Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs)
© Saeed and Iqtedar; licensee BioMed Central. 2015
Received: 13 June 2014
Accepted: 21 January 2015
Published: 30 January 2015
Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.
To examine these effects, Terc -/- BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr – known transcription regulators of osteogenesis. Similarly, in Terc -/- BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.
Taken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.
KeywordsTelomerase Telomeres BMSCs Mesenchymal stem cells Aging Osteoblast
Osteoporosis is a disease of increased bone/skeletal fragility common in the elderly and estrogen deficient postmenopausal women [1,2]. Bone homeostasis is strictly maintained by a well coordinated venture and steadiness between bone formation by osteoblasts and bone resorption by osteoclasts . However, disruption in any of these above mentioned processes leads to skeletal/bone fragility i-e., osteoporosis . In this regard, role of osteoclasts in bone resoption have extensively been studied and many treatments are targeted at preventing bone resorption, while little attention is paid towards bone anabolic effects by enhancing osteoblasts and osteocytes functions [3,4]. Therefore, equally important is to study the physical factors and instigators that can contribute towards a decline in osteoblast functions with advanced aging. Osteoblasts are derived from bone marrow stromal cells (BMSCs) also known as mesenchymal stem cells (MSCs) capable of differentiating into multiple lineages i-e., osteoblasts, chondrocytes and adipocytes , thus acting as the primary source of skeletal repair .
Tissue homeostasis is exceptionally maintained by a strict balance between cell loss and cell replacement during the course of tissue/organ life [7,8]. However, with aging and in degenerative diseases, this balance declines progressively resulting in reduced supply of new cells to compensate the lost/dead cells, resulting in impaired tissue integrity and function along with reduced regeneration capacity upon damage . Therefore, with aging, owing to several factors including telomerase deficiency, functional and numerical decline of BMSCs resulted in the compromised ability of BMSCs to repair the skeleton and maintain skeletal homeostasis [10-12].
Telomerase was first discovered by Greider et al.  in the extracts of the protozoan Tetrahymena thermophila. Telomerase plays an essential role in the maintenance of choromosomal ends that is telomeres - G-rich simple repeat sequences (TTAGGG) that are synthesized by a special reverse transcriptase called Telomerase [14,15]. This enzyme requires a template to act which is the RNA component of telomerase i.e, TERC [15,16]. Telomerase is inactive in most somatic cells but active in germ cells, stem cells and actively dividing cells . Telomerase deficient mice (Terc-/-) have been instrumental in delineating the impact of telomere shortening in context of whole organism . Disease states that appear in Terc -/- reiterate the disease states, with more or less same etiology, in humans characterized by short telomere in the cells as a result of excessive proliferation [19-21].
For clinical purposes, BSMCs are expanded in vitro to attain the desired number of cells which are not available from young marrow donors or through any other source, whereas the frequency and quality of BMSCs decline further with aging [22,23]. Yet, little attention is paid towards in vitro differentiation hitches encountered by BMSCs during the course of aging with telomerase deficiency. In this study, we performed osteogenic transcriptional profiling of telomerase deficient BMSCs during in vitro osteoblast differentiation and found significant disruption in normal osteogenic gene expression profiles. Data suggest that telomerase deficiency in BMSCs can cause noteworthy effect on the expression of genes vital for skeletal development, bone mineral metabolism, cell growth and differentiation, extra-cellular matrix production and transcription regulation to initiate and maintain normal osteogenesis.
Mice breeding, genotyping & handling
Terc deficient mice (Terc -/- Strain-004132) were purchased from Jackson laboratory and kept in a pathogen-free environment on standard chow. Terc -/- were inter-crossed to generate 3rd generation Terc -/- (Terc -/- - G3) mice, and were maintained in a C57BL/6J background. Wild type Terc +/+ mice were employed as controls. Genotyping was performed according to the protocol recommended by Jackson laboratory. NOD/MrkBomTac-Prkdc scid mice (NOD/SCID mice) were purchased from Taconic, Denmark. The Danish Animal ethical committee approved all the mouse experiments.
Bone marrow stromal cell isolation
Bone marrow stromal cells were harvested according to the protocol described by Peister , with some in house modifications . Media was changed for every 3rd day, subsequently after 1 to 2 weeks cells were dissociated using Trypsin/EDTA for 4 min at 37°C and plated according to the experimental setup.
Osteogenic differentiation of BMSCs; cells were plated at high densities; 20 × 103 cells/cm2 in 24 well plates for staining and in 6 well plates for RNA harvesting containing complete expansion media (CEM): Iscove modified Dulbecco medium (IMDM; GIBCO, Cat. No. 21980) containing 12% FBS (FBS; GIBCO), 100 U/ml penicillin (GIBCO), 100 μg/ml streptomycin (GIBCO) and 12 μM L-glutamine (GIBCO, Cat. No. 25030) supplemented with osteogenic cocktail; 10 nM dexamethasone (Sigma), 10 mM β – glycerol-phosphate (Sigma) & 50 μg/ml Vitamin C (Sigma). Media was changed every 3rd day until day 10 and then cells were stained for Alkaline phosphatase (ALP) and harvested for RNA isolation.
Alkaline phosphatase staining
Cells were fixed with acetone/citrate buffer pH 4.2 (11/2:1) for 5 min at room temperature and stained with Naphtol-AS-TR-phosphate solution for 1 h at room temperature. Naphtol-AS-TR-phosphate solution consists of Naphtol-AS-TR-phosphate (Sigma) diluted 1:5 in H2O and Fast Red TR (Sigma) diluted 1:1.2 in 0.1 M Tris buffer (OUH pharmacy), pH 9.0, where both solutions were mixed 1:1. Cells were counterstained with Mayers-Hematoxylin for 5 min at room temperature.
Alizarin red staining for mineralized matrix
Cells were fixed with 70% ice-cold ethanol for 1 h at −20°C, and stained with 40 mM alizarin red S (AR-S; Sigma), pH 4.2 for 10 min at room temperature.
Quantitative real time mouse osteogenesis RT2 profiler™ PCR array
Quantitative mRNA expression of 84 genes related to osteogenic differentiation was performed using Mouse osteogenesis RT2 profiler™ PCR array (Super array Bioscience Corporation, Frederick, MD, USA) [26,27]. RNA was isolated from WT and Terc -/- BMSCs (n = 3), at day 10 of osteoblast differentiation, using Trizol® and according to the manufacture’s protocol. Concentration of RNA was determined spectrophometrically by absorbance at 260 nm with GeneQuant pro (Biochrom Ltd.). First strand complementary DNA was synthesized from 2 μg of total RNA using a commercial revertAid H minus first strand cDNA synthesis kit (Fermentas, Helsingborg, Sweden), according to the manual’s instructions. cDNA from three biological replicates were pooled and PCR array analysis was done according to manufacturer instructions with RT2 Real Time™ SYBER GREEN PCR Master mix (Super array Biosciences, Corporation). Quantitative Real Time PCR array was performed in an iCycler IQ detection system (Bio-Rad, Herlev, Denmark).
Defective proliferation of Terc -/- BMSCs during in vitro osteoblast differentiation
Telomerase deficiency causes atypical osteoblast differentiation of BMSCs
We have previously shown that telomerase deficient BMSCs and bones exhibit signs of defective osteogenesis with more detailed data on long bones compared to BMSCs . Thus we aimed at delving into more detailed study of telomerase deficient BMSCs. Next task was to select a time point that could detect most of the super array genes to conclude, connect and delineate the functional dysregulation of genetic molecules during osteoblast differentiation due to telomerase deficiency. Thus we did a pilot experiment on WT BMSCs differentiation into osteoblasts considering three different time points (Additional file 1: Figure S1A); day 3, 7 and 10, and selected day 10 to perform super array based on observed data. We differentiated Terc -/- BMSCs into osteoblasts until day 10, where we analyzed ALP activity by ALP staining and matrix mineralization by Alizarin Red staining. Terc -/- BMSCs showed decreased in vitro osteoblast differentiation compared to WT BMSCs at day 10 (Figure 1C).
Osteogenic super array gene profiling displayed defective osteoblast differentiation and function in Terc -/- BMSCs
Transcription factors and regulators
Noteworthy genes that affected this category include, runt related transcription factor 2 (Runx2, -3.7), smad family member 3 (Smad3, -4.7), twist – basic helix loop helix transcription factor (Twist, -8.3) and vitamin D receptor (Vdr, -13.9) (Figure 2B).
Several genes were found to be differentially expressed - known to be involved in bone mineralization, cartilage condensation and ossification (Figure 2C-E). Notable genes that were down-regulated in Terc -/- BMSCs include fibroblast growth factor receptor 2 (Fgfr2, -2.4) important for bone mineralization, collagen type 11 alpha 1 (Col11a1, -14), collagen type 2 alpha 1 (Col2a1, -5.7), dentine matrix protein (Dmp1, -2.5) and phosphate regulating neutral endopeptidase on chromosome X (Phex, -3.8) involved in cartilage condensation (Figure 2C-E). Other noteworthy genes include runt related transcription factor 2 (Runx2, -3.7), bone morphogenetic protein 2 (Bmp4, -1.5), bone morphogenetic protein 6 (Bmp6, -2.3), transforming growth factor beta 1 (Tgfb1, -7.4) and vitamin D receptor (Vdr. -13.9) (Additional file 1: Figure S1B).
Bone mineral metabolism
Similarly, expression level of several genes known to be involved in calcium ion binding and phosphate transport were reduced in Terc -/- BMSCs compared to WT (Figure 3A & B). Most significantly affected (down-regulated) genes in calcium ion binding category include cartilage oligomatrix protein (Comp, -1.8), matrix metallopeptidase 2 (Mmp2, -2.3), bone morphogenetic protein 1 (Bmp1, -4.7) and vitamin D receptor (Vdr, -13.9). In phosphate transport category collagens were adversely affected – notable down-regulated genes include collagen type 1 alpha 1 (Col1a1, -40), collagen type 1 alpha 2 (Col2a1, -22), collagen type 2 alpha 1 (Col2a1, -5.7) and bone morphogenetic protein 5 (Bmp5, -3.9) (Figure 3A & B).
Cell growth and differentiation
Genes that were involved in cell cycle regulation and were down-regulated in Terc -/- BMSCs include integrin beta 1 (Itgb1, -2.4), transforming growth factor beta 1 (Tgfb1, -7.4) and platelet derived growth factor alpha (Pdgfa, -1.7) (Figure 3C & D). Several growth factors and receptors engaged in cell proliferation and differentiation were also down-regulated in Terc -/- BMSCs, such as colony stimulating factor 2 (Csf2, -2.5), colony stimulating factor 3 (Csf3, -1.9), bone morphogenetic protein 4 (Bmp4, -1.5), bone morphogenetic protein 6 (Bmp6, -2.3), insulin like-growth factor 1 (Igf1, -3.3), insulin like-growth factor receptor 1 (Igfr1, -1.7), transforming growth factor beta receptor 3 (Tgfbr3, -2.17) and growth differentiation factor 10, (Gdf10, -5.4) (Figure 4A and Additional file 1: Figure S1C). Similarly, genes important for cell differentiation were also markedly reduced in Terc -/- BMSCs compared to WT BMSCs, most conspicuous were bone morphogenetic protein 4 (Bmp4, -1.5), bone morphogenetic protein 6 (Bmp6,- 2.3), Insulin like-growth factor 1 (Igf1, -3.3), runt related transcription factor 2 (Runx2, -3.7) and twist – basic helix loop helix transcription factor (Twist, -8.4) (Figure 4A and Additional file 1: Figure S1C).
Extracellular matrix (ECM) protein
Among the ECM proteins, collagens were most significantly affected in Terc -/- BMSCs compared to WT BMSCs (Figures 4B-E & 5A). Most significant reduction was observed in collagen type 11 alpha 1 (Col11a1, -14), collagen type 1 alpha 1 (Col1a1, -40), collagen type 1 alpha 2 (Col1a2, -22) and collagen type 2 alpha 1 (Col2a1, -5.7). Among ECM proteases, matrix metallopeptidase 10 (Mmp10, -23) was markedly reduced in Terc -/- BMSCs (Figures 4B-E & 5A). Other most conspicuous molecules crucial for ECM production include, biglycan (Bgn, -4.2), alkaline phosphatase (Akp2, -3.6), cartilage oligomatrix protein (Comp, -1.8), dentine matrix protein 1 (Dmp1, -2.5), integrin alpha 2 (Itga2, -16.4), integrin alpha 2b (Itga2b, -8.5), collagen type 12 alpha 1 (Col12a1, -4.3) (Figures 4B-E & 5A).
Re-ascertainment of data analysis time point by real time PCR
We already compared genes expression at three different time points (day 3, 7 and 10) employing control samples, where suitable expression level can be ascertained to perform data analysis (Additional file 1: Figure S1A). However, next obvious question was to re-confirm the appropriateness of data analysis time point, using control and test samples, by comparing different time points; day 3, day 7 and day 10. Therefore, we selected two genes from each functional gene grouping category of osteogenic super array for comparative gene expression analysis using Real time PCR. Real time PCR data further suggest, that under available resources and time point options, day 10 provide better reckoning of osteoblast specific expression of genes (Runx2, Msx1, Bmp2, Dmp1, Igf1, Col1a1, Vdr, Gdf10 and Mmp8) pertinent for investigating the role of telomerase deficiency during in vitro osteoblast differentiation (Figure 5B).
Telomerase deficiency has been shown to effect BMSCs differentiation into osteoblast, adipocytes and chondrocytes . We have previously shown that telomerase deficiency causes reduced proliferation, enhanced senescence and up-regulation of cell cycle inhibitors in telomerase deficient BMSCs , while its over-expression has been shown to enhance BMSCs proliferation and osteogenic differentiation; both in vitro and in vivo, with no signs of in vitro senescence . In this study, we performed more detailed expression profiling of dysregulated genes affected during in vitro osteoblast differentiation in Terc -/- BMSCs. Data suggested that telomerase deficiency caused more significant dysregulation in genes involved in osteogenic commitment and extracellular matrix production during the differentiation process.
Osteoblastogenesis is a multifaceted and intricate process regulated by temporal and spatial expression of transcription factors, cytokines, growth factors, hormones and morphogens in a stage specific manner [30,31]. Subtle differences in any of these factors can affect the coordinated effort towards lineage commitment to lineage maturation. Our data demonstrated that telomerase deficiency caused differential gene expression profiles emanating from dysregulation of transcription regulation to extracellular matrix production and bone mineralization during the course of in vitro osteoblast differentiation. Reduction in Runx2, Twist and Vdr in Terc -/- BMSCs, possibly suggest initial inadequacy in osteogenic lineage commitment. Nevertheless, Msx1, another transcription regulator, was significantly up-regulated in Terc -/- BMSCs with documented role in cell proliferation and differentiation during embryonic development, affecting Runx2 expression in neural crest cells and during osteoblast differentiation . Thus, up-regulation of Msx1 in Terc -/- BMSCs could suggest a positive feedback signal to enhance Runx2 expression, since Runx2 lies downstream of Msx1 . Additionally, down-regulation of Twist in Terc -/- BMSCs presumably favour osteoblast specific genes expression because literature evidence suggests that down-regulation of Twist genes (Twist1 and Twist2) is required to initiate osteoblast specific gene expression . These data suggest that telomerase deficiency causes inadequate transcriptional control over BMSCs lineage commitment towards osteoblast. However, insensitivity of mesenchymal progenitors towards the differentiation signals could possibly come from the senescent cells. It is plausible that senescent cells secrete several inhibitory cytokines and mortifying enzymes affecting cell responses towards the inducing signals - ending up in the reduction of agile progenitor’s pool that could respond to give a more affirmative response towards the induction signals.
Furthermore, marked reduction in genes involved in cellular growth, differentiation and skeletal development such as, Pdgfa, Itgb1, Smad3, Dmp1, Phex, Fgfr2, Col11a1 and Tgfb1, suggest that telomerase deficiency resulted in defects from proliferative phase to mineralization phase of osteogenesis. For example, Itgb1 has been shown to promote and regulate cell spreading, proliferation and cytoskeleton integrity to influence cell differentiation , Dmp1 is vital for mineralization of bone and later functioning of osteocytes crucial for proper bone re-modelling process . The reduced levels of Fgfr2 mRNA, upon deletion of Twist, have been associated with decreased mRNA levels of Runx2, Bsp and Oc . Col11a1 has been shown to suppress terminal osteoblast differentiation which is activated by Lef1 via direct physical interaction of Lef1 with Col11a1 promoter sans b-catenin . Seemingly, there exist numbers of genetic signals, in Terc -/- BMSCs, opposing or affecting osteoblast differentiation processes. While in parallel there were signals with insisting support for osteoblast differentiation process as evident from various gene expression profiles, such as down-regulation of Col11a1, which suppresses terminal osteoblast differentiation , down-regulation of Tgfb1, known to muffle osteoblastic proteins, especially Runx2 via Smad3  to further suppress Alp and Oc expression [38,40] and up-regulation of Egf, Fgfr2 and Fgf1, known pro-mitogenic signals [41-43], vital for osteoprogenitors expansion to attain osteoblast maturation stage. Furthermore, these pro-osteoblastic efforts were reinforced by up-regulation of other genes such as Bmpr1b, Col5a1 and Cdh11 known to support osteoblast differentiation process at various phases and by distinct mechanisms [44-46]. Besides, these data should be interpreted with great care owing to cell culture heterogeneity and variation in telomere lengths that can result in muddled responses. Nevertheless, osteoblast suppressive responses are dominant over signals favouring osteoblast differentiation process. Moreover, it would be interesting to have different cell culture options with differences in telomerase inflection to better understand the dynamics of telomerase enzyme and it’s physiologically relevant enzymatic activity that could possibly favour optimal osteoblast differentiation.
Furthermore, extracellular matrix is a central component of cellular microenvironment that plays crucial role in cell behaviour and function by regulating cell adhesion, migration, apoptosis, proliferation and differentiation . Strikingly, several collagens associated with ECM, such as Col1a1, Col1a2, Col2a1 and Col11a1 were dysregulated in our data. Among all the collagens present in the ECM, Collagen type 1 (Col1a1) is a major protein present in the ECM of the bone and plays an important role in bone mineralization . Similarly, Itga2 deficiency in mice, another component of ECM - significantly reduced in Terc -/- BMSCs, has been shown to reduce joint pathology, such as reduction in pannus formation, joint inflammation and cartilage erosion . Our data and literature evidences suggest that telomerase deficiency causes severe modifications in ECM related collagens and integrins that could also synergise the unfavourable outcome regarding BMSCs proliferation, survival and differentiation, since ECM has much demanding role in osteoblast differentiation rather than any other cell differentiation process. Moreover, data also suggested the presence of mixed signals, ECM protective and EMC un-protective. For example, down-regulation of Serpinh1, an ECM protease inhibitor involved in ECM degradation, implicated in osteogenesis imperfecta and encodes collagen chaperone protein Hsp47  and up-regulation of Mmp8 - alleviating joint inflammation and bone erosion  coupled with down-regulation of Mmp10 involved in promoting cartilage degradation , respectively.
In conclusion, telomerase deficiency causes inadequate in vitro differentiation of BMSCs into osteoblasts manifested by dysregulation of genes involved in various phases of osteoblast differentiation. It is plausible that initial defect at transcriptional level ramify into regulatory changes in the expression of genes associated with specific phases of the differentiation process. It is also likely, that telomerase deficient cells undergoing senescence are not actively participating in the differentiation process, thus lacking optimal signal threshold levels suitable for osteoblast commitment and proliferation, thereby affecting osteoprogenitors pool size. Data also suggest that Terc -/- BMSCs seems quite perplexed in their response towards differentiating signals because osteoblast suppressive and osteoblast promoting signals were evident from the super array PCR data or dual responses suggest a positive and negative feedback loop mechanisms to assimilate a coordinated effort towards osteoblast differentiation. However, it is possible that Terc -/- BMSCs in culture exhibit differences in their telomere lengths, where cells having critically short telomeres undergoing senescence are associated with gene regulation oblivious of osteoblast differentiation, while cells having optimal lengths are giving due responses towards the differentiation signals. Other likely possibility is the conformational changes in the chromatin owing to telomere shortening, thus regulating undesirable gene expression pattern altering normal response of BMSCs towards osteogenic differentiation. Therefore, regulating telomerase expression at optimal physiological levels to attain uniform telomere lengths, using molecular, chemical and pharmacological ways, and to avoid senescence and malignant transformations could further improve our understanding of the mechanisms associated with defective differentiation due to telomerase deficiency and telomere length dynamics.
The authors are extremely grateful to Prof. Moustapha Kassem and Dr. Basem Abdallah for their kind supervision and support. The authors are also grateful to Bianca Jørgensen for excellent technical assistance. The study was supported by grants from the Lundbeck foundation, the NovoNordisk Foundation and the Danish Medical Research Council. H. Saeed has received PhD fellowship from the NovoNordisk Foundation.
- Pietschmann P, Rauner M, Sipos W, Kerschan-Schindl K. Osteoporosis: an age-related and gender-specific disease–a mini-review. Gerontol. 2009;55:3–12.View ArticleGoogle Scholar
- Seeman E. Bone quality: the material and structural basis of bone strength. J Bone Miner Metab. 2008;26:1–8.PubMedView ArticleGoogle Scholar
- Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev. 2000;21:115–37.PubMedGoogle Scholar
- Benisch P, Schilling T, Klein-Hitpass L, Frey SP, Seefried L, Raaijmakers N, et al. The transcriptional profile of mesenchymal stem cell populations in primary osteoporosis is distinct and shows overexpression of osteogenic inhibitors. PLoS One. 2012;7:e45142.PubMed CentralPubMedView ArticleGoogle Scholar
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7.PubMedView ArticleGoogle Scholar
- Fibbe WE. Mesenchymal stem cells. A potential source for skeletal repair. Ann Rheum Dis. 2002;61(2):ii29–31.PubMed CentralPubMedView ArticleGoogle Scholar
- Rando TA. Stem cells, ageing and the quest for immortality. Nature. 2006;441:1080–6.PubMedView ArticleGoogle Scholar
- Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell. 2000;100:157–68.PubMedView ArticleGoogle Scholar
- Artegiani B, Calegari F. Age-related cognitive decline: can neural stem cells help us? Aging (Albany NY). 2012;4:176–86.Google Scholar
- Bergman RJ, Gazit D, Kahn AJ, Gruber H, McDougall S, Hahn TJ. Age-related changes in osteogenic stem cells in mice. J Bone Miner Res. 1996;11:568–77.PubMedView ArticleGoogle Scholar
- D’Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res. 1999;14:1115–22.PubMedView ArticleGoogle Scholar
- Saeed H, Abdallah BM, Ditzel N, Catala-Lehnen P, Qiu W, Amling M, et al. Telomerase-deficient mice exhibit bone loss owing to defects in osteoblasts and increased osteoclastogenesis by inflammatory microenvironment. J Bone Miner Res. 2011;26:1494–505.PubMedView ArticleGoogle Scholar
- Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985;43:405–13.PubMedView ArticleGoogle Scholar
- Blackburn EH, Chiou SS. Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena. Proc Natl Acad Sci U S A. 1981;78:2263–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Blackburn EH, Gall JG. A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J Mol Biol. 1978;120:33–53.PubMedView ArticleGoogle Scholar
- Blackburn EH. Telomeres: do the ends justify the means? Cell. 1984;37:7–8.PubMedView ArticleGoogle Scholar
- Kassem M, Abdallah BM, Yu Z, Ditzel N, Burns JS. The use of hTERT-immortalized cells in tissue engineering. Cytotechnology. 2004;45:39–46.PubMed CentralPubMedView ArticleGoogle Scholar
- Blasco MA. Telomeres and human disease: ageing, cancer and beyond. Nat Rev Genet. 2005;6:611–22.PubMedView ArticleGoogle Scholar
- Ju Z, Jiang H, Jaworski M, Rathinam C, Gompf A, Klein C, et al. Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment. Nat Med. 2007;13:742–7.PubMedView ArticleGoogle Scholar
- Lee HW, Blasco MA, Gottlieb GJ, Horner JW, Greider CW, DePinho RA. Essential role of mouse telomerase in highly proliferative organs. Nature. 1998;392:569–74.PubMedView ArticleGoogle Scholar
- Vulliamy T, Marrone A, Dokal I, Mason PJ. Association between aplastic anaemia and mutations in telomerase RNA. Lancet. 2002;359:2168–70.PubMedView ArticleGoogle Scholar
- Fan M, Chen W, Liu W, Du GQ, Jiang SL, Tian WC, et al. The effect of age on the efficacy of human mesenchymal stem cell transplantation after a myocardial infarction. Rejuvenation Res. 2010;13:429–38.PubMedView ArticleGoogle Scholar
- Zhang H, Fazel S, Tian H, Mickle DA, Weisel RD, Fujii T, et al. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy. Am J Physiol Heart Circ Physiol. 2005;289:H2089–96.PubMedView ArticleGoogle Scholar
- Peister A, Mellad JA, Larson BL, Hall BM, Gibson LF, Prockop DJ. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood. 2004;103:1662–8.PubMedView ArticleGoogle Scholar
- Post S, Abdallah BM, Bentzon JF, Kassem M. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells. Bone. 2008;43:32–9.PubMedView ArticleGoogle Scholar
- Gouttenoire J, Valcourt U, Bougault C, Aubert-Foucher E, Arnaud E, Giraud L, et al. Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish. J Biol Chem. 2007;282:30960–73.PubMedView ArticleGoogle Scholar
- Xue Y, Xing Z, Hellem S, Arvidson K, Mustafa K. Endothelial cells influence the osteogenic potential of bone marrow stromal cells. Biomed Eng Online. 2009;8:34.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu L, DiGirolamo CM, Navarro PA, Blasco MA, Keefe DL. Telomerase deficiency impairs differentiation of mesenchymal stem cells. Exp Cell Res. 2004;294:1–8.PubMedView ArticleGoogle Scholar
- Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, Rattan SI, et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol. 2002;20:592–6.PubMedView ArticleGoogle Scholar
- Hughes FJ, Turner W, Belibasakis G, Martuscelli G. Effects of growth factors and cytokines on osteoblast differentiation. Periodontol 2000. 2006;41:48–72.PubMedView ArticleGoogle Scholar
- Stein GS, Lian JB, Stein JL, van Wijnen AJ, Montecino M. Transcriptional control of osteoblast growth and differentiation. Physiol Rev. 1996;76:593–629.PubMedGoogle Scholar
- Han J, Ishii M, Bringas Jr P, Maas RL, Maxson Jr RE, Chai Y. Concerted action of Msx1 and Msx2 in regulating cranial neural crest cell differentiation during frontal bone development. Mech Dev. 2007;124:729–45.PubMed CentralPubMedView ArticleGoogle Scholar
- Aberg T, Wang XP, Kim JH, Yamashiro T, Bei M, Rice R, et al. Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis. Dev Biol. 2004;270:76–93.PubMedView ArticleGoogle Scholar
- Bialek P, Kern B, Yang X, Schrock M, Sosic D, Hong N, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell. 2004;6:423–35.PubMedView ArticleGoogle Scholar
- Chen HM, Lin YH, Cheng YM, Wing LY, Tsai SJ. Overexpression of integrin-beta1 in leiomyoma promotes cell spreading and proliferation. J Clin Endocrinol Metab. 2013;98:E837–46.PubMedView ArticleGoogle Scholar
- Kalajzic I, Braut A, Guo D, Jiang X, Kronenberg MS, Mina M, et al. Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. Bone. 2004;35:74–82.PubMedView ArticleGoogle Scholar
- Guenou H, Kaabeche K, Mee SL, Marie PJ. A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre-Chotzen syndrome. Hum Mol Genet. 2005;14:1429–39.PubMedView ArticleGoogle Scholar
- Kahler RA, Yingst SM, Hoeppner LH, Jensen ED, Krawczak D, Oxford JT, et al. Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation. Matrix Biol. 2008;27:330–8.PubMed CentralPubMedView ArticleGoogle Scholar
- Alliston T, Choy L, Ducy P, Karsenty G, Derynck R. TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. EMBO J. 2001;20:2254–72.PubMed CentralPubMedView ArticleGoogle Scholar
- Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai S, Gallea S, et al. Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone. 2001;29:323–30.PubMedView ArticleGoogle Scholar
- Fang MA, Kujubu DA, Hahn TJ. The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells. Endocrinology. 1992;131:2113–9.PubMedGoogle Scholar
- Miraoui H, Oudina K, Petite H, Tanimoto Y, Moriyama K, Marie PJ. Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J Biol Chem. 2009;284:4897–904.PubMedView ArticleGoogle Scholar
- Raucci A, Bellosta P, Grassi R, Basilico C, Mansukhani A. Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways. J Cell Physiol. 2008;215:442–51.PubMedView ArticleGoogle Scholar
- Di BA, Watkins M, Grimston S, Salazar V, Donsante C, Mbalaviele G, et al. N-cadherin and cadherin 11 modulate postnatal bone growth and osteoblast differentiation by distinct mechanisms. J Cell Sci. 2010;123:2640–8.View ArticleGoogle Scholar
- Singhatanadgit W, Olsen I. Endogenous BMPR-IB signaling is required for early osteoblast differentiation of human bone cells. In Vitro Cell Dev Biol Anim. 2011;47:251–9.PubMedView ArticleGoogle Scholar
- Wu YF, Matsuo N, Sumiyoshi H, Yoshioka H. Sp7/Osterix is involved in the up-regulation of the mouse pro-alpha1 (V) collagen gene (Col5a1) in osteoblastic cells. Matrix Biol. 2010;29:701–6.PubMedView ArticleGoogle Scholar
- Mathews S, Bhonde R, Gupta PK, Totey S. Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells. Differentiation. 2012;84:185–92.PubMedView ArticleGoogle Scholar
- Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem. 2001;129:133–8.PubMedView ArticleGoogle Scholar
- Peters MA, Wendholt D, Strietholt S, Frank S, Pundt N, Korb-Pap A, et al. The loss of alpha2beta1 integrin suppresses joint inflammation and cartilage destruction in mouse models of rheumatoid arthritis. Arthritis Rheum. 2012;64:1359–68.PubMedView ArticleGoogle Scholar
- Christiansen HE, Schwarze U, Pyott SM, AlSwaid A, Al BM, Alrasheed S, et al. Homozygosity for a missense mutation in SERPINH1, which encodes the collagen chaperone protein HSP47, results in severe recessive osteogenesis imperfecta. Am J Hum Genet. 2010;86:389–98.PubMed CentralPubMedView ArticleGoogle Scholar
- Garcia S, Forteza J, Lopez-Otin C, Gomez-Reino JJ, Gonzalez A, Conde C. Matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the K/BxN serum-transfer arthritis model. Arthritis Res Ther. 2010;12:R224.PubMed CentralPubMedView ArticleGoogle Scholar
- Barksby HE, Milner JM, Patterson AM, Peake NJ, Hui W, Robson T, et al. Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis. Arthritis Rheum. 2006;54:3244–53.PubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.