TY - JOUR
AU1 - Kang, Soo Kyung
AU2 - Putnam, Lorna
AU3 - Dufour, Jason
AU4 - Ylostalo, Joni
AU5 - Jung, Jin Sup
AU6 - Bunnell, Bruce A.
AB - 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. Telomerase, Adipose tissue stromal cell, Osteogenic differentiation, Microarray 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 [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 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 [17–19]. 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 [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 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 [24–30]. 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, 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 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 1. Open in new tabDownload slide 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 1. Open in new tabDownload slide 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 2. Open in new tabDownload slide 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. Figure 2. Open in new tabDownload slide 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 3. Open in new tabDownload slide 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. Figure 3. Open in new tabDownload slide 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 4. Open in new tabDownload slide 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. Figure 4. Open in new tabDownload slide 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 5. Open in new tabDownload slide 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. Figure 5. Open in new tabDownload slide 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 6. Open in new tabDownload slide 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. Figure 6. Open in new tabDownload slide 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 7. Open in new tabDownload slide 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. Figure 7. Open in new tabDownload slide 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 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 (cholinesterase‐related cell division controller) AA576621 8621 2.8     S‐phase response (cyclin‐related) NM_006542.1 10638 3.22     Ceroid‐lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) NM_018941.1 2055 7.24     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 (transforming growth factor beta superfamily) NM_003240.1 7044 5.69     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, X. laevis, cdk‐activating kinase) L20320.1 1022 2.44     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 (activating enhancer binding protein 2 alpha) BF343007 7020 25.47     Centromere protein A, 17 kDa NM_001809.2 1058 42.93     Zinc finger protein 45 (a Kruppel‐associated box [KRAB] domain polypeptide) NM_003425.1 7596 112.1     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 (melanoma‐associated) NM_001897.1 1464 3.25     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) activation‐associated factor NM_003580.1 8439 2.29     Insulin‐like 4 (placenta) NM_002195.1 3641 3.37     Calcium channel, voltage‐dependent, alpha 2/delta subunit 1 NM_000722.1 781 4.86     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 Motor activity genes     Actin, gamma 2, smooth muscle, enteric NM_001615.2 72 2.09     Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 AA046411 10513 2.29     Elastin (supravalvular aortic stenosis, Williams‐Beuren syndrome) AA479278 2006 7.91 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 (peptidyl‐dipeptidaseA) 2 AK026461.1 59272 7.65     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 (ovalbumin), member 2 NM_002575.1 5055 70.72     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, member 6b, decoy AK000485.1 8771 2.02     Wingless‐type MMTV integration site family, member 5A AI968085 7474 4.22     Cholinergic receptor, nicotinic, alpha polypeptide 3 BC000513.1 1136 5.25     Transient receptor potential cation channel, subfamilyV, member 2 NM_015930.1 51393 8.24     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 super family, member 11b (osteoprotegerin) BF433902 4982 21.87     Likely ortholog of mouse gene rich cluster, A gene NM_019858.1 27239 18.35     CD36 antigen (collagen type I receptor, thrombospondin receptor) M98399.1 948 61.74 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 Cell adhesion molecule activity genes     Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) NM_002204.1 3675 2.29     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), human rhinovirus receptor AI608725 3383 2.56     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, typeVI, alpha 2 AL531750 1292 2.52     Collagen, type IV, alpha 6 AI889941 1288 6.15     Collagen, typeVIII, alpha 1 BE877796 1295 2.34     Collagen, typeVII, 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), member 2 AF034102.1 3177 439.87     Adaptor‐related protein complex 4, epsilon 1 subunit NM_007347.1 23431 45.52     Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 BC006252.1 6533 8.79     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 polypeptide AF150882.1 11280 8.14 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 (cholinesterase‐related cell division controller) AA576621 8621 2.8     S‐phase response (cyclin‐related) NM_006542.1 10638 3.22     Ceroid‐lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) NM_018941.1 2055 7.24     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 (transforming growth factor beta superfamily) NM_003240.1 7044 5.69     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, X. laevis, cdk‐activating kinase) L20320.1 1022 2.44     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 (activating enhancer binding protein 2 alpha) BF343007 7020 25.47     Centromere protein A, 17 kDa NM_001809.2 1058 42.93     Zinc finger protein 45 (a Kruppel‐associated box [KRAB] domain polypeptide) NM_003425.1 7596 112.1     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 (melanoma‐associated) NM_001897.1 1464 3.25     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) activation‐associated factor NM_003580.1 8439 2.29     Insulin‐like 4 (placenta) NM_002195.1 3641 3.37     Calcium channel, voltage‐dependent, alpha 2/delta subunit 1 NM_000722.1 781 4.86     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 Motor activity genes     Actin, gamma 2, smooth muscle, enteric NM_001615.2 72 2.09     Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 AA046411 10513 2.29     Elastin (supravalvular aortic stenosis, Williams‐Beuren syndrome) AA479278 2006 7.91 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 (peptidyl‐dipeptidaseA) 2 AK026461.1 59272 7.65     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 (ovalbumin), member 2 NM_002575.1 5055 70.72     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, member 6b, decoy AK000485.1 8771 2.02     Wingless‐type MMTV integration site family, member 5A AI968085 7474 4.22     Cholinergic receptor, nicotinic, alpha polypeptide 3 BC000513.1 1136 5.25     Transient receptor potential cation channel, subfamilyV, member 2 NM_015930.1 51393 8.24     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 super family, member 11b (osteoprotegerin) BF433902 4982 21.87     Likely ortholog of mouse gene rich cluster, A gene NM_019858.1 27239 18.35     CD36 antigen (collagen type I receptor, thrombospondin receptor) M98399.1 948 61.74 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 Cell adhesion molecule activity genes     Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) NM_002204.1 3675 2.29     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), human rhinovirus receptor AI608725 3383 2.56     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, typeVI, alpha 2 AL531750 1292 2.52     Collagen, type IV, alpha 6 AI889941 1288 6.15     Collagen, typeVIII, alpha 1 BE877796 1295 2.34     Collagen, typeVII, 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), member 2 AF034102.1 3177 439.87     Adaptor‐related protein complex 4, epsilon 1 subunit NM_007347.1 23431 45.52     Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 BC006252.1 6533 8.79     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 polypeptide AF150882.1 11280 8.14 Abbreviation: ATSC, adipose stromal cell line. Open in new tab 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 (cholinesterase‐related cell division controller) AA576621 8621 2.8     S‐phase response (cyclin‐related) NM_006542.1 10638 3.22     Ceroid‐lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) NM_018941.1 2055 7.24     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 (transforming growth factor beta superfamily) NM_003240.1 7044 5.69     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, X. laevis, cdk‐activating kinase) L20320.1 1022 2.44     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 (activating enhancer binding protein 2 alpha) BF343007 7020 25.47     Centromere protein A, 17 kDa NM_001809.2 1058 42.93     Zinc finger protein 45 (a Kruppel‐associated box [KRAB] domain polypeptide) NM_003425.1 7596 112.1     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 (melanoma‐associated) NM_001897.1 1464 3.25     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) activation‐associated factor NM_003580.1 8439 2.29     Insulin‐like 4 (placenta) NM_002195.1 3641 3.37     Calcium channel, voltage‐dependent, alpha 2/delta subunit 1 NM_000722.1 781 4.86     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 Motor activity genes     Actin, gamma 2, smooth muscle, enteric NM_001615.2 72 2.09     Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 AA046411 10513 2.29     Elastin (supravalvular aortic stenosis, Williams‐Beuren syndrome) AA479278 2006 7.91 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 (peptidyl‐dipeptidaseA) 2 AK026461.1 59272 7.65     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 (ovalbumin), member 2 NM_002575.1 5055 70.72     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, member 6b, decoy AK000485.1 8771 2.02     Wingless‐type MMTV integration site family, member 5A AI968085 7474 4.22     Cholinergic receptor, nicotinic, alpha polypeptide 3 BC000513.1 1136 5.25     Transient receptor potential cation channel, subfamilyV, member 2 NM_015930.1 51393 8.24     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 super family, member 11b (osteoprotegerin) BF433902 4982 21.87     Likely ortholog of mouse gene rich cluster, A gene NM_019858.1 27239 18.35     CD36 antigen (collagen type I receptor, thrombospondin receptor) M98399.1 948 61.74 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 Cell adhesion molecule activity genes     Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) NM_002204.1 3675 2.29     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), human rhinovirus receptor AI608725 3383 2.56     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, typeVI, alpha 2 AL531750 1292 2.52     Collagen, type IV, alpha 6 AI889941 1288 6.15     Collagen, typeVIII, alpha 1 BE877796 1295 2.34     Collagen, typeVII, 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), member 2 AF034102.1 3177 439.87     Adaptor‐related protein complex 4, epsilon 1 subunit NM_007347.1 23431 45.52     Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 BC006252.1 6533 8.79     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 polypeptide AF150882.1 11280 8.14 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 (cholinesterase‐related cell division controller) AA576621 8621 2.8     S‐phase response (cyclin‐related) NM_006542.1 10638 3.22     Ceroid‐lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) NM_018941.1 2055 7.24     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 (transforming growth factor beta superfamily) NM_003240.1 7044 5.69     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, X. laevis, cdk‐activating kinase) L20320.1 1022 2.44     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 (activating enhancer binding protein 2 alpha) BF343007 7020 25.47     Centromere protein A, 17 kDa NM_001809.2 1058 42.93     Zinc finger protein 45 (a Kruppel‐associated box [KRAB] domain polypeptide) NM_003425.1 7596 112.1     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 (melanoma‐associated) NM_001897.1 1464 3.25     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) activation‐associated factor NM_003580.1 8439 2.29     Insulin‐like 4 (placenta) NM_002195.1 3641 3.37     Calcium channel, voltage‐dependent, alpha 2/delta subunit 1 NM_000722.1 781 4.86     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 Motor activity genes     Actin, gamma 2, smooth muscle, enteric NM_001615.2 72 2.09     Amyloid beta precursor protein (cytoplasmic tail) binding protein 2 AA046411 10513 2.29     Elastin (supravalvular aortic stenosis, Williams‐Beuren syndrome) AA479278 2006 7.91 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 (peptidyl‐dipeptidaseA) 2 AK026461.1 59272 7.65     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 (ovalbumin), member 2 NM_002575.1 5055 70.72     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, member 6b, decoy AK000485.1 8771 2.02     Wingless‐type MMTV integration site family, member 5A AI968085 7474 4.22     Cholinergic receptor, nicotinic, alpha polypeptide 3 BC000513.1 1136 5.25     Transient receptor potential cation channel, subfamilyV, member 2 NM_015930.1 51393 8.24     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 super family, member 11b (osteoprotegerin) BF433902 4982 21.87     Likely ortholog of mouse gene rich cluster, A gene NM_019858.1 27239 18.35     CD36 antigen (collagen type I receptor, thrombospondin receptor) M98399.1 948 61.74 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 Cell adhesion molecule activity genes     Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA‐3 receptor) NM_002204.1 3675 2.29     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), human rhinovirus receptor AI608725 3383 2.56     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, typeVI, alpha 2 AL531750 1292 2.52     Collagen, type IV, alpha 6 AI889941 1288 6.15     Collagen, typeVIII, alpha 1 BE877796 1295 2.34     Collagen, typeVII, 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), member 2 AF034102.1 3177 439.87     Adaptor‐related protein complex 4, epsilon 1 subunit NM_007347.1 23431 45.52     Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 BC006252.1 6533 8.79     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 polypeptide AF150882.1 11280 8.14 Abbreviation: ATSC, adipose stromal cell line. Open in new tab Table 2. Expressed gene profile of ATSCs and ATSC‐TERT cells and partial list of genes that were downregulated in ATSC‐TERT cells Gene Accession Locus link Fold change DNA replication and chromosome cycle genes     ATP‐binding cassette, sub‐family C (CFTR/MRP), member 9 NM_020297 10060 −14.49     E2F transcription factor 1 NM_005225 1869 −3.58     Protamine 1 NM_002761. 5619 −10.3     Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform AA974416 5521 −26.24 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     NeuropeptideY receptorY1 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 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 homolog B (avian) NM_005461 9935 −51.91     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 (interleukin 1, beta, convertase) AI719655 834 −2.38     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‐ sialyltransferase) NM_003033 6482 −5.92     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 gelatinase, 92kDa type IV collagen NM_004994 4318 −5.55     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) with thrombospondin type AB002364.1 9508 −4.91     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     PTK9 protein tyrosine kinase 9 AW665024 5756 −16.83     Fibroblast growth factor receptor 1 (fms‐related tyrosine kinase 2, Pfeiffer syndrome) AK024388.1 2260 −2.46     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), member 1 NM_005025 5274 −15.48     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 (betaglycan, 300 kDa) NM_003243 7049 −3.36     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 Gene Accession Locus link Fold change DNA replication and chromosome cycle genes     ATP‐binding cassette, sub‐family C (CFTR/MRP), member 9 NM_020297 10060 −14.49     E2F transcription factor 1 NM_005225 1869 −3.58     Protamine 1 NM_002761. 5619 −10.3     Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform AA974416 5521 −26.24 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     NeuropeptideY receptorY1 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 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 homolog B (avian) NM_005461 9935 −51.91     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 (interleukin 1, beta, convertase) AI719655 834 −2.38     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‐ sialyltransferase) NM_003033 6482 −5.92     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 gelatinase, 92kDa type IV collagen NM_004994 4318 −5.55     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) with thrombospondin type AB002364.1 9508 −4.91     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     PTK9 protein tyrosine kinase 9 AW665024 5756 −16.83     Fibroblast growth factor receptor 1 (fms‐related tyrosine kinase 2, Pfeiffer syndrome) AK024388.1 2260 −2.46     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), member 1 NM_005025 5274 −15.48     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 (betaglycan, 300 kDa) NM_003243 7049 −3.36     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. Open in new tab Table 2. Expressed gene profile of ATSCs and ATSC‐TERT cells and partial list of genes that were downregulated in ATSC‐TERT cells Gene Accession Locus link Fold change DNA replication and chromosome cycle genes     ATP‐binding cassette, sub‐family C (CFTR/MRP), member 9 NM_020297 10060 −14.49     E2F transcription factor 1 NM_005225 1869 −3.58     Protamine 1 NM_002761. 5619 −10.3     Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform AA974416 5521 −26.24 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     NeuropeptideY receptorY1 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 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 homolog B (avian) NM_005461 9935 −51.91     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 (interleukin 1, beta, convertase) AI719655 834 −2.38     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‐ sialyltransferase) NM_003033 6482 −5.92     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 gelatinase, 92kDa type IV collagen NM_004994 4318 −5.55     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) with thrombospondin type AB002364.1 9508 −4.91     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     PTK9 protein tyrosine kinase 9 AW665024 5756 −16.83     Fibroblast growth factor receptor 1 (fms‐related tyrosine kinase 2, Pfeiffer syndrome) AK024388.1 2260 −2.46     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), member 1 NM_005025 5274 −15.48     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 (betaglycan, 300 kDa) NM_003243 7049 −3.36     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 Gene Accession Locus link Fold change DNA replication and chromosome cycle genes     ATP‐binding cassette, sub‐family C (CFTR/MRP), member 9 NM_020297 10060 −14.49     E2F transcription factor 1 NM_005225 1869 −3.58     Protamine 1 NM_002761. 5619 −10.3     Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform AA974416 5521 −26.24 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     NeuropeptideY receptorY1 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 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 homolog B (avian) NM_005461 9935 −51.91     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 (interleukin 1, beta, convertase) AI719655 834 −2.38     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‐ sialyltransferase) NM_003033 6482 −5.92     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 gelatinase, 92kDa type IV collagen NM_004994 4318 −5.55     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) with thrombospondin type AB002364.1 9508 −4.91     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     PTK9 protein tyrosine kinase 9 AW665024 5756 −16.83     Fibroblast growth factor receptor 1 (fms‐related tyrosine kinase 2, Pfeiffer syndrome) AK024388.1 2260 −2.46     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), member 1 NM_005025 5274 −15.48     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 (betaglycan, 300 kDa) NM_003243 7049 −3.36     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. Open in new tab 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. References 1 Zhu J , Wang H, Bishop JM, et al. . Telomerase extends the lifespan of virus‐transformed human cells without net telomere lengthening . Proc Natl Acad Sci U S A 1999 ; 96 : 3723 – 3728 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Blackburn EH . Structure and function of telomerase . Nature 1991 ; 350 : 569 – 573 . 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TI - Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differentiation of Adipose Tissue–Derived Stromal Cells
JF - Stem Cells
DO - 10.1634/stemcells.2004-0023
DA - 2004-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/expression-of-telomerase-extends-the-lifespan-and-enhances-osteogenic-L2x7AdLa1h
SP - 1356
EP - 1372
VL - 22
IS - 7
DP - DeepDyve
ER -