The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID−γ null mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID−γ null monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.
LindroosB, SuuronenR, MiettinenS. 2011. The potential of adipose stem cells in regenerative medicine. Stem Cell Rev, 7:269–291.
6.
KernS, EichlerH, StoeveJ, KlüterH, BiebackK. 2006. Comparative analysis of mesenchymal stem cells from bone marrow umbilical cord blood or adipose tissue. Stem Cells, 24:1294–1301.
7.
ToyodaM, MatsubaraY, LinK, SugimachiK, FurueM. 2009. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. Cell Biochem Funct, 27:440–447.
8.
NakanishiC, NagayaN, OhnishiS, YamaharaK, TakabatakeS, KonnoT, HayashiK, KawashiriMA, TsubokawaT, YamagishiM. 2011. Gene and protein expression analysis of mesenchymal stem cells derived from rat adipose tissue and bone marrow. Circ J, 75:2260–2268.
9.
HelderMN, KnippenbergM, Klein-NulendJ, WuismanPI. 2007. Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue Eng, 13:1799–1808.
10.
LiuTM, MartinaM, HutmacherDW, HuiJH, LeeEH, LimB. 2007. Identification of common pathways mediating differentiation of bone marrow and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells, 25:750–760.
11.
RocheR, HoareauL, MounetF, FestyF. 2007. Adult stem cells for cardiovascular diseases: the adipose tissue potential. Expert Opin Biol Ther, 7:791–798.
12.
SaulnierN, PuglisiMA, LattanziW, CastelliniL, PaniG, LeoneG, AlfieriS, MichettiF, PiscagliaAC, GasbarriniA. 2011. Gene profiling of bone marrow and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation. Cytotherapy, 13:329–340.
13.
GronthosS, FranklinDM, LeddyHA, RobeyPG, StormsRW, GimbleJM. 2001. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol, 189:54–63.
14.
GimbleJM, GuilakF, BunnellBA. 2010. Clinical and preclinical translation of cell-based therapies using adipose tissue-derived cells. Stem Cell Res Ther, 1:19–29.
15.
MizunoH, TobitaM, UysalAC. 2012. Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells, 30:804–810.
16.
CasalboreP, BudoniM, Ricci-VitianiL, CenciarelliC, PetrucciG, MilazzoL, MontanoN, TabolacciE, MairaG, LaroccaLM, PalliniR. 2009. Tumorigenic potential of olfactory bulb-derived human adult neural stem cells associates with activation of TERT and NOTCH1. PLoS One, 4:e4434.
17.
LiH, FanX, KoviRC, JoY, MoquinB, KonzR, StoicovC, Kurt-JonesE, GrossmanSRet al.2007. Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice. Cancer Res, 67:10889–10898.
18.
ChenBY, WangX, ChenLW, LuoZJ. 2012. Molecular targeting regulation of proliferation and differentiation of the bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells. Curr Drug Targets, 13:561–571.
19.
QinY, JiH, WuY, LiuH. 2009. Chromosomal instability of murine adipose tissue-derived mesenchymal stem cells in long-term culture and development of cloned embryos. Cloning Stem Cells, 11:445–452.
20.
YuanHF, ZhaiC, YanXL, ZhaoDD, WangJX, ZengQ, ChenL, NanX, HeLJet al.2012. SIRT1 is required for long-term growth of human mesenchymal stem cells. J Mol Med (Berl), 90:389–400.
21.
NingH, LiuG, LinG, GarciaM, LiLC, LueTF, LinCS. 2009. Identification of an aberrant cell line among human adipose tissue-derived stem cell isolates. Differentiation, 77:172–180.
22.
WilliamsBR, PrabhuVR, HunterKE, GlazierCM, WhittakerCA, HousmanDE, AmonA. 2008. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science, 322:703–709.
23.
KnoepflerPS. 2009. Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine. Stem Cells, 27:1050–1056.
AsakawaN, ShimizuT, TsudaY, SekiyaS, SasagawaT, YamatoM, FukaiF, OkanoT. 2010. Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering. Biomaterials, 31:3903–3909.
30.
LundAW, YenerB, StegemannJP, PlopperGE. 2009. The natural and engineered 3D microenvironment as a regulatory cue during stem cell fate determination. Tissue Eng Part B Rev, 15:371–380.
31.
ForteG, PietronaveS, NardoneG, ZamperoneA, MagnaniE, PagliariS, PagliariF, GiacintiC, NicolettiCet al.2011. Human cardiac progenitor cell grafts as unrestricted source of supernumerary cardiac cells in healthy murine hearts. Stem Cells, 29:2051–2061.
32.
PagliariS, Vilela-SilvaAC, ForteG, PagliariF, MandoliC, VozziG, PietronaveS, PratM, LicocciaSet al.2011. Cooperation of biological and mechanical signals in cardiac progenitor cell differentiation. Adv Mater, 23:514–518.
33.
NeofytouEA, ChangE, PatlolaB, JoubertLM, RajadasJ, GambhirSS, ChengZ, RobbinsRC, BeyguiRE. 2011. Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds. J Biomed Mater Res A, 98:383–393.
34.
SantoVE, DuarteAR, PopaEG, GomesME, ManoJF, ReisRL. 2012. Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming. J Control Release, 162:19–27.
35.
RodriguesAI, GomesME, LeonorIB, ReisRL. 2012. Bioactive starch-based scaffolds and human adipose stem cells are a good combination for bone tissue engineering. Acta Biomater, 8:3765–3776.
36.
ShiZ, NeohKG, KangET, PohCK, WangW. 2012. Enhanced endothelial differentiation of adipose-derived stem cells by substrate nanotopography. J Tissue Eng Regen Med[Epub ahead of print]10.1002/term1496.
37.
ShiJG, FuWJ, WangXX, XuYD, LiG, HongBF, WangY, DuZY, ZhangX. 2012. Tissue engineering of ureteral grafts by seeding urothelial differentiated hADSCs onto biodegradable ureteral scaffolds. J Biomed Mater Res A, 100:2612–2622.
38.
LiH, XuY, FuQ, LiC. 2012. Effects of multiple agents on epithelial differentiation of rabbit adipose-derived stem cells in 3D. Culture Tissue Eng Part A, 18:1760–1770.
39.
OgushiY, SakaiS, KawakamiK. 2012. Adipose tissue engineering using adipose-derived stem cells enclosed within an injectable carboxymethylcellulose-based hydrogel. J Tissue Eng Regen Med[Epub ahead of print]10.1002/term1480.
40.
ChoiJS, KimBS, KimJD, ChoiYC, LeeHY, ChoYW. 2012. In vitro cartilage tissue engineering using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells. Tissue Eng Part A, 18:80–92.
41.
von FiguraG, HartmannD, SongZ, RudolphKL. 2009. Role of telomere dysfunction in aging and its detection by biomarkers. J Mol Med (Berl), 87:1165–1171.
42.
CristofaloVJ, AllenRG, PignoloRJ, MartinBG, BeckJC. 1998. Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc Natl Acad Sci U S A, 95:10614–10619.
43.
MerlinS, PietronaveS, LocarnoD, ValenteG, FollenziA, PratM. 2009. Deletion of the ectodomain unleashes the transforming invasive and tumorigenic potential of the MET oncogene. Cancer Sci, 100:633–638.
44.
CallicottRJ, WomackJE. 2006. Real-time PCR assay for measurement of mouse telomeres. Comp Med, 56:17–22.
45.
YamakawaK, IwasakiH, MasudaI, OhjimiY, HondaI, SaekiK, ZhangJ, ShonoE, NaitoM, KikuchiM. 2003. The utility of alizarin red s staining in calcium pyrophosphate dihydrate crystal deposition disease. J Rheumatol, 30:1032–1035.
46.
SprioAE, Di ScipioF, RaimondoS, SalamoneP, PagliariF, PagliariS, FolinoA, ForteG, GeunaS, Di NardoP, BertaGN. 2012. Self-renewal and multipotency coexist in a long-term cultured adult rat dental pulp stem cell line: an exception to the rule?Stem Cells Dev[Epub ahead of print]10.1089/scd20120141.
47.
Committee on Standardized Genetic Nomenclature For Mice—Standard Karyotype of the Mouse Mus musculus. 1972. J Hered, 63:69–72.
48.
FollenziA, BentenD, NovikoffP, FaulknerL, Raut SS, GuptaS. 2008. Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice. J Clin Invest, 118:935–945.
49.
ThiMM, Urban-MaldonadoM, SprayDC, SuadicaniSO. 2010. Characterization of hTERT-immortalized osteoblast cell lines generated from wild-type and connexin43-null mouse calvaria. Am J Physiol Cell Physiol, 299:994–1006.
50.
KoganI, GoldfingerN, MilyavskyM, CohenM, ShatsI, DoblerG, KlockerH, WasylykB, VollerMet al.2006. hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis. Cancer Res, 66:3531–3540.
51.
BoklanJ, NanjangudG, MacKenzieKL, MayC, SadelainM, MooreMA. 2002. Limited proliferation and telomere dysfunction following telomerase inhibition in immortal murine fibroblasts. Cancer Res, 62:2104–2114.
52.
YamaokaE, HiyamaE, SotomaruY, OnitakeY, FukubaI, SudoT, SuedaT, HiyamaK. 2011. Neoplastic transformation by TERT in FGF-2-expanded human mesenchymal stem cells. Int J Oncol, 39:5–11.
53.
CollinsK, MitchellJR. 2002. Telomerase in the human organism. Oncogene, 21:564–579.
54.
KimNW, PiatyszekMA, ProwseKR, HarleyCB, WestMD, HoPL, CovielloGM, WrightWE, WeinrichSL, ShayJW. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science, 266:2011–2015.
55.
PazdroR, HarrisonDE. Murine adipose tissue-derived stromal cell apoptosis and susceptibility to oxidative stress in vitro are regulated by genetic background. PLoS One, 2013; 8:e61235.
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