Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differentiation of Adipose...

IntroductionThe finite proliferation of mammalian cells is considered to

be the result of a reduction of telomere length [1, 2]. The

telomere contains repeated sequences of six nucleotide bases,

TTAGGG, located at the termini of individual chromosomes,

and has been shown to be shortened by 33–120 bp at each cell

division in human fibroblastic cells and lymphocytes, thus

causing aging and finite mitotic capability [3, 4]. Telomere

Expression of Telomerase Extends the Lifespan and

Enhances Osteogenic Differentiation of Adipose

Tissue–Derived Stromal Cells

Soo Kyung Kang,a,c Lorna Putnam,c Jason Dufour,b Joni Ylostalo,c

Jin Sup Jung,e Bruce A. Bunnella,c,d

aDivision of Gene Therapy and bDivision of Veterinary Medicine, Tulane National Primate Center,

Tulane University Health Sciences Center, Covington, Louisiana, USA; cCenter of Gene Therapy and dDepartment

of Pharmacology, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA;eDepartment of Physiology, College of Medicine, Busan National University, Busan, South Korea

Key Words. Telomerase • Adipose tissue stromal cell • Osteogenic differentiation • Microarray

Abstract Expression of TERT, the catalytic protein subunit of thetelomerase complex, can be used to generate cell lines thatexpand indefinitely and retain multilineage potential. Wehave created immortal adipose stromal cell lines (ATSCs)by stably transducing nonhuman primate-derivedATSCs with a retroviral vector expressing TERT. Trans-duced cells (ATSC-TERT) had an increased level oftelomerase activity and increased mean telomere lengthin the absence of malignant cellular transformation.Long-term culture of the ATSC-TERT cells demon-strated that the cells retain the ability to undergo differ-entiation along multiple lineages such as adipogenic,chondrogenic, and neurogenic. Untransduced cellsdemonstrated markedly reduced multilineage and self-renewal potentials after 12 passages in vitro. To deter-mine the functional role of telomerase during osteogene-sis, we examined osteogenic differentiation potential of

ATSC-TERT cells in vitro. Compared with naive ATSCs,which typically begin to accumulate calcium after 3–4weeks of induction by osteogenic differentiation medium,ATSC-TERT cells were found to accumulate significantamounts of calcium after only 1 week of culture inosteogenic induction medium. The cells have increasedproduction of osteoblastic markers, such as AP2,osteoblast-specific factor 2, chondroitin sulfate proteo-glycan 4, and the tumor necrosis factor receptor super-family, compared with control ATSCs, indicating thattelomerase expression may aid in maintaining theosteogenic stem cell pool during in vitro expansion. Theseresults show that ectopic expression of the telomerasegene in nonhuman primate ATSCs prevents senescence-associated impairment of osteoblast functions and thattelomerase therapy may be a useful strategy for boneregeneration and repair. Stem Cells 2004;22:1356–1372

STEM CELLS 2004;22:1356–1372 www.StemCells.com

Correspondence: Bruce A. Bunnell, Ph.D., Center for Gene Therapy, Department of Pharmacology, Division of Gene Therapy,Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA70433, USA. Telephone: 985-871-6594; Fax: 985-871-6587; e-mail: [email protected] Received February 6, 2004;accepted for publication June 17, 2004. 1066-5099/2004/$12.00/0 doi: 10.1634/stemcells.2004-0023

Original Article

Stem Cells®

Matthew Holt

length is maintained by telomerase, a ribonuclear protein

complex consisting of an integral RNA (hTR), which serves

as the telomeric template; a catalytic subunit (hTERT), which

has reverse transcriptase activity; and associated protein com-

ponents [5–11]. In the absence of hTERT, telomeres shorten

during cell division because the DNA replication complex

cannot completely copy telomeric DNA. Cellular senescence

and growth arrest are proposed to occur when telomere

lengths in germ cells and most cancer cells are decreased.

However, ectopic expression of hTERT leads to telomere

elongation and extended lifespan in several cell types [1,

12–14].

Possible mechanisms of age-dependent bone loss may be

attributed, at least in part, to a deficiency of osteoblast func-

tion or a decrease in the number of osteogenic progenitor cells

rather than to an increase in bone resorption by osteoclasts

[15, 16]. It has been suggested that telomere-associated cellu-

lar senescence may contribute to various age-related disor-

ders. Recent studies reported that the introduction of hTERT

into osteoblasts isolated from human trabeculae induced

telomerase activity and extended the lifespan of these cells

[5, 6]. However, the role of telomerase in bone formation,

particularly with respect to maintenance of the osteogenic

precursor cell population, is largely unknown. Pluripotent

human bone marrow stromal cells (BMSCs) were originally

described as progenitors of osteoblasts because of their

capacity to form normal bone in vivo [5, 6, 13]. Mesenchymal

stem cells, including BMSCs and adipose stromal cell lines

(ATSCs), are being analyzed as new therapeutic agents for

repairing large bone defects that cannot undergo spontaneous

healing [17–19].

The regeneration of diseased or damaged tissue is the

principle goal of the emerging discipline of tissue engineer-

ing. A key requirement in tissue regeneration is the availabil-

ity of the constituent cells. Adipose tissue stromal cells have

been defined as multipotential adult stem cells, capable of

differentiating into a variety of cell types such as osteoblasts,

chondrocytes, adipocytes, muscle cells, and neural cells

[20–23]. Recently, our group and others reported that human

and nonhuman primate-derived ATSCs and BMSCs can

propagate in vitro and contain detectable levels of telo-

merase activity. Forced division of ATSCs in vitro may cause

excessive telomere shortening in the descendent lineages,

although ATSCs themselves possess telomerase activity.

Indeed, recent studies have demonstrated that the telomerase

activity of mesenchymal stem cells is not sufficient to com-

pletely compensate for the reduction of telomere length dur-

ing continuous in vitro subculture. To extend the prolifera-

tive lifespan of ATSCs, supplementation with transduced

exogenous hTERT may be necessary, because the self-

renewal and replicative potential of these cells may depend

on sufficient telomerase activity to maintain stable telo-

meres. Reconstitution of telomerase activity through expres-

sion of exogenous hTERT enables normal human fibro-

blasts, as well as retinal epithelial, myometrial, and endo-

thelial cells, to avoid senescence [24–30]. After ectopic

expression of telomerase, the lifespan of BMSCs was signif-

icantly increased, and proliferative capacity was extended in

vitro. The cells were demonstrated to have an enhanced

capacity for bone formation in vitro and in vivo [5, 6, 31–33].

The enhanced formation and normal morphology of the

ectopically formed bone strongly suggest that ATSC-TERT

cells represent a highly useful candidate cell source for bone

tissue regeneration and engineering.

Materials and Methods

Construction of Retroviral Vectors and Production of TERT DNA-Carrying RetrovirusesThe retrovirus vectors pBabe-hTERT (generously provided

by Robert Weinberg, Whitehead Institute for Biomedical

Research, Cambridge, MA) were used for construction of

BamH1-EcoR1 TERT DNA-ligated murine stem cell virus

neovector. The final construct was transfected to the mouse

amphotropic cell line FLYA13 using polybrene (5 µg/ml,

1,5-dimethyl-1,5-diazaundecamethylene polymethylene-

bromide [Sigma, St. Louis]). After transfection, the FLYA13

cells were placed under G418 selection (300 µg/ml [Sigma]),

and after 10 days of selection, culture supernatants contain-

ing amphotropic viruses were collected and then used to

infect primary cultures of ATSCs.

Isolation, Culture, and Transduction of ATSCsNonhuman primate adipose tissue was obtained under local

anesthesia. The raw adipose tissue was processed according

to established methodologies to obtain a stromal vascular

fraction. To isolate stromal cells, samples were washed exten-

sively with equal volumes of phosphate-buffered saline

(PBS) and digested at 37°C for 30 minutes with 0.075% colla-

genase IV (Sigma). Enzyme activity was neutralized with α-

modified Eagle’s medium (MEM; Invitrogen, Carlsbad, CA)

containing 10% fetal bovine serum (FBS) and centrifuged at

1200 g for 10 minutes. For obtaining a high-density cell pel-

let, the pellet was resuspended in red blood cell (RBC) lysis

buffer (BioWhittaker, Walkersville, MD) and incubated at

room temperature for 10 minutes to lyse contaminating

RBCs. The stromal cell pellet was collected by centrifuga-

tion, as described above, and incubated overnight at 37ºC/5%

CO2 in 10% FBS containing α-MEM medium. After 1 day of

culture, cells were subcultured onto six-well plates and

Kang, Putnam, Dufour et al. 1357

allowed to attach overnight. Cells were then infected for 2 or 4

hours with the amphotropic hTERT retroviral vectors in the

presence of polybrene (5 µg/ml) and selected in G418 (400

ng/ml) for 10 days. Following infection with virus, G418 was

used to select transduced cells and permit colony formation of

the hTERT-infected cells. Cell lines were generated and

maintained in α-MEM medium with 10% FBS (Atlanta Bio-

logicals, Lawrenceville, GA).

Nonradioisotopic Telomerase AssayTelomerase activity was analyzed using a modified telomeric

repeat amplification protocol (TRAP) assay according to the

manufacturer’s instructions (BD Science, San Diego). Pro-

tein extracts were prepared from ATSC controls (population

doubling [PD] 5, 10, and 15) and ATSC-TERT (PD 5, 10, and

15). Protein extract (0.5 µg) prepared from each cell line was

incubated in the presence of synthetic oligonucleotide

(telomerase-specific primer, 5'-AATCCGTCGAGCAGAG

TT-3') that could be the substrate for the addition of telomeric

repeats by telomerase. If telomerase activity was present in

the extracts, the oligonucleotide was elongated and could

work as a template in subsequent polymerase chain reaction

(PCR). PCR was performed in the presence of nucleotides,

and the formation of the amplification product was analyzed

by monitoring the telomerase repeat amplification. PCR reac-

tion products were separated on 12.5% nondenaturing

acrylamide gels and stained using Syber-Gold dye (Molecu-

lar Probes, Eugene, OR). Quantification of telomerase for

comparisons with telomerase activity in ATSC-TERT and

ATSCs was performed by the PCR enzyme-linked immuno-

sorbent assay procedure suggested by the manufacturer (BD

Science).

Telomere Length Analysis and Telomerase Reverse Transcription–PCRTelomere length was analyzed by determining the mean

length of the terminal restriction fragments (TRFs) by South-

ern blotting according to the manufacturer’s protocol

(Roche, Grenzacherstrasse, Basel, Switzerland). Genomic

DNA from cultured cells was isolated by using a Wizard

genomic DNA extraction kit (Promega, Madison, WI). Each

DNA sample was digested with Rsa I and Hinf I, resolved on

a 0.6% agarose gel, and, after transfer onto a positively

charged nylon membrane (Amersham, Piscataway, NJ),

hybridized with probe for the telomeric TTAGGG repeats.

The probe was constituted by a mixture of synthetic

(TTAGGG)n fragments ranging in size from 1–20 kb. The

mean TRF length was determined from the intensity of the

hybridization signal using chemiluminescence (Bio-Rad,

Philadelphia). For detection of hTERT by reverse transcrip-

tion (RT)-PCR, total RNA of cells (1 µg) was analyzed using

the human-specific TERT primers for analysis of ectopic

telomerase and endogeneous telomerase as a control.

Induction of Adipogenic, Osteogenic, andChondrogenic DifferentiationTo verify the multipotential differentiation of mesenchymal

characteristics of ATSC-TERT, cells were subjected to dif-

ferentiation in conditions known to induce adipogenic,

osteogenic, and chondrogenic lineages in human cells.

Before culture in the induction media, cultures were grown to

confluence.

For adipogenic differentiation,ATSCs were induced by

passaging cells at a 1:10 dilution in control media supple-

mented with 10 ng/ml insulin and 10–9 M dexamethasone

(Sigma). Adipogenic differentiation was visualized by the

presence of highly refractory intracellular lipid droplets

using phase-contrast microscopy. To induce osteogenic dif-

ferentiation, the cultures were fed daily with control

medium to which was added 10 mM β-glycerophosphate,

50 ng/ml ascorbic acid, and 10–9 M dexamethasone for 3

weeks. Mineralization of the extracellular matrix was visu-

alized by staining of the cultures with Alizarin red S (2%

[wt/vol] Alzarin red S adjusted to pH 4 using ammonium

hydroxide) for 5 minutes at room temperature followed by a

wash with water. Von Kossa staining was performed by

using an aqueous 5% AgNO3 solution, followed by fixation

for 2 minutes in 5% Na2S2O3 solution. For chondrocyte dif-

ferentiation, a pellet culture system was performed. Approx-

imately 3 ✕ 106 ATSC-TERT and ATSCs controls (PD 10)

were placed in a well of a 96-well plate. The pellet was cul-

tured at 37ºC with 5% CO2 in 500 µl of chonodrogenic

media containing 6.25 µg/ml insulin, 10 ng/ml transforming

growth factor β1, and 50 ng ascorbate-2-phosphate in con-

trol media for 2–3 weeks. The medium was replaced every 2

days for 15 days. For calcium deposit and chondrocyte

analysis in paraffin-embedded tissue, we stained with

0.03% Toluidine blue sodium borate and von Kossa staining

solution. For immunohistochemistry, paraffin sections (5

µm) were dried, deparaffinized using XEM-200 (Vogel,

Giessen, Germany), rehydrated in alcohol, and pretreated

with 2 mg/ml of hyaluronidase (Merck, Darmstadt, Ger-

many) for 15 minutes at 37ºC and subsequently with 1

mg/ml of pronase (Roche, Grenzacherstrasse, Basel,

Switzerland) for 30 minutes at 37ºC. Nonspecific back-

ground was blocked using PBS containing 10% goat serum

for 1 hour. Sections were incubated overnight at 4ºC with a

monoclonal mouse anti-human type I and II collagen

(Chemicon, Temecula, CA) in blocking solution. After

washing with PBS, reactivity was detected using fluorescein

1358 Enhanced Osteogenic Differentiation of ATSCs

isothiocyanate–conjugated anti-mouse secondary antibody

(1:200; Molecular Probes) for 30 minutes at room tempera-

ture and TO-PRO 3 (1:1000; Molecular Probes) for 3 min-

utes at room temperature for nuclear staining, and sections

were examined using fluorescence microscopy (Nikon,

Tokyo). Immunohistochemical studies were repeated at

least three times.

Induction of Neural Lineage Differentiation of ATSC-TERTUndifferentiated ATSCs and ATSC-TERT (passage 5) were

cultured and trypsinized in 0.25% trypsin (Invitrogen,

Gaithersburg, MD). Undifferentiated ATSCs cultured at high

densities spontaneously formed spherical clumps of cells

that were collected as they became free-floating masses of

cells that were released from the cell culture surface into the

culture media. We cultured the neurospheres in Petri dishes

using Neurobasal medium (NB; Invitrogen) supplemented

with B27 (Invitrogen), 20 ng/ml basic fibroblast growth fac-

tor (bFGF), and 20 ng/ml epidermal growth factor (EGF;

Sigma) for 4–7 days. The culture density of the spheroid

bodies was maintained at 10–20 cells/cm2 to prevent self-

aggregation. For neural lineage differentiation, neurospheres

derived from ATSCs were layered on poly-D-lysine (PDL)-

laminin double-coated chamber slides (Lab Tek, Nalge/

Nunc, Rochester, NY). Spheres were cultured and main-

tained for 10 days in NB media containing only the B27 sup-

plement. During differentiation, 70% of the media was

replaced every 4 days.

In Vivo Bone Formation and Tumorigenesis AssayFor transplantation,ATSCs and ATSC-TERTs at PD 15 were

immobilized and cultured in calcium phosphate scaffold

(Bio-Rad, Hercules, CA) or Matrigel (BD Bioscience, San

Jose, CA). Approximately 2 ✕ 106 ATSC-TERT or ATSC

control cells were mixed with Matrigel or scaffold and

implanted subcutaneously in 8-week-old immunodeficient

beige mice (NIH III/bg/nu/xid; Charles River Laboratories,

Wilmington, MA). The procedures were performed in accor-

dance with specifications of an approved protocol. The trans-

plants were recovered at 6 weeks after transplantation, fixed

with 4% formalin, and decalcified with 10% EDTA (pH 8.0)

for paraffin embedding. The paraffin-embedded sections

were deparaffinized and stained with toluidine blue O, von

Kossa, and hematoxylin-eosin.

Oligonucleotide Microarray AnalysisControl cells (ATSCs) and ATSC-TERT cells were har-

vested, and total RNA was isolated for microarray analysis.

RNA samples were then reverse transcribed to prepare cDNA

probes for hybridization to membranes as described. We

used newly available Affymetrix HG-U133A oligonu-

cleotide arrays. This gene array contains 22,000 expressed

genes and expressed sequence tags (ESTs). Affymetrix Suite

5.0 and dChip 1.3+ were used to analyze the data as described

in Materials and Methods. Gene expression that exceeded

the criteria for perfect match/mismatch was called present;

gene expression that failed to meet the criteria was called

absent. Genes were filtered as followed: The gene expression

level had to be at least 1.5 times higher compared with the

control data, and the gene had to be called present. Frag-

mented cRNA (15 µg) was hybridized for 16 hours at 45ºC to

the HG-U95A array for a comparison study (Affymetrix,

Santa Clara, CA). After hybridization, the gene chips were

automatically washed and stained with streptavidine-phyco-

erythrin by using a fluidics station. Finally, the probe arrays

were scanned at a 3-µm resolution using the Genechip Sys-

tem confocal scanner made for Affymetrix by Aligent.

Data AnalysisAffymetrix Microarray Suite 4 was used to scan and analyze

the relative abundance of each gene as derived from the aver-

age difference of intensities. Analysis parameters used by the

software were set to values corresponding to moderate strin-

gency. The threshold values to determine the present or

absent call were set as follows: α1 = 0.05, α2 = 0.065, τ =

0.015. Fluorescence intensity was measured for each chip

and normalized to average fluorescence intensity for the

entire chip. Output from the microarray analysis was merged

with the Unigene or GeneBank descriptor and stored as a

Microsoft Excel (Redmond, WA) data spreadsheet. The defi-

nition of increase or decrease or no change of expression for

individual genes was based on ranking the difference call

from two comparisons (2 ✕ 1); namely, no change of expres-

sion for individual genes was merged with the Unigene or

GeneBank descriptor and stored as an Excel data spread-

sheet. The definition of increase or no change of expression

for individual genes was based on ranking the difference call

from the two comparisons (2 ✕ 1, namely, no change = 0, mar-

ginal increase/decrease = 1/–1, increase/decrease = 2/–2).

The final rank referred to summing up the two values corre-

sponding to the difference calls, and the value varied from –6

to 6. The cutoff value for the final determination of increase/

decrease was set as 3/–3. Evaluation of the reproducibility of

paired experiments was based on calculation of the coeffi-

cient of variation (CV; standard deviation/mean) for fold

change (FC). The CV of FC must be less than or equal to 1.0.

Finally, genes with an FC greater than 1.5 were considered

significant. These cutoff values represented a conservative

estimate of the numbers of genes whose expression levels


differed between samples. Gene categorization was based on

a literature review.

Results

Growth Characteristics and TelomeraseExpression in ATSC-TERT CellsDuring prolonged periods of culture, the population of con-

trol ATSCs isolated from nonhuman primate fat underwent a

progressive decrease in proliferative potential, and finally

cells underwent senescence after passage 20 (80–90 days in

culture). At the end of their proliferative lifespan, the cells

were flatter and larger in morphology in a monolayer similar

to that described for senescent fibroblasts (data not shown).

hTERT-transduced ATSCs grow continuously for more than

9 months (>50 passages) without diminished cell expansion

or rate of proliferation (Fig. 1). Their rate of proliferation

resembled that of control ATSCs, and ATSC-TERTs retained

their inhibition of cellular proliferation by cell-to-cell con-

tact. The results indicate that the immortalization of the stro-

mal cells by telomerase expression does not alter cell growth.

The TRAP assay and telomerase immunocytochemistry

method were used to examine telomerase activity in primary

cultures of ATSCs (passage 0) and hTERT retrovirus-

infected cells (passages 5, 10, 15, and 20). Naive ATSCs have

low levels of protein and enzymatic telomerase activity

(Figs. 2A–2C). Whereas ATSCs transduced with the hTERT

retrovirus had reconstituted telomerase activity, the ATSC-

TERTs continuously expressed high levels of telomerase

over time (Figs. 2B, 2C).

Telomere Length Analysis of ATSC-TERT CellsIt has been proposed previously that critically shortened

telomeres mediate massive genomic instability and con-

tribute to M2 crisis. We assessed telomere lengths in cells at

different stages of proliferation. Unexpectedly, in cells

expressing only endogenous hTERT (control ATSCs), the

TRFs continued to shorten for 30 cell doublings after entering

crisis. In ATSC-TERT cells, telomere length was maintained

at least 50 doublings beyond the expected crisis point, and the

bulk of the TRFs increased slightly in length and became

more clustered at approximately a mean length of 23 kb,

which is the length of telomeres in passage-0 ATSCs (Fig. 3).

Homeostasis of telomere length was essentially achieved in

ATSC control cells, presumably by the balance between

telomere synthesis by telomerase and the erosions of telo-

meres during proliferation. In ATSC-TERTs, telomere length

did not increase even after 50 doublings past the normal crisis

point. Average telomere length was approximately 15 kb at

the last time point analyzed. Finally, telomeres continued to

shorten in hTERT-expressing cells as the cells proliferated

beyond the expected crisis point, with the average telomere

length in these late-passage hTERT-expressing cells consid-

erably shorter than in control cells in crisis (data not shown).


Figure 1. Ectopic expression of hTERT induces immortalization of ATSCs. (A): ATSCs overexpressing TERT were generated bytransduction with a TERT-expressing oncoretrovirus vector followed by neomycin selection to obtain stable clones. Control ATSCs atlow passage (5) and late passage (20) (left). Untransduced cells demonstrated markedly reduced self-renewal potentials after 12 pas-sages in vitro. Cells expressing hTERT (right) at low passage (5) and higher passage (20) maintained their thin spindle fibroblast mor-phology and growth rate. (B): Growth kinetics of control and TERT-expressing ATSCs. ATSC control cells showed markedly reducedexpansion after 50 days in vitro. hTERT-overexpressing cells underwent continuous expansion without a lag growth phase and havebeen in continuous culture for more than 9 months with no marked alterations in their growth characteristics. Abbreviation:ATSC, adi-pose stromal cell line.

Ectopic Expression of hTERT in ATSCs Does Not Alter Functional Characteristics We examined whether ectopic expression of hTERT affected

the multipotent characteristics of ATSCs. No marked differ-

ences between ATSC control and ATSC-TERT cells were

detected. ATSC-TERT cells retained the ability to accumu-

late lipid droplets typical for the adipocyte phenotype, and

they maintained osteogenic and chondrogenic differentia-

tion potential (Fig. 4, adipogenesis [Oil red O], osteogenic

[Alizarin red], and chondrogenesis [von Kossa]). Also,

ATSC-TERTs were induced toward the neurogenic lineage

through neurosphere formation and final differentiation on

PDL-laminin–coated substrate in NB media supplemented

with B27, bFGF, and EGF. During neurogenic induction in

NB media, both cell populations undergo a marked morpho-

logic change from elongated fibroblast morphology to com-

pact, spheroid bodies, which expand to larger spheroid bod-

ies as the total cell number expands (Fig. 4, neurospheres).

After detachment of the spheroid bodies from substrate, we

performed neural induction for 4 days through suspension

culture in Petri dishes, and then the intact neurospheres or

dissociated neurospheres were layered on PDL-laminin–

coated chamber slide and cultured for an additional 10 days.

As soon as the cells were layered on the laminin-coated sur-

face, the spheroid cell mass began to adhere and spread

across the growth surface, forming long chains of cellular

processes and, finally, the cell processes began to exhibit sec-

ondary branching with multiple extensions (Fig. 4, Tuj/

DAPI).

Enhanced In Vitro Osteogenesis and Chondrogenesis by ATSC-TERTATSCs are known progenitors of skeletal tissues and differ-

entiate into osteoblast-like cells in culture supplemented

with ascorbic acid and a source of glucocorticoid. However,

ATSCs lose their osteogenic capacity during continuous sub-

culture in vitro. This may limit their therapeutic use because

of effective treatment of extensive bone defects that require

the transplantation of large numbers of ex vivo–expanded

ATSCs. To determine the functional role of telomerase dur-

ing osteogenesis, we examined the osteogenic differentiation

potential of ATSC-TERT cells in vitro. ATSCs typically


Figure 2. Telomerase expression in control and hTERT-expressing ATSCs. (A): Immunocytochemistry of hTERT in ATSC-TERTclones and control cells. (B): Telomeric repeat amplification protocol assay for telomerase activity measurement in control and TERT-expressing ATSCs. The negative control assay was performed, omitting the TERT enzyme extract from the reaction mixture. (C):Quantification of telomerase activity in ATSC-TERT cells and ATSCs was performed by polymerase chain reaction enzyme-linkedimmunosorbent assay procedure. Abbreviation:ATSC, adipose stromal cell line.

begin to accumulate at calcium after 2–4 weeks of induction

in osteogenic differentiation medium. However, ATSC-

TERT cells were found to accumulate significant amounts of

calcium after only 1 week of osteogenic induction in vitro

(Fig. 5). We quantified the differences in the efficiency of

nodule formation between the naive and TERT-expressing

ATSCs by determining the number of stained nodules in 25

random fields. As shown in Figure 5C, there are more than

three times more nodules in the TERT-expressing ATSCs

compared with control ATSCs. After differentiation, an

extensive number of calcium deposits derived from ATSC-

TERT cells accumulated on the cell surface and ultimately

were released into culture supernatant. We identified and

quantified calcium deposits from culture supernatant after

calcium-specific Fluo-3 staining and flow cytometry analy-

sis (data not shown).

Also, after culture of ATSCs-TERT cells (passage 5) in

the pellet culture system for chondrogenic differentiation,

we stained von Kossa for calcium deposit and Toluidine blue

O for proteoglycan, a chondrocyte marker. ATSCs-TERT

had a highly formed calcium deposit and proteoglycan

matrix (Fig. 6). Immunohistochemistry results showed that

ATSC-TERT cells highly expressed collagen I and II in the

matrix (Fig. 6). In contrast to ATSC-TERT, we failed to

detect collagen-positive cells in control ATSCs.

In Vivo Osteogenesis after Engraftment of ATSC-TERT CellsWe studied in vivo osteogenesis effects after implantation of

ATSC-TERT subcutaneously with Matrigel and hydroxyap-

atite scaffolds in immune-deficient mice. Five weeks after

transplantation of ATSC-TERT cells, we analyzed paraffin-

embedded tissue using hematoxylin-eosin, Toluidine blue O

for proteoglycan, and von Kossa for calcium deposition. The

results of hematoxilin-eosin and von Kossa staining of

implant tissue section revealed highly enhanced bone forma-

tion by ATSC-TERT cells compared with control ATSCs.

Control ATSC-implanted tissue section failed to show any

hematoxylin-eosin and von Kossa–positive staining (data

not shown). Toluidine blue O staining showed that both

implants did not express proteoglycan (Fig. 7).

cDNA Expression Profile of ATSC-TERT To analyze the gene expression pattern, we performed oligo-

nucleotide microarray analysis. The gene expression profile

in ATSC-TERT cells was compared with ATSC controls.

Total RNA was harvested from both cultures, and gene

expression profiles were compared using Affymetrix HG-

U95a microarray (22,000 genes and ESTs). Affymetrix

Microarray Suite 5.0 was used to scan and analyze the rela-

tive abundance of each gene. The signal output from each

gene from the ATSC control profile was plotted against the

ATSC-TERT profile (data not shown), and the correlation

coefficient (r) was calculated for each comparison. The

analysis of the gene expression levels demonstrated that

fewer than 1% of the total genes were expressed at greater

than 2.2-fold different levels in ATSCs and ATSC-TERT, as

indicated by the r value (0.8). Tables 1 and 2 give a partial list

assembled into gene function of upregulated (total number of

genes = 288) or downregulated (total number of genes = 580)

genes expressed in ATSC-TERT compared with naive

ATSCs. Relative expression of telomerase,AP2, BDNF, and

MAP2 were examined by real-time RT-PCR. Comparing

expression of those genes in ATSCs and ATSC-TERT

revealed that some neural lineage-related genes are highly


Figure 3. Expression of vector-derived hTERT and telomerelength analysis. (A): Detection of hTERT by reverse transcrip-tion–PCR. Total cellular RNA was isolated from TERT-express-ing ATSCs at passage 5 or 15 (p5, p15). One microgram of RNAwas then analyzed for TERT expression using PCR primers spe-cific for human and endogenous rhesus TERT mRNA. The PCRproduct was separated on 1.5% agarose gel and visualized byethidium bromide staining. (B): Mean telomere length assessedby telomeric restriction fragment analysis of ATSCs at passage 5and ATSC-TERT cells at passage 15. Abbreviations:ATSC, adi-pose stromal cell line; PCR, polymerase chain reaction.


Figure 4. Confirmation of multipotential differentiation of TERT-expressing ATSCs. Cells (passage 5) were subjected to differentia-tion along adipogenic, osteogenic, chondrogenic, and neurogenic lineages in vitro. Adipogenic differentiation was induced, and accu-mulation of lipid vacuoles was visualized under the microscope after Oil red O staining (adipogenic). Mineralization of the extracellu-lar matrix was visualized by staining of the cultures with Alizarin red S and von Kossa reagents (osteogenic and chondrogenic). Toassess neural differentiation, the ATSC-TERT cells were tested for their ability to form neurospheres after plating density (neu-rospheres). The neurospheres were collected and underwent extensive neural differentiation when cultured on PDL-laminin for 10days and immunostained using neuronal lineage-specific antibody (TuJ1/DAPI). Abbreviations: ATSC, adipose stromal cell line;DAPI, 4',6'-diamidino-2-phenylindole.

Figure 5. Telomerase expression enhances osteoblast differentiation in vitro. Osteoblast differentiation of ATSC-TERT cells (A) andcontrol ATSCs (B), both at passage 6. Osteoblast differentiation was induced with L-ascorbate-2-phosphate, dexamethasone, and inor-ganic phosphate for 7–14 days. Cells were stained with Alzarin red S and von Kossa for detection of calcified deposits in both cell popu-lations after 7 days of osteoinduction. (C): The numbers of stained nodules in each population were counted in 25 random fields forquantification of differences in osteogenic differentiation potential. Abbreviation:ATSC, adipose stromal cell line.


Figure 6. Synthesis of bone and cartilage by ATSC-TERT cells and control ATSCs by immunohistochemistry for collagen I and II. (A):For chondrocyte differentiation, a pellet culture system was used. Approximately 3 ✕106 ATSC-TERT cells (passage 6) and controlATSCs (passage 6) were cultured in medium containing insulin, ascorbic acid, β-glycerophosphate, dexamethasone, and transforminggrowth factor β1 for 14–21 days. For microscopic analysis, the pellets were embedded in paraffin, cut into 5-µm sections, and stainedwith HE, von Kossa (calcium), and Toluidine blue (purple color is proteoglycan and blue color is background). (B): Paraffin-embeddedsections were stained for collagen synthesis using anti-collagen human type I and II antibodies, which were detected using fluoresceinisothiocyanate–conjugated anti-mouse secondary antibody. The nucleus was counterstained with TO-PRO 3. Abbreviations:ATSC,adipose stromal cell line; HE, hematoxylin-eosin.

Figure 7. In vivo bone formation by ATSC-TERT transplants. Cross-section of transplanted calcium phosphate scaffold or Matrigelscaffolds seeded with TERT-expressing ATSCs after 6 weeks. Left: Sections were stained with stained with HE. ATSC-TERT cellsgenerated higher amounts of bone formation at 6 weeks after transplantation. Right:Toluidine blue O staining for detection of chondro-cyte differentiation in a section from a ATSC-TERT implant. We were not able to detect newly formed bone in any sections from trans-planted control ATSCs. Abbreviations:ATSC, adipose stromal cell line; B, bone; HE, hematoxylin-eosin; M, marrow.


Table 1. Expressed gene profile of ATSCs and ATSC-TERT cells and partial list of genes that were upregulated in ATSC-TERT cells

Gene Accession Locus link Fold change

Regulation of cell-cycle genes

CDC28 protein kinase regulatory subunit 1B BC001425.1 1163 2.46

CDC42 effector protein (Rho GTPase binding) 3AL136842.1 10602 33.15

Cyclin-dependent kinase (CDC2-like) 10 NM_003674.1 8558 3.36

Cell division cycle 2-like 5 AA576621 8621 2.8(cholinesterase-related cell division controller)

S-phase response (cyclin-related) NM_006542.1 10638 3.22

Ceroid-lipofuscinosis, neuronal 8 NM_018941.1 2055 7.24(epilepsy, progressive with mental retardation)

Histone methyltransferase DOT1L AC004490 84444 73.29

BCL2-like 1 NM_001191.1 598 4.56

BCL2/adenovirus E1B 19-kDa interacting protein 3 U15174.1 664 8.72

Dickkopf homolog 1 (Xenopus laevis) NM_012242.1 22943 3.4

Hippocalcin-like 1 BE617588 3241 2.21

Endometrial bleeding–associated factor NM_003240.1 7044 5.69(transforming growth factor beta superfamily)

Islet amyloid polypeptide NM_000415.1 3375 2.36

Dedicator of cyto-kinesis 2 D86964.1 1794 6.75

Cyclin-dependent kinase 7 (MO15 homolog, L20320.1 1022 2.44X. laevis, cdk-activating kinase)

Enolase 1, (alpha) U88968.1 2023 2.28

Nucleotide binding genes

Ribosomal protein S4,Y-linked NM_001008.1 6192 84.02

Ribosomal protein S19 BC000023.1 6223 10.45

Transcription factor AP-2 alpha BF343007 7020 25.47(activating enhancer binding protein 2 alpha)

Centromere protein A, 17 kDa NM_001809.2 1058 42.93

Zinc finger protein 45 NM_003425.1 7596 112.1(a Kruppel-associated box [KRAB]

domain polypeptide)

Peroxisome proliferative-activated receptor, gamma NM_015869.1 5468 4.27

Zinc finger protein 272 X78931.1 10794 16.57

Leucyl-tRNA synthetase, mitochondrial NM_015340.1 23395 73.88

Mitogen-activated protein kinase 4 BF115223 5596 2.31

Chondroitin sulfate proteoglycan 4 NM_001897.1 1464 3.25(melanoma-associated)

DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9 BE910323 1660 2.33

Ras homolog enriched in brain 2 BF033683 6009 2.03

Cell fraction genes

Neutral sphingomyelinase (N-SMase) NM_003580.1 8439 2.29activation-associated factor

Insulin-like 4 (placenta) NM_002195.1 3641 3.37

Calcium channel, voltage-dependent, NM_000722.1 781 4.86alpha 2/delta subunit 1

Calmin (calponin-like, transmembrane) NM_024734.1 79789 3.04

Interferon, gamma-inducible protein 30 NM_006332.1 10437 23.57

src family–associated phosphoprotein 2 AB014486.1 8935 13.45

Complement component 3 NM_000064.1 718 11.19

(continued)


Motor activity genes

Actin, gamma 2, smooth muscle, enteric NM_001615.2 72 2.09

Amyloid beta precursor protein (cytoplasmic tail) AA046411 10513 2.29binding protein 2

Elastin (supravalvular aortic stenosis, AA479278 2006 7.91Williams-Beuren syndrome)

Catalytic activity genes

Nicotinamide N-methyltransferase NM_006169.1 4837 8.89

Procollagen-proline, 2-oxoglutarate 4-dioxygenase NM_004199.1 8974 2.45

Mitogen-activated protein kinase 9 W37431 5601 7.73

Angiotensin I converting enzyme AK026461.1 59272 7.65(peptidyl-dipeptidase A) 2

Protein phosphatase 1, regulatory subunit 3D AL109928 5509 15.23

4-Hydroxyphenylpyruvate dioxygenase NM_002150.1 3242 9.51

Gamma-glutamyltransferase-like 4 L20490.1 91227 3.16

Cathepsin Z AF073890.1 1522 6.79

Sialyltransferase 9 AF119418.1 8869 3.23

Enzyme inhibitor activity genes

Serine (or cysteine) proteinase inhibitor, clade B NM_002575.1 5055 70.72(ovalbumin), member 2

Calcium-binding protein 1 (calbrain) NM_004276.1 9478 3.99

Receptor activity genes

Leptin receptor U50748.1 3953 6.61

Tumor necrosis factor receptor superfamily, AK000485.1 8771 2.02member 6b, decoy

Wingless-type MMTV integration site family, AI968085 7474 4.22member 5A

Cholinergic receptor, nicotinic, alpha polypeptide 3 BC000513.1 1136 5.25

Transient receptor potential cation channel, NM_015930.1 51393 8.24subfamily V, member 2

Interleukin 21 receptor NM_021798.1 50615 6.97

Purinergic receptor P2X, ligand-gated ion channel, 3 NM_002559.1 5024 10.74

Retinoic acid receptor responder 1 NM_002888.1 5918 2.29

Tumor necrosis factor receptor superfamily, BF433902 4982 21.87

member 11b (osteoprotegerin)

Likely ortholog of mouse gene rich cluster,A gene NM_019858.1 27239 18.35

CD36 antigen (collagen type I receptor, M98399.1 948 61.74thrombospondin receptor)

Cytokine activity genes

Transforming growth factor, beta 1 NM_000660.1 7040 2.57

Transforming growth factor, beta 2 M19154.1 7042 3.85

Platelet-derived growth factor C NM_016205.1 56034 2.09

Platelet-derived growth factor beta polypeptide NM_002608.1 5155 4.81

Brain-derived neurotrophic factor NM_001709.1 627 2.68

Neurotrophin 3 NM_002527.2 4908 2.12

Fibroblast growth factor 2 (basic) M27968.1 2247 2.09

(continued)

Table 1. (continued)


upregulated in ATSC-TERT, and that was consistent with our

Affymetrix Microarray result (data not shown).

DiscussionTelomerase is not an oncogene product, and as such its pres-

ence permits proliferation but does not cause an uncontrolled

proliferation or immortalization [12, 34]. Our study has iden-

tified that ectopic hTERT extends the ATSCs lifespan in vitro

and highly enhances osteogenic differentiation of ATSCs.

ATSC-TERT cells were capable of maintaining their prolifer-

ation rate through passage 50 in vitro. This result suggests that

activation of telomerase in ATSC-TERT cells may trigger

internal signals that contribute to maintenance of the prolifer-

ative capability of ATSC-TERT cells over time. The mecha-

nism by which these cell-cycle genes regulate proliferation

and differentiation of ATSCs is largely unknown. However,

the ability of ectopic hTERT to extend lifespan may be related

to the site of integration and the levels of telomere or telom-

erase-associated proteins in a cell type–specific manner. Pre-

viously researchers demonstrated that ectopic expression

of telomerase could increase the osteo-genic capacity of

BMSCs and correlate with a significant elevation in number of

cells expressing the surface antigen, STRO-1, an early marker

of osteogenic precursor cells. Also, human BMSC-telomerase

(BMSC-Ts) displayed an accelerated capacity for osteogenic

differentiation in vivo [5, 31, 32]. BMSC-Ts consistently

exhibited high expression levels for the osteoblastic-associ-

ated markers CBFA1, osterix, and osteocalcin [31]. In our


Cell adhesion molecule activity genes

Integrin, alpha 3 (antigen CD49C, alpha 3 subunit NM_002204.1 3675 2.29of VLA-3 receptor)

Laminin, beta 1 NM_002291.1 3912 2.91

Laminin, gamma 2 NM_005562.1 3918 2.6

Tenascin C (hexabrachion) NM_002160.1 3371 3.33

Intercellular adhesion molecule 1 (CD54), AI608725 3383 2.56human rhinovirus receptor

Vascular cell adhesion molecule 1 NM_001078.1 7412 7.01

Neuronal cell adhesion molecule NM_005010.1 4897 2.01

BH-protocadherin (brain-heart) NM_002589.1 5099 2.44

Osteoblast-specific factor 2 (fasciclin I-like) D13665.1 10631 14.12

Biglycan NM_001711.1 633 2.23

Collagen, type VI, alpha 2 AL531750 1292 2.52

Collagen, type IV, alpha 6 AI889941 1288 6.15

Collagen, type VIII, alpha 1 BE877796 1295 2.34

Collagen, type VII, alpha 1 NM_000094.1 1294 13.93

Protocadherin beta 8 NM_019120.1 56128 328.47

Transporter activity genes

Solute carrier family 29 (nucleoside transporters), AF034102.1 3177 439.87member 2

Adaptor-related protein complex 4, epsilon 1 NM_007347.1 23431 45.52subunit

Solute carrier family 6 (neurotransmitter BC006252.1 6533 8.79transporter, taurine), member 6

Tight junction protein 3 (zona occludens 3) AC005954 27134 10.75

Syntaxin-binding protein 2 AB002559.1 6813 3.23

Synaptic transmission genes

Glutamate receptor, ionotrophic,AMPA 3 NM_007325.1 2892 2.66

Sodium channel, voltage-gated, type XI, alpha AF150882.1 11280 8.14polypeptide

Abbreviation:ATSC, adipose stromal cell line.




Table 2. Expressed gene profile of ATSCs and ATSC-TERT cells and partial list of genes that were downregulated in ATSC-TERTcells


DNA replication and chromosome cycle genes

ATP-binding cassette, sub-family C NM_020297 10060 -14.49(CFTR/MRP), member 9

E2F transcription factor 1 NM_005225 1869 -3.58

Protamine 1 NM_002761. 5619 -10.3

Protein phosphatase 2 (formerly 2A), regulatory AA974416 5521 -26.24subunit B (PR 52), beta isoform

Nucleotide-binding genes

Adrenergic, beta, receptor kinase 1 NM_001619.2 156 -14.84

Short-chain dehydrogenase/reductase 1 NM_004753 9249 -13.68

Phosphodiesterase 10A AF127480.1 10846 -10.07

ATPase, class II, type 9A AB014511.1 10079 -15.86

Chemokine (C-X-C motif) receptor 4 L01639.1 7852 -12.52

Cell fraction genes

Tyrosinase-related protein 1 NM_000550 7306 -9.06

CDW52 antigen (CAMPATH-1 antigen) NM_001803 1043 -12.17

WNT1-inducible signaling pathway protein 2 NM_003881 8839 -14.23

Myelin-associated oligodendrocyte basic protein NM_006501 4336 -9.26

Peripheral myelin protein 22 L03203.1 5376 -4.05

Phosphodiesterase 4D, cAMP-specific AF012074.1 5144 -13.09

Calcitonin/calcitonin-related polypeptide, alpha X15943 796 -18.17

Proline arginine-rich end leucine-rich repeat protein U41344 5549 -93.73

msh homeo box homolog 2 (Drosophila) NM_002449.2 4488 -288.74

Distal-less homeo box 6 NM_005222 1750 -25.33

Receptor activity genes

Neuropeptide Y receptor Y1 NM_000909 4886 -6.8

Opiate receptor-like 1 NM_000913 4987 -115.68

5-Hydroxytryptamine (serotonin) receptor 2B NM_000867 3357 -28.68

Nuclear receptor subfamily 4, group A, member 3 U12767.1 8013 -12.13

Nuclear receptor subfamily 4, group A, member 1 D49728.1 3164 -12.89

Follicle-stimulating hormone receptor M95489.1 2492 -15.23

GDNF family receptor alpha 3 NM_001496 2676 -4.01

Transferrin receptor 2 AK022002.1 7036 -5.08

Proline-rich protein BstNI subfamily 4 X07882 5545 -20.48

T-cell receptor alpha locus AE000659 6955 -63.9

Interphotoreceptor matrix proteoglycan 2 NM_016247 50939 -71.3

Cannabinoid receptor 1 (brain) U73304 1268 -8.11

G-protein–coupled receptor 88 NM_022049 54112 -34.07

Neuropeptide FF 1; RFamide-related peptide receptor NM_022146 64106 -3.46

Tetraspan 5 AA748177 10098 -14.49

Transcription factor activity genes

Basic transcription element binding protein 1 NM_001206 687 -11.62

Early growth response 3 NM_004430 1960 -17.56

Zinc finger protein 141 (clone pHZ-44) NM_003441 7700 -14.73

Zinc finger protein, X-linked NM_003410 7543 -13.74

(continued)


RAD23 homolog B (Saccharomyces cerevisiae) T93562 5887 -105.51

Zinc finger protein 236 AK000847.1 7776 -22.56

Tumor necrosis factor, alpha-induced protein 3 NM_006290 7128 -5.48

v-maf musculoaponeurotic fibrosarcoma oncogene NM_005461 9935 -51.91homolog B (avian)

Zinc finger protein, multitype 2 NM_012082.2 23414 -33.29

Motor activity genes

Myosin, heavy polypeptide 2, skeletal muscle, adult NM_017534 4620 -42.91

Myosin, heavy polypeptide 1, skeletal muscle, adult NM_005963.2 4619 -285.94

Myosin, heavy polypeptide 4, skeletal muscle NM_017533 4622 -18.85

Myosin ID AA621962 4642 -10.6

Dynamin 1 L07810.1 1759 -20.53

Supervillin NM_003174.2 6840 -13.43

Syntrophin, gamma 2 NM_018968 54221 -105.24

Catalytic activity genes

Glutamate-ammonia ligase (glutamine synthase) NM_002065 2752 -16.26

Creatine kinase, brain NM_001823 1152 -2.67

Aldehyde dehydrogenase 1 family, member A3 NM_000693 220 -12.6

Prostaglandin-endoperoxide synthase 1 NM_000962 5742 -39.38

Acylphosphatase 1, erythrocyte (common) type NM_001107 97 -66.02

Acetylcholinesterase (YT blood group) AI190022 43 -3.51

Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 NM_003654 8534 -52.51

Caspase 1, apoptosis-related cysteine protease AI719655 834 -2.38(interleukin 1, beta, convertase)

Alcohol dehydrogenase 1C (class I), gamma polypeptide NM_000669.2 126 -12.64

Sialyltransferase 8E (alpha-2, 8-polysialyltransferase) NM_013305 29906 -5.9

Sialyltransferase 4A (beta-galactoside alpha-2,3- NM_003033 6482 -5.92sialyltransferase)

PI-3-kinase-related kinase SMG-1 U32581.2 23049 -10.11

Retinol dehydrogenase 5 (11-cis and 9-cis) U43559.1 5959 -11.54

Monoglyceride lipase BC006230.1 11343 -14.12

NADPH oxidase 1 NM_007052.2 27035 -19.56

Peptidase activity genes

Matrix metalloproteinase 9 (gelatinase B, 92kDa NM_004994 4318 -5.55

gelatinase, 92kDa type IV collagen

Matrix metalloproteinase 1 (interstitial collagenase) NM_002421.2 4312 -108.59

Matrix metalloproteinase 3 (stromelysin 1, progelatinase) NM_002422.2 4314 -15.58

A disintegrin and metalloproteinase domain 20 AF029899.1 8748 -12.51

A disintegrin and metalloproteinase domain 19 (meltrin beta) Y13786.2 8728 -4.92

A disintegrin-like and metalloprotease (reprolysin type) AB002364.1 9508 -4.91with thrombospondin type

Transmembrane protease, serine 4 NM_016425 56649 -26.59

Kallikrein 14 NM_022046 43847 -46.32

Protein kinase activity genes

Neuropilin 1 BE620457 8829 -7.86

Mitogen-activated protein kinase kinase 2 AI762811 5605 -9.48

(continued)



study,ATSC-TERT cells showed markedly increased expres-

sion levels for the osteogenesis-related markers sialyltrans-

ferase, osteoprotegerin, osteoblast specific factor 2, and bigly-

can. Affymetrix cDNA gene expression analysis revealed that

fewer than 1% of the total genes were expressed at greater than

2.2-fold different levels in ATSCs and ATSC-TERT cells.

These included regulation of cell cycle, cell fraction, receptor

activity, transcription factor, motor activity, catalytic activity,

enzyme inhibitor, receptor and cytokine activity, cell adhesion

molecule, signal transducer, and transporter activity–related

genes (Tables 1, 2). Interestingly, ATSC-TERT retained the

ability to undergo neurogenic, osteogenic, adipogenic, and

chondrogenic differentiation in vitro (Fig. 4).

hTERT has been increasingly recognized to effect cellu-

lar functions other than proliferation. We found that ATSC-

TERT maintained both expression of osteoblastic markers

and differentiation potential. hTERT expression also

enhanced the bone-forming ability of ATSCs (Figs. 5, 6). In

agreement with this, ectopic expression of hTERT in senes-

cent fibroblasts restored their functional capacity in a dermal

reconstitution model [35]. Similarly, hTERT expression in

human endothelial cells did not affect their phenotype and




PTK9 protein tyrosine kinase 9 AW665024 5756 -16.83

Fibroblast growth factor receptor 1 AK024388.1 2260 -2.46(fms-related tyrosine kinase 2, Pfeiffer syndrome)

Chemokine (C-C motif) ligand 2 S69738.1 6347 -5.05

Neurotrophic tyrosine kinase, receptor, type 2 AA707199 4915 -5.4

Enzyme inhibitor activity genes

Serine (or cysteine) proteinase inhibitor, clade I (neuroserpin), NM_005025 5274 -15.48member 1

Tissue factor pathway inhibitor 2 L27624.1 7980 -18.79

Alpha-2-macroglobulin NM_000014.3 2 -30.19

Serine (or cysteine) proteinase inhibitor NM_016186. 51156 -11.07

Signal transducer activity genes

Transforming growth factor, beta receptor III NM_003243 7049 -3.36(betaglycan, 300 kDa)

SH3 domain-binding glutamic acid-rich protein NM_007341 6450 -18.17

Growth hormone receptor NM_000163 2690 -8.98

Sialophorin (gpL115, leukosialin, CD43) X52075 6693 -7.19

Gamma-aminobutyric acid receptor, rho 1 NM_002042 2569 -6.48

Glutamate receptor, metabotropic 7 NM_000844 2917 -7.73

Ephrin-A3 AW189015 1944 -2.93

Glucagon receptor U03469.1 2642 -3.02

CD44 antigen (homing function and Indian blood group system) AV700298 960 -2.67

Regulator of G-protein signaling 5 NM_025226 8490 -115.68

Insulin-like 5 NM_005478.2 10022 -173.37

Neuroepithelial cell-transforming gene 1 NM_005863 10276 -10.88

Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6 D25304.1 9459 -63.9

RAS guanyl releasing protein 2 (calcium and DAG-regulated) AI688812 10235 -5.08

Regulator of G-protein signaling 12 AF030110.1 6002 -26.59

Osteoglycin (osteoinductive factor, mimecan) NM_014057 4969 -5.25

Cell adhesion molecule activity genes

Thrombospondin 2 NM_003247 7058 -9.01

Cadherin 2, type 1, N-cadherin (neuronal) NM_001792 1000 -2.86

Thrombospondin 4 NM_003248 7060 -4.82

Abbreviation:ATSC, adipose stromal cell line.

enhanced their ability to form microvascular structures [26].

These findings suggest that cellular dysfunction associated

with replicative senescence is linked to telomere shortening.

Like other cell types that ectopically express hTERT,ATSCs-

TERT did not form tumors when implanted in immune-defi-

cient mice. Furthermore, chromosomal analysis showed a

normal karyotype and no evidence of abnormalities associ-

ated with malignancy [9]. In our study,ATSC-TERT implan-

tation into immunodeficient mice also showed enhanced bone

formation capacity compared with control ATSCs, and they

did not form tumors subcutaneously. The nonclonal and tran-

sient chromosomal abnormalities reported in some cell lines

that ectopically express hTERT therefore seem to be rare

events. Assuming that ATSC-TERT cells maintain a normal

phenotype, hTERT reactivation may be useful for tissue

regeneration and engineering [36, 37]. Understanding of

the hTERT-activated pathways controlling bone formation

may also lead therapeutic approaches for preventing bone

loss during aging and in osteoporosis. TERT-expressing cells

also maintain their adipogenic and chondrogenic abilities

[38]; they may be useful in engineering other connective

tissues.

AcknowledgmentsWe are grateful to Cynthia Trygg for supporting basic experi-

ments. The work was supported by grant RR00164 from the

National Center for Research Resources, National Institutes

of Health, and a grant from the State of Louisiana Millen-

nium Health Excellence Fund and the Louisiana Gene Ther-

apy Research Consortium.


References

1 Zhu J, Wang H, Bishop JM et al. Telomerase extends thelifespan of virus-transformed human cells without nettelomere lengthening. Proc Natl Acad Sci U S A 1999;96:3723–3728.

2 Blackburn EH. Structure and function of telomerase.Nature 1991;350:569–573.

3 Yamada O,Akiyama M, Kawauchi K et al. Overexpressionof telomerase confers a survival advantage through suppres-sion of TRF1 gene expression while maintaining differenti-ation characteristics in K562 cells. Cell Transplant 2003;12:365–377.

4 Moyzis RK, Buckingham JM, Cram LS et al. A highly con-served repetitive DNA sequence, (TTAGGG)n, present atthe telomeres of human chromosomes. Proc Natl Acad SciU S A 1988;85:6623–6626.

5 Shi S, Gronthos S, Chen S et al. Bone formation by humanpostnatal bone marrow stromal stem cells is enhanced bytelomerase expression. Nat Biotechnol 2002;20:587–591.

6 Simonsen JL, Rosada C, Serakini N et al. Telomeraseexpression extends the proliferative life-span and maintainsthe osteogenic potential of human bone marrow stromalcells. Nat Biotechnol 2002;20:592–596.

7 Saldanha SN,Andrews LG, Tollefsbol TO et al. Analysis oftelomerase activity and detection of its catalytic subunit,hTERT. Anal Biochem 2003;315:1–21.

8 Harley CD, Futcher AB, Greider CW et al. Telomeresshorten during aging of human fibroblasts. Nature 1990;345:458–460.

9 Vaziri H, Schachter F, Uchida I et al. Loss of telomeric DNAduring aging of normal and trisomy 21 human lymphocytes.Am J Hum Genet 1993;52:661–667.

10 Prescott JC, Blackburn EH. Telomerase: Dr. Jekyll or Mr.Hyde? Curr Opin Genet Dev 1999;9:368–373.

11 Meyerson M, Counter CM, Eaton EN et al. hEST2, the puta-tive human telomerase catalytic subunit gene, is up-regu-lated in tumor cells and during immortalization. Cell1997;90:785–795.

12 Urqidi V, Tarin D, Goodison S et al. Role of telomerase incell senescence and oncogenesis. Annu Rev Med 2000;51:65–79.

13 Vaziri H, Benchimol S. Reconstitution of telomerase activ-ity in normal human cells leads to elongation of telomeresand extended replicative life span. Curr Biol 1998;8:279–282.

14 Yang J, Chang E, Cherry AM et al. Human endothelial celllife extension by telomerase expression. J Biol Chem1999;274:26141–26148.

16 Kassemm M,Ankersen L, Eriksen EF et al. Demonstrationof cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. OsteoporosInt 1997;7:514–524.

17 Caplan AI, Bruder SP. Mesenchymal stem cells: buildingblocks for molecular medicine in the 21st century. TrendsMol Med 2001;7:259–264.

18 Krebsbach PH, Kuznetsov SA, Satomura K et al. Bone for-mation in vivo: comparison of osteogenesis by transplantedmouse and human marrow stromal fibroblasts. Transplanta-tion 1997;63:1059–1069.

19 Reddi AH. Morphogenesis and tissue engineering of boneand cartilage: inductive signals, stem cells, and biomimeticbiomaterials. Tissue Eng 2000;6:351–359.

20 Kang SK, Lee DH, Bae YC et al. Improvement of neurologi-cal deficits by intracerebral transplantation of human adi-pose tissue-derived stromal cells after cerebral ischemia inrats. Exp Neurol 2003;183:355–366.

21 Safford KM, Hicok KC, Safford SD et al. Neurogenic dif-ferentiation of murine and human adipose-derived stromalcells. Biochem Biophys Res Commun 2002;294:371–379.

21 Parfitt AM. Bone forming cells in clinical conditions. In:Hall BK, ed. The Osteoblast and Osteocyte. London:Telford, 1990:351–426.

22 Zuk PA, Zuh M, Mizuno H et al. Multilineage cells fromhuman adipose tissue: implications for cell-based therapies.Tissue Eng 2001;7:211–228.

23 Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is asource of multipotent stem cells. Mol Biol Cell 2002;13:4279–4295.

24 Bodnar AG, Ouellette M, Frolkis M et al. Extension of life-span by introduction of telomerase into normal human cells.Science 1998;279:349–352.

25 Dickson MA, Hahn WC, Ino Y et al. Human keratinocytesthat express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortalyet retain normal growth and differentiation characteristics.Mol Cell Biol 2000;20:1436–1447.

26 Yang J, Nagavarapu U, Relloma K et al. Telomerized humanmicrovasculature is functional in vivo. Nat Biotechnol2001;19:219–224.

27 Ilsopp RC, Morin GB, Horner JW et al. Effect of TERTover-expression on the long-term transplantation capacityof hematopoietic stem cells. Nat Med 2003;9:369–371.

28 Condon J,Yin S, Mayhew B et al. Telomerase immortaliza-tion of human myometrial cells. Biol Reprod 2002;67:506–514.

29 Di Donna S, Mamchaoui K, Cooper RN et al. Telomerasecan extend the proliferative capacity of human myoblasts,but does not lead to their immortalization. Mol Cancer Res2003;1:643–653.

30 Funk WD, Wang CK, Shelton DN et al. Telomerase expres-sion restores dermal integrity to in vitro-aged fibroblasts in areconstituted skin model. Exp Cell Res 2000;258:270–278.

31 Gronthos S, Chen S, Wang CY et al. Telomerase acceleratesosteogenesis of bone marrow stromal stem cells by upregu-lation of CBFA1, Osterix, and Osteocalcin. J Bone MinerRes 2003;18:716–722.

32 Gronthos S, Mankani M, Brahim J et al. Postnatal humandental pulp stem cells (DPSCs) in vitro and in vivo. ProcNatl Acad Sci U S A 2000;97:13625–13630.

33 Harada S, Rodan GA. Control of osteoblast function andregulation of bone mass. Nature 2003;423:349–355.

34 Jiang XR, Jimenez G, Chang E et al. Telomerase expressionin human somatic cells does not induce changes associatedwith a transformed phenotype. Nat Genet 1999;21:111–114.

35 Morales CP, Holt SE, Ouellette M et al. Absence of cancer-associated changes in human fibroblasts immortalized withtelomerase. Nat Genet 1999;21:115–118.

36 Shay JW,Wright WE. The use of telomerized cells for tissueengineering. Nat Biotechnol 2000;18:22–23.

37 Tuan R. Boning up on telomerase. Nat Biotechnol 2002;20:560–561.

38 Zheng Q, Zhou G, Morello R et al. Type X collagen generegulation by Runx2 contributes directly to its hypertrophicchondrocytes-specific expression in vivo. J Cell Biol2003;162:833–842.


Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differentiation of Adipose...

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