Dynamics of adipogenic promoter DNA methylation during clonal culture of human adipose stem cells to senescence

Agate Noer1, Andrew C. Boquest1, Philippe Collas1
1Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Blindern, Norway

Tóm tắt

Abstract Background

Potential therapeutic use of mesenchymal stem cells (MSCs) is likely to require large-scale in vitro expansion of the cells before transplantation. MSCs from adipose tissue can be cultured extensively until senescence. However, little is known on the differentiation potential of adipose stem cells (ASCs) upon extended culture and on associated epigenetic alterations. We examined the adipogenic differentiation potential of clones of human ASCs in early passage culture and upon senescence, and determined whether senescence was associated with changes in adipogenic promoter DNA methylation.

Results

ASC clones cultured to senescence display reduced adipogenic differentiation capacity in vitro, on the basis of limited lipogenesis and reduced transcriptional upregulation of FABP4 and LPL, two adipogenic genes, while LEP and PPARG2 transcription remains unaffected. In undifferentiated senescent cells, PPARG2 and LPL expression is unaltered, whereas LEP and FABP4 transcript levels are increased but not in all clones. Bisulfite sequencing analysis of DNA methylation reveals overall relative stability of LEP, PPARG2, FABP4 and LPL promoter CpG methylation during senescence and upon differentiation. Mosaicism in methylation profiles is maintained between and within ASC clones, and any CpG-specific methylation change detected does not necessarily relate to differentiation potential. One exception to this contention is CpG No. 21 in the LEP promoter, whose senescence-related methylation may impair upregulation of the gene upon adipogenic stimulation.

Conclusion

Senescent ASCs display reduced in vitro differentiation ability and transcriptional activation of adipogenic genes upon differentiation induction. These restrictions, however, cannot in general be attributed to specific changes in DNA methylation at adipogenic promoters. There also seems to be a correlation between CpGs that are hypomethylated and important transcription factor binding sites.

Từ khóa


Tài liệu tham khảo

Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001, 7: 211-228. 10.1089/107632701300062859.

Fraser JK, Wulur I, Alfonso Z, Hedrick MH: Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol. 2006, 24: 150-154. 10.1016/j.tibtech.2006.01.010.

Boquest AC, Shahdadfar A, Fronsdal K, Sigurjonsson O, Tunheim SH, Collas P, Brinchmann JE: Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell. 2005, 16: 1131-1141. 10.1091/mbc.E04-10-0949.

Cowan CM, Shi YY, Aalami OO, Chou YF, Mari C, Thomas R, Quarto N, Contag CH, Wu B, Longaker MT: Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol. 2004, 22: 560-567. 10.1038/nbt958.

Kang SK, Lee DH, Bae YC, Kim HK, Baik SY, Jung JS: Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp Neurol. 2003, 183: 355-366. 10.1016/S0014-4886(03)00089-X.

Cousin B, Andre M, Arnaud E, Penicaud L, Casteilla L: Reconstitution of lethally irradiated mice by cells isolated from adipose tissue. Biochem Biophys Res Commun. 2003, 301: 1016-1022. 10.1016/S0006-291X(03)00061-5.

Prunet-Marcassus B, Cousin B, Caton D, Andre M, Penicaud L, Casteilla L: From heterogeneity to plasticity in adipose tissues: site-specific differences. Exp Cell Res. 2006, 312: 727-736. 10.1016/j.yexcr.2005.11.021.

Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC: Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005, 23: 412-423. 10.1634/stemcells.2004-0021.

Kern S, Eichler H, Stoeve J, Kluter H, Bieback K: Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood or adipose tissue. Stem Cells. 2006, 24: 1294-1301. 10.1634/stemcells.2005-0342.

Noer A, Sørensen AL, Boquest AC, Collas P: Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured and differentiated mesenchymal stem cells from adipose tissue. Mol Biol Cell. 2006, 17: 3543-3556. 10.1091/mbc.E06-04-0322.

Boquest AC, Noer A, Collas P: Epigenetic programming of mesenchymal stem cells from human adipose tissue. Stem Cell Rev. 2006, In press:

Antequera F: Structure, function and evolution of CpG island promoters. Cell Mol Life Sci. 2003, 60: 1647-1658. 10.1007/s00018-003-3088-6.

Boquest AC, Noer A, Sorensen AL, Vekterud K, Collas P: CpG methylation profiles of endothelial cell-specific gene promoter regions in adipose tissue stem cells suggest limited differentiation potential toward the endothelial cell lineage. Stem Cells. 2007, 25: 852-861. 10.1634/stemcells.2006-0428.

Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I: Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells. 2004, 22: 675-682. 10.1634/stemcells.22-5-675.

Bruder SP, Jaiswal N, Haynesworth SE: Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997, 64: 278-294. 10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-F.

Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R: Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp Hematol. 2000, 28: 707-715. 10.1016/S0301-472X(00)00160-0.

Rombouts WJ, Ploemacher RE: Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia. 2003, 17: 160-170. 10.1038/sj.leu.2402763.

Wright WE, Shay JW: Historical claims and current interpretations of replicative aging. Nat Biotechnol. 2002, 20: 682-688. 10.1038/nbt0702-682.

Jun ES, Lee TH, Cho HH, Suh SY, Jung JS: Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 2004, 14: 261-268. 10.1159/000080335.

Sethe S, Scutt A, Stolzing A: Aging of mesenchymal stem cells. Ageing Res Rev. 2006, 5: 91-116. 10.1016/j.arr.2005.10.001.

Stolzing A, Scutt A: Age-related impairment of mesenchymal progenitor cell function. Aging Cell. 2006, 5: 213-224. 10.1111/j.1474-9726.2006.00213.x.

Shay JW, Wright WE: Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis. 2005, 26: 867-874. 10.1093/carcin/bgh296.

Rando TA: Stem cells, ageing and the quest for immortality. Nature. 2006, 441: 1080-1086. 10.1038/nature04958.

Laird PW: Cancer epigenetics. Hum Mol Genet. 2005, 14: R65-R76. 10.1093/hmg/ddi113.

Bird A: DNA methylation patterns and epigenetic memory. Genes Dev. 2002, 16: 6-21. 10.1101/gad.947102.

Graff JR, Gabrielson E, Fujii H, Baylin SB, Herman JG: Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression. J Biol Chem. 2000, 275: 2727-2732. 10.1074/jbc.275.4.2727.

Zhu X, Deng C, Kuick R, Yung R, Lamb B, Neel JV, Richardson B, Hanash S: Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome. Proc Natl Acad Sci U S A. 1999, 96: 8058-8063. 10.1073/pnas.96.14.8058.

Ross SR, Graves RA, Greenstein A, Platt KA, Shyu HL, Mellovitz B, Spiegelman BM: A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. Proc Natl Acad Sci U S A. 1990, 87: 9590-9594. 10.1073/pnas.87.24.9590.

Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29: e45-10.1093/nar/29.9.e45.

Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ: Identification and resolution of artifacts in bisulfite sequencing. Methods. 2002, 27: 101-107. 10.1016/S1046-2023(02)00060-9.

Jabbari K, Bernardi G: Cytosine methylation and CpG, TpG (CpA) and TpA frequencies. Gene. 2004, 333:143-9.: 143-149. 10.1016/j.gene.2004.02.043.

Duret L, Galtier N: The covariation between TpA deficiency, CpG deficiency, and G+C content of human isochores is due to a mathematical artifact. Mol Biol Evol. 2000, 17: 1620-1625.

Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B: Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 2006, 7: 14-21. 10.1186/1471-2121-7-14.

Farmer SR: Transcriptional control of adipocyte formation. Cell Metab. 2006, 4: 263-273. 10.1016/j.cmet.2006.07.001.

Bowers RR, Kim JW, Otto TC, Lane MD: Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene. Proc Natl Acad Sci U S A. 2006, 103: 13022-13027. 10.1073/pnas.0605789103.

Schoonjans K, Gelman L, Haby C, Briggs M, Auwerx J: Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes. J Mol Biol. 2000, 304: 323-334. 10.1006/jmbi.2000.4218.

Wu Z, Rosen ED, Brun R, Hauser S, Adelmant G, Troy AE, McKeon C, Darlington GJ, Spiegelman BM: Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity. Mol Cell. 1999, 3: 151-158. 10.1016/S1097-2765(00)80306-8.

Linhart HG, Ishimura-Oka K, DeMayo F, Kibe T, Repka D, Poindexter B, Bick RJ, Darlington GJ: C/EBPalpha is required for differentiation of white, but not brown, adipose tissue. Proc Natl Acad Sci U S A. 2001, 98: 12532-12537. 10.1073/pnas.211416898.

Feve B: Adipogenesis: cellular and molecular aspects. Best Pract Res Clin Endocrinol Metab. 2005, 19: 483-499. 10.1016/j.beem.2005.07.007.

Silva AJ, Ward K, White R: Mosaic methylation in clonal tissue. Dev Biol. 1993, 156: 391-398. 10.1006/dbio.1993.1086.

Daniel JM, Spring CM, Crawford HC, Reynolds AB, Baig A: The p120(ctn)-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides. Nucleic Acids Res. 2002, 30: 2911-2919. 10.1093/nar/gkf398.

Yoon HG, Chan DW, Reynolds AB, Qin J, Wong J: N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso. Mol Cell. 2003, 12: 723-734. 10.1016/j.molcel.2003.08.008.

Mason MM, He Y, Chen H, Quon MJ, Reitman M: Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor. Endocrinology. 1998, 139: 1013-1022. 10.1210/en.139.3.1013.

Melzner I, Scott V, Dorsch K, Fischer P, Wabitsch M, Bruderlein S, Hasel C, Moller P: Leptin gene expression in human preadipocytes is switched on by maturation-induced demethylation of distinct CpGs in its proximal promoter. J Biol Chem. 2002, 277: 45420-45427. 10.1074/jbc.M208511200.

Bey L, Etienne J, Tse C, Brault D, Noe L, Raisonnier A, Arnault F, Hamilton MT, Galibert F: Cloning, sequencing and structural analysis of 976 base pairs of the promoter sequence for the rat lipoprotein lipase gene. Comparison with the mouse and human sequences. Gene. 1998, 209: 31-38. 10.1016/S0378-1119(98)00003-1.

Merkel M, Eckel RH, Goldberg IJ: Lipoprotein lipase: genetics, lipid uptake, and regulation. J Lipid Res. 2002, 43: 1997-2006. 10.1194/jlr.R200015-JLR200.

Tuncman G, Erbay E, Hom X, De V, Campos H, Rimm EB, Hotamisligil GS: A genetic variant at the fatty acid-binding protein aP2 locus reduces the risk for hypertriglyceridemia, type 2 diabetes, and cardiovascular disease. Proc Natl Acad Sci U S A. 2006, 103: 6970-6975. 10.1073/pnas.0602178103.

Kim JY, Siegmund KD, Tavare S, Shibata D: Age-related human small intestine methylation: evidence for stem cell niches. BMC Med. 2005, 3: 10-16. 10.1186/1741-7015-3-10.

Yatabe Y, Tavare S, Shibata D: Investigating stem cells in human colon by using methylation patterns. Proc Natl Acad Sci U S A. 2001, 98: 10839-10844. 10.1073/pnas.191225998.

Esteller M: Aberrant DNA methylation as a cancer-inducing mechanism. Annu Rev Pharmacol Toxicol. 2005, 45: 629-656. 10.1146/annurev.pharmtox.45.120403.095832.

Ushijima T, Okochi-Takada E: Aberrant methylations in cancer cells: where do they come from?. Cancer Sci. 2005, 96: 206-211. 10.1111/j.1349-7006.2005.00035.x.

Bahar R, Hartmann CH, Rodriguez KA, Denny AD, Busuttil RA, Dolle ME, Calder RB, Chisholm GB, Pollock BH, Klein CA, Vijg J: Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature. 2006, 441: 1011-1014. 10.1038/nature04844.

Hasty P, Campisi J, Hoeijmakers J, van SH, Vijg J: Aging and genome maintenance: lessons from the mouse?. Science. 2003, 299: 1355-1359. 10.1126/science.1079161.

Roura S, Farre J, Soler-Botija C, Llach A, Hove-Madsen L, Cairo JJ, Godia F, Cinca J, Bayes-Genis A: Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells. Eur J Heart Fail. 2006, 8: 555-563. 10.1016/j.ejheart.2005.11.006.

Fajas L, Auboeuf D, Raspe E, Schoonjans K, Lefebvre AM, Saladin R, Najib J, Laville M, Fruchart JC, Deeb S, Vidal-Puig A, Flier J, Briggs MR, Staels B, Vidal H, Auwerx J: The organization, promoter analysis, and expression of the human PPARgamma gene. J Biol Chem. 1997, 272: 18779-18789. 10.1074/jbc.272.30.18779.

e! Ensembl Human. 2007, [http://www.ensembl.org/Homo_sapiens]

MethPrimer - Design Primers for Methylation PCRs. 2007, [http://www.urogene.org/methprimer/index1.html]