technical assistance.
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