Prolonged Expansion of Human Nucleus Pulposus Cells Expressing
Human Telomerase Reverse Transcriptase Mediated by
Jianhong Wu, Deli Wang, Dike Ruan, Qing He, Yan Zhang, Chaofeng Wang, Hongkui Xin, Cheng Xu,
Department of Orthopaedic Surgery, Navy General Hospital, No.6 Fu-cheng Road, Beijing 100048, PR China
Received 5 May 2013; accepted 31 July 2013
Published online 27 August 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22474
extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated
discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has
been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or
recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells
to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a
gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly
isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral
infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere
length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study
suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD. ? 2013 Orthopaedic Research
Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:159–166, 2014.
Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and
cells; human telomerase reverse transcriptase
degenerative disc diseases; nucleus pulposus cells; telomerase; lentiviral vector; gene therapy; human nucleus pulposus
Lower back pain is a major public health problem in
modern society. The most common cause for lower
back pain is degeneration of intervertebral disc, which
is often called degenerative disc disease (DDD). The
DDD is characterized by progressive loss of human
nucleus pulposus (HNP) cells and extracellular ma-
trix.1The HNP cells secrete aggrecan and collagen II,
which are the massive deposition of the extracellular
matrix during development.2Therefore, cell therapy to
supplement HNP cells to degenerated disc has been
thought as a promising strategy to treat DDD. This
strategy requires a large quantity of fully functional
HNP cells can be expanded in vitro with limited life
span. After serial passages in vitro, HNP cells undergo
replicative senescence marked with the changes on
cell morphology, gene expression, and metabolism; and
eventually they also lose their ability to synthesize
and secret collagen II and aggrecan.3,4
One of strategies to extend the expansion of func-
tional HNP cells in vitro is to ectopically express
human telomerase reverse transcriptase (hTERT) in
HNP cells. The hTERT is a catalytic component of the
telomerase. The telomerase maintains the length of
telomeres of chromosomes by adding the telomere
repeat sequence (TTAGGG). Ectopic expression of
hTERT reconstitutes the telomerase activity, stabilizes
telomere length, and extends the life span of various
types of human cells,5,6including HNP cells.
Previous studies have shown that delivering hTERT
gene into HNP cells using lipofectamine and recombi-
nant adeno-associated viral (rAAV) vector can extend
ovine nucleus pulposus cells7and HNP cells.8The
lipofectamine has some drawbacks such as transient
expression of transgene and high cellular toxicity.9
Treating DDD, a chronic disease, needs long-term and
continuous supplement of functional HNP cells. Thus,
lipofectamine-mediated hTERT cannot meet its need.
For these reasons, we previously developed a rAAV-
hTERT vector to deliver hTERT to HNP cells. The
rAAV-hTERT effectively mediates hTERT into HNP
cells and extends the life-span of HNP in vitro up to
150 days. However, rAAV has a limited transgene
packaging capacity (up to 5kb). In addition, because
this replication-deficient rAAV-hTERT exists in HNP
cells as episomal concatamers,10,11which cannot repli-
cate during cell divisions, the expression of hTERT
120 days post viral infection.
Lentiviral vectors had been used to deliver genes
into various types of cells, including both dividing and
non-dividing cells.12When infecting cells, lentiviral
vectors incorporate themselves into the genome of
transduced cells, allowing for stable transgene expres-
sion. In addition, lentiviral vectors have transgene
packaging capacity of 8–9kb. The self-inactivating
lentiviral transfer vectors and the third generation
packaging system which separates viral genes in three
plasmids make lentiviral vector safer.13In this study,
we constructed a lentiviral vector bearing the hTERT
coding sequence and DNA sequence coding for a green
cannotbe detected at
Grant sponsor: National Natural Science Foundation of China;
Grant number: 81171740.
Correspondence to: Deli Wang (T: þ86-10-66958615; F: þ86-10-
68780323; E-mail: email@example.com)
# 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
JOURNAL OF ORTHOPAEDIC RESEARCH JANUARY 2014
5. Zhdanova NS, Minina IuM, Rubtsov NB. 2012. Mammalian
telomere biology. Mol Biol (Mosk) 46:539–555.
6. Gomez DE, Armando RG, Farina HG. 2012. Telomere
structure and telomerase in health and disease (review). Int
J Oncol 41:1561–1569.
7. Chung SA, Wei AQ, Connor DE. 2007. Nucleus pulposus
cellular longevity by telomerase gene therapy. Spine 32:
8. Wu J, Wang D, Zhang C. 2011. Extending the activities of
human nucleus pulposus cells with recombinant adeno-associ-
ated virus vector-mediated human telomerase reverse tran-
scriptase gene transfer. Tissue Eng Part A 17:2407–2415.
9. Perez-Martinez FC, Guerra J, Posadas I. 2011. Barriers to
non-viral vector-mediated gene delivery in the nervous
system. Pharm Res 28:1843–1858.
10. Grieger JC, Samulski RJ. 2005. Adeno-associated virus as a
gene therapy vector:vector development, production and clini-
cal applications. Adv Biochem Eng Biotechnol 99:119–145.
11. Goncalves MA. 2005. Adeno-associated virus: from defective
virus to effective vector. Virol J 2:43.
12. Durand S, Cimarelli A. 2011. The inside out of lentiviral
vectors. Viruses 3:132–159.
13. Murakami Y, Ikeda Y, Yonemitsu Y. 2010. Inhibition of
choroidal neovascularization via brief subretinal exposure
with Sendai viral envelope proteins. Hum Gene Ther 21:
14. Copreni E, Nicolis E, Tamanini A. 2009. Late generation
lentiviral vectors: evaluation of inflammatory potential in
human airway epithelial cells. Virus Res 144:8–17.
15. Rossi JJ. 2009. Dotting the I’s and crossing the T’s: integra-
tion analyses in transduced patient T cells. Mol Ther
16. Sinn PL, Sauter SL, McCray PB Jr. 2005. Gene therapy
progress and prospects: development of improved lentiviral
and retroviral vectors—design, biosafety, and production.
Gene Ther 12:1089–1098.
17. Throm RE, Ouma AA, Zhou S. 2009. Efficient construction
of producer cell lines for a SIN lentiviral vector for SCID-X1
gene therapy by concatemeric array transfection. Blood
18. Song H, Yang PC. 2010. Construction of shRNA lentiviral
vector. N Am J Med Sci 2:598–601.
19. Mayshar Y, Ben-David U, Lavon N. 2010. Identification and
classification of chromosomal aberrations in human induced
pluripotent stem cells. Cell Stem Cell 7:521–531.
20. Taghizadeh RR, Sherley JL. 2008. CFP and YFP, but not
GFP, provide stable fluorescent marking of rat hepatic adult
stem cells. J Biomed Biotechnol 2008:453590.
21. Liu HS, Jan MS, Chou CK. 1999. Is green fluorescent
protein toxic to the living cells? Biochem Biophys Res
22. Herbst F, Ball CR, Tuorto F. 2012. Extensive methylation of
promoter sequences silences lentiviral transgene expression
during stem cell differentiation in vivo. Mol Ther 20:
23. Cong Y, Shay JW. 2008. Actions of human telomerase
beyond telomeres. Cell Res 18:725–732.
24. Melin BS, Nordfjall K, Andersson U. 2012. hTERT cancer
risk genotypes are associated with telomere length. Genet
25. Danet-Desnoyers GA, Luongo JL, Bonnet DA. 2005. Telome-
rase vaccination has no detectable effect on SCID-repopulat-
ing and colony-forming activities in the bone marrow of
cancer patients. Exp Hematol 33:1275–1280.
26. Nourbakhsh M, Golestani A, Zahrai M. 2010. Androgens
stimulate telomerase expression, activity and phosphoryla-
tion in ovarian adenocarcinoma cells. Mol Cell Endocrinol
27. Zhao CF, Hu HY, Meng L. 2010. Immortalization of bovine
mammary epithelial cells alone by human telomerase
reverse transcriptase. Cell Biol Int 34:579–586.
28. Tsuruga Y, Kiyono T, Matsushita M. 2008. Establishment of
immortalized human hepatocytes by introduction of HPV16
E6/E7 and hTERT as cell sources for liver cell-based
therapy. Cell Transplant 17:1083–1094.
29. Raty JK, Lesch HP, Wirth T. 2008. Improving safety of gene
therapy. Curr Drug Saf 3:46–53.
WU ET AL.
JOURNAL OF ORTHOPAEDIC RESEARCH JANUARY 2014