Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differentiation of Adipose Tissue–Derived Stromal Cells

Authors

  • Soo Kyung Kang,

    1. Division of Gene Therapy, Tulane National Primate Center, Tulane University Health Sciences Center, Covington, Louisiana, USA
    2. Center of Gene Therapy, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA
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  • Lorna Putnam,

    1. Center of Gene Therapy, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA
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  • Jason Dufour,

    1. Division of Veterinary Medicine, Tulane National Primate Center, Tulane University Health Sciences Center, Covington, Louisiana, USA
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  • Joni Ylostalo,

    1. Center of Gene Therapy, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA
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  • Jin Sup Jung,

    1. Department of Physiology, College of Medicine, Busan National University, Busan, South Korea
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  • Bruce A. Bunnell Ph.D.

    Corresponding author
    1. Division of Gene Therapy, Tulane National Primate Center, Tulane University Health Sciences Center, Covington, Louisiana, USA
    2. Center of Gene Therapy, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA
    3. Department of Pharmacology, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA
    • 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, LA 70433, USA. Telephone: 985-871-6594; Fax: 985-871-6587
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Abstract

Expression of TERT, the catalytic protein subunit of the telomerase complex, can be used to generate cell lines that expand indefinitely and retain multilineage potential. We have created immortal adipose stromal cell lines (ATSCs) by stably transducing nonhuman primate-derived ATSCs with a retroviral vector expressing TERT. Transduced cells (ATSC-TERT) had an increased level of telomerase activity and increased mean telomere length in the absence of malignant cellular transformation. Long-term culture of the ATSC-TERT cells demonstrated that the cells retain the ability to undergo differentiation along multiple lineages such as adipogenic, chondrogenic, and neurogenic. Untransduced cells demonstrated markedly reduced multilineage and self-renewal potentials after 12 passages in vitro. To determine the functional role of telomerase during osteogenesis, we examined osteogenic differentiation potential of ATSC-TERT cells in vitro. Compared with naive ATSCs, which typically begin to accumulate calcium after 3–4 weeks of induction by osteogenic differentiation medium, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of culture in osteogenic induction medium. The cells have increased production of osteoblastic markers, such as AP2, osteoblast-specific factor 2, chondroitin sulfate proteoglycan 4, and the tumor necrosis factor receptor superfamily, compared with control ATSCs, indicating that telomerase expression may aid in maintaining the osteogenic stem cell pool during in vitro expansion. These results show that ectopic expression of the telomerase gene in nonhuman primate ATSCs prevents senescence-associated impairment of osteoblast functions and that telomerase therapy may be a useful strategy for bone regeneration and repair.

Introduction

The 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 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 components [511]. 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, 1214].

Possible mechanisms of age-dependent bone loss may be attributed, at least in part, to a deficiency of osteoblast function 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 cellular senescence may contribute to various age-related disorders. 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 [1719].

The regeneration of diseased or damaged tissue is the principle goal of the emerging discipline of tissue engineering. A key requirement in tissue regeneration is the availability 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 [2023]. Recently, our group and others reported that human and nonhuman primate-derived ATSCs and BMSCs can propagate in vitro and contain detectable levels of telomerase 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 completely compensate for the reduction of telomere length during continuous in vitro subculture. To extend the proliferative 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 telomeres. Reconstitution of telomerase activity through expression of exogenous hTERT enables normal human fibro-blasts, as well as retinal epithelial, myometrial, and endothelial cells, to avoid senescence [2430]. After ectopic expression of telomerase, the lifespan of BMSCs was significantly 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, 3133]. 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 Retroviruses

The 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 containing amphotropic viruses were collected and then used to infect primary cultures of ATSCs.

Isolation, Culture, and Transduction of ATSCs

Nonhuman 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 extensively with equal volumes of phosphate-buffered saline (PBS) and digested at 37°C for 30 minutes with 0.075% collagenase 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 pellet, the pellet was resuspended in red blood cell (RBC) lysis buffer (Bio Whittaker, Walkersville, MD) and incubated at room temperature for 10 minutes to lyse contaminating RBCs. The stromal cell pellet was collected by centrifugation, as described above, and incubated overnight at 37°C/5% CO2 in 10% FBS containing α-MEM medium. After 1 day of culture, cells were subcultured onto six-well plates and 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 Biologicals, Lawrenceville, GA).

Nonradioisotopic Telomerase Assay

Telomerase activity was analyzed using a modified telomeric repeat amplification protocol (TRAP) assay according to the manufacturer's instructions (BD Science, San Diego). Protein 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 reaction products were separated on 12.5% nondenaturing acrylamide gels and stained using Syber-Gold dye (Molecular Probes, Eugene, OR). Quantification of telomerase for comparisons with telomerase activity in ATSC-TERT and ATSCs was performed by the PCR enzyme-linked immunosorbent assay procedure suggested by the manufacturer (BD Science).

Telomere Length Analysis and Telomerase Reverse Transcription–PCR

Telomere length was analyzed by determining the mean length of the terminal restriction fragments (TRFs) by Southern 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 transcription (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, and Chondrogenic Differentiation

To verify the multipotential differentiation of mesenchymal characteristics of ATSC-TERT, cells were subjected to differentiation 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 supplemented 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 differentiation, 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 visualized 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 differentiation, a pellet culture system was performed. Approximately 3 × 106 ATSC-TERT and ATSCs controls (PD 10) were placed in a well of a 96-well plate. The pellet was cultured at 37°C 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 control 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, Germany) for 15 minutes at 37°C and subsequently with 1 mg/ml of pronase (Roche, Grenzacherstrasse, Basel, Switzerland) for 30 minutes at 37°C. Nonspecific background was blocked using PBS containing 10% goat serum for 1 hour. Sections were incubated overnight at 4°C 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 isothiocyanate–conjugated anti-mouse secondary antibody (1:200; Molecular Probes) for 30 minutes at room temperature and TO-PRO 3 (1:1000; Molecular Probes) for 3 minutes 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-TERT

Undifferentiated 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 factor (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 maintained for 10 days in NB media containing only the B27 supplement. During differentiation, 70% of the media was replaced every 4 days.

In Vivo Bone Formation and Tumorigenesis Assay

For 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 accordance with specifications of an approved protocol. The transplants 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 Analysis

Control cells (ATSCs) and ATSC-TERT cells were harvested, 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 oligonucleotide 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. Fragmented cRNA (15 μg) was hybridized for 16 hours at 45°C 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-phycoerythrin by using a fluidics station. Finally, the probe arrays were scanned at a 3-μm resolution using the Genechip System confocal scanner made for Affymetrix by Aligent.

Data Analysis

Affymetrix Microarray Suite 4 was used to scan and analyze the relative abundance of each gene as derived from the average difference of intensities. Analysis parameters used by the software were set to values corresponding to moderate stringency. 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 definition 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 expression for individual genes was merged with the Unigene or Gene Bank descriptor and stored as an Excel data spreadsheet. 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, marginal increase/decrease = 1/–1, increase/decrease = 2/–2). The final rank referred to summing up the two values corresponding 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 coefficient 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 Telomerase Expression in ATSC-TERT Cells

During prolonged periods of culture, the population of control 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 contact. The results indicate that the immortalization of the stromal 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).

Figure Figure 1..

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

Figure Figure 2..

Telomerase expression in control and hTERT-expressing ATSCs. (A): Immunocytochemistry of hTERT in ATSC-TERT clones 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-linked immunosorbent assay procedure. Abbreviation: ATSC, adipose stromal cell line.

Telomere Length Analysis of ATSC-TERT Cells

It has been proposed previously that critically shortened telomeres mediate massive genomic instability and contribute 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 considerably shorter than in control cells in crisis (data not shown).

Figure Figure 3..

Expression of vector-derived hTERT and telomere length analysis. (A): Detection of hTERT by reverse transcription–PCR. Total cellular RNA was isolated from TERT-expressing ATSCs at passage 5 or 15 (p5, p15). One microgram of RNA was then analyzed for TERT expression using PCR primers specific for human and endogenous rhesus TERT mRNA. The PCR product was separated on 1.5% agarose gel and visualized by ethidium bromide staining. (B): Mean telomere length assessed by telomeric restriction fragment analysis of ATSCs at passage 5 and ATSC-TERT cells at passage 15. Abbreviations: ATSC, adipose stromal cell line; PCR, polymerase chain reaction.

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 differences between ATSC control and ATSC-TERT cells were detected. ATSC-TERT cells retained the ability to accumulate lipid droplets typical for the adipocyte phenotype, and they maintained osteogenic and chondrogenic differentiation 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 morphologic change from elongated fibroblast morphology to compact, spheroid bodies, which expand to larger spheroid bodies 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 surface, 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 secondary branching with multiple extensions (Fig. 4, Tuj/ DAPI).

Figure Figure 4..

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

Enhanced In Vitro Osteogenesis and Chondrogenesis by ATSC-TERT

ATSCs are known progenitors of skeletal tissues and differentiate into osteoblast-like cells in culture supplemented with ascorbic acid and a source of glucocorticoid. However, ATSCs lose their osteogenic capacity during continuous subculture 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 during osteogenesis, we examined the osteogenic differentiation potential of ATSC-TERT cells in vitro. ATSCs typically 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 analysis (data not shown).

Figure Figure 5..

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

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.

Figure 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 control ATSCs (passage 6) were cultured in medium containing insulin, ascorbic acid, β-glycerophosphate, dexamethasone, and transforming growth factor β1 for 14–21 days. For microscopic analysis, the pellets were embedded in paraffin, cut into 5-μm sections, and stained with HE, von Kossa (calcium), and Toluidine blue (purple color is proteoglycan and blue color is background). (B): Paraffin-embedded sections were stained for collagen synthesis using anti-collagen human type I and II antibodies, which were detected using fluorescein isothiocyanate–conjugated anti-mouse secondary antibody. The nucleus was counterstained with TO-PRO 3. Abbreviations: ATSC, adipose stromal cell line; HE, hematoxylin-eosin.

In Vivo Osteogenesis after Engraftment of ATSC-TERT Cells

We studied in vivo osteogenesis effects after implantation of ATSC-TERT subcutaneously with Matrigel and hydroxyapatite scaffolds in immunedeficient 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 formation 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).

Figure Figure 7..

In vivo bone formation by ATSC-TERT transplants. Cross-section of transplanted calcium phosphate scaffold or Matrigel scaffolds seeded with TERT-expressing ATSCs after 6 weeks. Left: Sections were stained with stained with HE. ATSC-TERT cells generated 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 transplanted control ATSCs. Abbreviations: ATSC, adipose stromal cell line; B, bone; HE, hematoxylin-eosin; M, marrow.

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 relative 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 upregulated in ATSC-TERT, and that was consistent with our Affymetrix Microarray result (data not shown).

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

    Abbreviation: ATSC, adipose stromal cell line.

GeneAccessionLocus linkFold change
    CDC28 protein kinase regulatory subunit 1BBC001425.111632.46
    CDC42 effector protein (Rho GTPase binding)3AL136842.11060233.15
    Cyclin-dependent kinase (CDC2-like)10 NM_003674.185583.36
    Cell division cycle 2-like 5 (cholinesterase-related cell division controller)AA57662186212.8
    S-phase response (cyclin-related)NM_006542.1106383.22
    Ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation)NM_018941.120557.24
    Histone methyltransferase DOT1LAC0044908444473.29
    BCL2-like 1NM_001191.15984.56
    BCL2/adenovirus E1B 19-kDa interacting protein 3U15174.16648.72
    Dickkopf homolog 1 (Xenopus laevis)NM_012242.1229433.4
    Hippocalcin-like 1BE61758832412.21
    Endometrial bleeding–associated factor (transforming growth factor beta superfamily)NM_003240.170445.69
    Islet amyloid polypeptideNM_000415.133752.36
    Dedicator of cyto-kinesis 2D86964.117946.75
    Cyclin-dependent kinase 7 (MO15 homolog, X. laevis, cdk-activating kinase)L20320.110222.44
    Enolase 1, (alpha)U88968.120232.28
    Ribosomal protein S4, Y-linkedNM_001008.1619284.02
    Ribosomal protein S19BC000023.1622310.45
    Transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)BF343007702025.47
    Centromere protein A, 17 kDaNM_001809.2105842.93
    Zinc finger protein 45 (a Kruppel-associated box [KRAB] domain polypeptide)NM_003425.17596112.1
    Peroxisome proliferative-activated receptor, gammaNM_015869.154684.27
    Zinc finger protein 272X78931.11079416.57
    Leucyl-tRNA synthetase, mitochondrialNM_015340.12339573.88
    Mitogen-activated protein kinase 4BF11522355962.31
    Chondroitin sulfate proteoglycan 4 (melanoma-associated)NM_001897.114643.25
    DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9BE91032316602.33
    Ras homolog enriched in brain 2BF03368360092.03
    Neutral sphingomyelinase (N-SMase) activation-associated factorNM_003580.184392.29
    Insulin-like 4 (placenta)NM_002195.136413.37
    Calcium channel, voltage-dependent, alpha 2/delta subunit 1NM_000722.17814.86
    Calmin (calponin-like, transmembrane)NM_024734.1797893.04
    Interferon, gamma-inducible protein30 NM_006332.11043723.57
    src family–associated phosphoprotein 2AB014486.1893513.45
    Complement component 3NM_000064.171811.19
    Actin, gamma 2, smooth muscle, entericNM_001615.2722.09
    Amyloid beta precursor protein (cytoplasmic tail) binding protein 2AA046411105132.29
    Elastin (supravalvular aortic stenosis, Williams-Beuren syndrome)AA47927820067.91
    Nicotinamide N-methyltransferaseNM_006169.148378.89
    Procollagen-proline, 2-oxoglutarate 4-dioxygenaseNM_004199.189742.45
    Mitogen-activated protein kinase 9W3743156017.73
    Angiotensin I converting enzyme (peptidyl-dipeptidaseA) 2AK026461.1592727.65
    Protein phosphatase 1, regulatory subunit 3DAL109928550915.23
    4-Hydroxyphenylpyruvate dioxygenaseNM_002150.132429.51
    Gamma-glutamyltransferase-like 4L20490.1912273.16
    Cathepsin ZAF073890.115226.79
    Sialyltransferase 9AF119418.188693.23
    Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2NM_002575.1505570.72
    Calcium-binding protein 1 (calbrain)NM_004276.194783.99
    Leptin receptorU50748.139536.61
    Tumor necrosis factor receptor superfamily, member 6b, decoyAK000485.187712.02
    Wingless-type MMTV integration site family, member 5AAI96808574744.22
    Cholinergic receptor, nicotinic, alpha polypeptide 3BC000513.111365.25
    Transient receptor potential cation channel, subfamilyV, member 2NM_015930.1513938.24
    Interleukin 21 receptorNM_021798.1506156.97
    Purinergic receptor P2X, ligand-gated ion channel, 3NM_002559.1502410.74
    Retinoic acid receptor responder 1NM_002888.159182.29
    Tumor necrosis factor receptor super family, member 11b (osteoprotegerin)BF433902498221.87
    Likely ortholog of mouse gene rich cluster, A geneNM_019858.12723918.35
    CD36 antigen (collagen type I receptor, thrombospondin receptor)M98399.194861.74
    Transforming growth factor, beta 1NM_000660.170402.57
    Transforming growth factor, beta 2M19154.170423.85
    Platelet-derived growth factor CNM_016205.1560342.09
    Platelet-derived growth factor beta polypeptideNM_002608.151554.81
    Brain-derived neurotrophic factorNM_001709.16272.68
    Neurotrophin 3NM_002527.249082.12
    Fibroblast growth factor 2 (basic)M27968.122472.09
    Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)NM_002204.136752.29
    Laminin, beta 1NM_002291.139122.91
    Laminin, gamma 2NM_005562.139182.6
    Tenascin C (hexabrachion)NM_002160.133713.33
    Intercellular adhesion molecule 1 (CD54), human rhinovirus receptorAI60872533832.56
    Vascular cell adhesion molecule 1NM_001078.174127.01
    Neuronal cell adhesion moleculeNM_005010.148972.01
    BH-protocadherin (brain-heart)NM_002589.150992.44
    Osteoblast-specific factor 2 (fasciclin I-like)D13665.11063114.12
    BiglycanNM_001711.16332.23
    Collagen, typeVI, alpha 2AL53175012922.52
    Collagen, type IV, alpha 6AI88994112886.15
    Collagen, typeVIII, alpha 1BE87779612952.34
    Collagen, typeVII, alpha 1NM_000094.1129413.93
    Protocadherin beta 8NM_019120.156128328.47
    Solute carrier family 29 (nucleoside transporters), member 2AF034102.13177439.87
    Adaptor-related protein complex 4, epsilon 1 subunitNM_007347.12343145.52
    Solute carrier family 6 (neurotransmitter transporter, taurine), member 6BC006252.165338.79
    Tight junction protein 3 (zona occludens 3)AC0059542713410.75
    Syntaxin-binding protein 2AB002559.168133.23
    Glutamate receptor, ionotrophic, AMPA 3NM_007325.128922.66
    Sodium channel, voltage-gated, type XI, alpha polypeptideAF150882.1112808.14
Table Table 2.. Expressed gene profile of ATSCs and ATSC-TERT cells and partial list of genes that were downregulated in ATSC-TERT cells
  1. a

    Abbreviation: ATSC, adipose stromal cell line.

GeneAccessionLocus linkFold change
    ATP-binding cassette, sub-family C (CFTR/MRP), member 9NM_02029710060−14.49
    E2F transcription factor 1NM_0052251869−3.58
    Protamine 1NM_002761.5619−10.3
    Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoformAA9744165521−26.24
    Adrenergic, beta, receptor kinase 1NM_001619.2156−14.84
    Short-chain dehydrogenase/reductase 1NM_0047539249−13.68
    Phosphodiesterase 10AAF127480.110846−10.07
    ATPase, class II, type 9AAB014511.110079−15.86
    Chemokine (C-X-C motif) receptor 4L01639.17852−12.52
    Tyrosinase-related protein 1NM_0005507306−9.06
    CDW52 antigen (CAMPATH-1 antigen)NM_0018031043−12.17
    WNT1-inducible signaling pathway protein 2NM_0038818839−14.23
    Myelin-associated oligodendrocyte basic proteinNM_0065014336−9.26
    Peripheral myelin protein 22L03203.15376−4.05
    Phosphodiesterase 4D, cAMP-specificAF012074.15144−13.09
    Calcitonin/calcitonin-related polypeptide, alphaX15943796−18.17
    Proline arginine-rich end leucine-rich repeat proteinU413445549−93.73
    msh homeo box homolog 2 (Drosophila)NM_002449.24488−288.74
    Distal-less homeo box 6NM_0052221750−25.33
    NeuropeptideY receptorY1NM_0009094886−6.8
    Opiate receptor-like 1NM_0009134987−115.68
    5-Hydroxytryptamine (serotonin) receptor 2BNM_0008673357−28.68
    Nuclear receptor subfamily 4, group A, member 3U12767.18013−12.13
    Nuclear receptor subfamily 4, group A, member 1D49728.13164−12.89
    Follicle-stimulating hormone receptorM95489.12492−15.23
    GDNF family receptor alpha 3NM_0014962676−4.01
    Transferrin receptor 2AK022002.17036−5.08
    Proline-rich protein BstNI subfamily 4X078825545−20.48
    T-cell receptor alpha locusAE0006596955−63.9
    Interphotoreceptor matrix proteoglycan 2NM_01624750939−71.3
    Cannabinoid receptor 1 (brain)U733041268−8.11
    G-protein–coupled receptor 88NM_02204954112−34.07
    Neuropeptide FF 1; RFamide-related peptide receptorNM_02214664106−3.46
    Tetraspan 5AA74817710098−14.49
    Basic transcription element binding protein 1NM_001206687−11.62
    Early growth response 3NM_0044301960−17.56
    Zinc finger protein 141 (clone pHZ-44)NM_0034417700−14.73
    Zinc finger protein, X-linkedNM_0034107543−13.74
RAD23 homolog B (Saccharomyces cerevisiae)T935625887−105.51
    Zinc finger protein 236AK000847.17776−22.56
    Tumor necrosis factor, alpha-induced protein 3NM_0062907128−5.48
    v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)NM_0054619935−51.91
    Zinc finger protein, multitype 2NM_012082.223414−33.29
    Myosin, heavy polypeptide 2, skeletal muscle, adultNM_0175344620−42.91
    Myosin, heavy polypeptide 1, skeletal muscle, adultNM_005963.24619−285.94
    Myosin, heavy polypeptide 4, skeletal muscleNM_0175334622−18.85
    Myosin IDAA6219624642−10.6
    Dynamin 1L07810.11759−20.53
    SupervillinNM_003174.26840−13.43
    Syntrophin, gamma 2NM_01896854221−105.24
    Glutamate-ammonia ligase (glutamine synthase)NM_0020652752−16.26
    Creatine kinase, brainNM_0018231152−2.67
    Aldehyde dehydrogenase 1 family, member A3NM_000693220−12.6
    Prostaglandin-endoperoxide synthase 1NM_0009625742−39.38
    Acylphosphatase 1, erythrocyte (common) typeNM_00110797−66.02
    Acetylcholinesterase (YT blood group)AI19002243−3.51
    Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1NM_0036548534−52.51
    Caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase)AI719655834−2.38
    Alcohol dehydrogenase 1C (class I), gamma polypeptideNM_000669.2126−12.64
    Sialyltransferase 8E (alpha-2, 8-polysialyltransferase)NM_01330529906−5.9
    Sialyltransferase 4A (beta-galactoside alpha-2,3- sialyltransferase)NM_0030336482−5.92
    PI-3-kinase-related kinase SMG-1U32581.223049−10.11
    Retinol dehydrogenase 5 (11-cis and 9-cis)U43559.15959−11.54
    Monoglyceride lipaseBC006230.111343−14.12
    NADPH oxidase 1NM_007052.227035−19.56
    Matrix metalloproteinase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenNM_0049944318−5.55
    Matrix metalloproteinase 1 (interstitial collagenase)NM_002421.24312−108.59
    Matrix metalloproteinase 3 (stromelysin 1, progelatinase)NM_002422.24314−15.58
    A disintegrin and metalloproteinase domain 20AF029899.18748−12.51
    A disintegrin and metalloproteinase domain 19 (meltrin beta)Y13786.28728−4.92
    A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin typeAB002364.19508−4.91
    Transmembrane protease, serine 4NM_01642556649−26.59
    Kallikrein 14NM_02204643847−46.32
    Neuropilin 1BE6204578829−7.86
    Mitogen-activated protein kinase kinase 2AI7628115605−9.48
    PTK9 protein tyrosine kinase 9AW6650245756−16.83
    Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome)AK024388.12260−2.46
    Chemokine (C-C motif) ligand 2S69738.16347−5.05
    Neurotrophic tyrosine kinase, receptor, type 2AA7071994915−5.4
    Serine (or cysteine) proteinase inhibitor, clade I (neuroserpin), member 1NM_0050255274−15.48
    Tissue factor pathway inhibitor 2L27624.17980−18.79
    Alpha-2-macroglobulinNM_000014.32−30.19
    Serine (or cysteine) proteinase inhibitorNM_016186.51156−11.07
    Transforming growth factor, beta receptor III (betaglycan, 300 kDa)NM_0032437049−3.36
    SH3 domain-binding glutamic acid-rich proteinNM_0073416450−18.17
    Growth hormone receptorNM_0001632690−8.98
    Sialophorin (gpL115, leukosialin, CD43)X520756693−7.19
    Gamma-aminobutyric acid receptor, rho 1NM_0020422569−6.48
    Glutamate receptor, metabotropic 7NM_0008442917−7.73
    Ephrin-A3AW1890151944−2.93
    Glucagon receptorU03469.12642−3.02
    CD44 antigen (homing function and Indian blood group system)AV700298960−2.67
    Regulator of G-protein signaling 5NM_0252268490−115.68
    Insulin-like 5NM_005478.210022−173.37
    Neuroepithelial cell-transforming gene 1NM_00586310276−10.88
    Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6D25304.19459−63.9
    RAS guanyl releasing protein 2 (calcium and DAG-regulated)AI68881210235−5.08
    Regulator of G-protein signaling 12AF030110.16002−26.59
    Osteoglycin (osteoinductive factor, mimecan)NM_0140574969−5.25
    Thrombospondin 2NM_0032477058−9.01
    Cadherin 2, type 1, N-cadherin (neuronal)NM_0017921000−2.86
    Thrombospondin 4NM_0032487060−4.82

Discussion

Telomerase is not an oncogene product, and as such its presence permits proliferation but does not cause an uncontrolled proliferation or immortalization [12, 34]. Our study has identified that ectopic hTERT extends the ATSCs lifespan in vitro and highly enhances osteogenic differentiation of ATSCs. ATSC-TERT cells were capable of maintaining their proliferation 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 proliferative capability of ATSC-TERT cells over time. The mechanism 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 telomerase-associated proteins in a cell type–specific manner. Previously researchers demonstrated that ectopic expression of telomerase could increase the osteogenic 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-associated markers CBFA1, osterix, and osteocalcin [31]. In our study, ATSC-TERT cells showed markedly increased expression levels for the osteogenesis-related markers sialyltransferase, 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 cellular 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 senescent fibroblasts restored their functional capacity in a dermal reconstitution model [35]. Similarly, hTERT expression in human endothelial cells did not affect their phenotype and 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-deficient mice. Furthermore, chromosomal analysis showed a normal karyotype and no evidence of abnormalities associated with malignancy [9]. In our study, ATSC-TERT implantation into immunodeficient mice also showed enhanced bone formation capacity compared with control ATSCs, and they did not form tumors subcutaneously. The nonclonal and transient 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.

Acknowledgements

We are grateful to Cynthia Trygg for supporting basic experiments. 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 Millennium Health Excellence Fund and the Louisiana Gene Therapy Research Consortium.

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