Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality
2012; Elsevier BV; Volume: 54; Issue: 3 Linguagem: Inglês
10.1194/jlr.m030650
ISSN1539-7262
AutoresLotta Kilpinen, Feven Tigistu‐Sahle, Sofia Oja, Dario Greco, Amarjit Parmar, Päivi Saavalainen, Janne Nikkilä, Matti Korhonen, Petri Lehenkari, Reijo Käkelä, Saara Laitinen,
Tópico(s)Adipose Tissue and Metabolism
ResumoHuman mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor's age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality. Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor's age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality. arachidonic acid 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester CoA-independent transacylase cyclooxygenase cytosolic phospholipases A2 diacylglycerol docosahexaenoic acid glyserophospholipid gene ontology human bone marrow mesenchymal stromal cell human mesenchymal stromal cell lipoxygenase micro ribonucleic acid mesenchymal stromal cell peripheral blood mononuclear cell phosphatidylcholine phosphatidylethanolamine prostaglandin prostaglandin E2 phosphatidylinositol phosphorylated PI phosphatidylserine saturated fatty acid, TRF, terminal restriction fragment Human mesenchymal stem/stromal cells (hMSC) are currently being studied in a number of clinical applications, for example, to improve the engraftment of hematopoietic stem cell transplant, to promote myocardial repair, and to control immunological responses in graft versus host diseases, autoimmune diseases, and solid organ transplantations (1Ball L.M. Bernardo M.E. Roelofs H. Lankester A. Cometa A. Egeler R.M. Locatelli F. Fibbe W.E. Cotransplantation of ex vivo–expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem-cell transplantation.Blood. 2007; 110: 2764-2767Crossref PubMed Scopus (436) Google Scholar–5Hare J.M. Traverse J.H. Henry T.D. Dib N. Strumpf R.K. Schulman S.P. Gerstenblith G. DeMaria A.N. Denktas A.E. Gammon R.S. et al.A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction.J. Am. Coll. Cardiol. 2009; 54: 2277-2286Crossref PubMed Scopus (1093) Google Scholar). In addition to being immunologically privileged, these cells can modulate both innate and adaptive immune responses in vitro and in vivo. hMSCs have been shown to be able to inhibit T-cell proliferation, inhibit dendritic cell maturation (6Chiesa S. Morbelli S. Morando S. Massollo M. Marini C. Bertoni A. Frassoni F. Bartolome S.T. Sambuceti G. Traggiai E. et al.Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 17384-17389Crossref PubMed Scopus (213) Google Scholar), recruit regulatory T-cells (7Aggarwal S. Pittenger M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105: 1815-1822Crossref PubMed Scopus (3648) Google Scholar, 8English K. Ryan J.M. Tobin L. Murphy M.J. Barry F.P. Mahon B.P. Cell contact, prostaglandin E2 and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25Highforkhead box P3+ regulatory T cells.Clin. Exp. Immunol. 2009; 156: 149-160Crossref PubMed Scopus (530) Google Scholar), and modulate B-cell functions (9Corcione A. Benvenuto F. Ferretti E. Giunti D. Cappiello V. Cazzanti F. Risso M. Gualandi F. Mancardi G.L. Pistoia V. et al.Human mesenchymal stem cells modulate B-cell functions.Blood. 2006; 107: 367-372Crossref PubMed Scopus (1458) Google Scholar). The mechanisms by which these cells exert their immune modulatory functions are still unclear, but it is likely that both direct cell-cell contacts and the secretion of soluble factors are needed. Several cytokines, growth factors, enzymes, and lipid mediators, such as transforming growth factor β1, the tryptophan degrading enzyme indoleamine 2,3-dioxygenase, and prostaglandin E2 (PGE2), have been indicated as key players in the immunomodulatory process (7Aggarwal S. Pittenger M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105: 1815-1822Crossref PubMed Scopus (3648) Google Scholar, 10Di Nicola M. Carlo-Stella C. Magni M. Milanesi M. Longoni P.D. Matteucci P. Grisanti S. Gianni A.M. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2688) Google Scholar, 11Meisel R. Zibert A. Laryea M. Göbel U. Däubener W. Dilloo D. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase–mediated tryptophan degradation.Blood. 2004; 103: 4619-4621Crossref PubMed Scopus (1391) Google Scholar). A long expansion period has been considered to have an adverse effect on the proliferation and differentiation potential and other functional properties of progenitor cells (12Tekkatte C. Gunasingh G.P. Cherian K.M. Sankaranarayanan K. "Humanized" stem cell culture techniques: the animal serum controversy.Stem Cells Int. 2011; 2011: 504723Crossref PubMed Scopus (130) Google Scholar, 13Fuchs B. Schiller J. Cross M.A. Apoptosis-associated changes in the glycerophospholipid composition of hematopoietic progenitor cells monitored by 31P NMR spectroscopy and MALDI-TOF mass spectrometry.Chem. Phys. Lipids. 2007; 150: 229-238Crossref PubMed Scopus (27) Google Scholar). One of the key questions in MSC therapy is, how many cell doublings the cells can undergo before the risks of cellular malfunction or even malignant transformation surface? The number of safe cell doublings may be dependent on the cell source, donor age, and culture conditions. To date, only a few studies on stem cell membrane lipids have been performed (13Fuchs B. Schiller J. Cross M.A. Apoptosis-associated changes in the glycerophospholipid composition of hematopoietic progenitor cells monitored by 31P NMR spectroscopy and MALDI-TOF mass spectrometry.Chem. Phys. Lipids. 2007; 150: 229-238Crossref PubMed Scopus (27) Google Scholar, 14Park H. Haynes C.A. Nairn A.V. Kulik M. Dalton S. Moremen K. Merrill Jr, A.H. Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies.J. Lipid Res. 2010; 51: 480-489Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and the changes in lipid composition during the expansion have not been studied systematically. Although glycosphingolipids and signaling lipids have been studied in relation to the cell identity and functions (15Yu R.K. Suzuki Y. Yanagisawa M. Membrane glycolipids in stem cells.FEBS Lett. 2010; 584: 1694-1699Crossref PubMed Scopus (38) Google Scholar–18Ratajczak M.Z. Kim C.H. Abdel-Latif A. Schneider G. Kucia M. Morris A.J. Laughlin M.J. Ratajczak J. A novel perspective on stem cell homing and mobilization: review on bioactive lipids as potent chemoattractants and cationic peptides as underappreciated modulators of responsiveness to SDF-1 gradients.Leukemia. 2012; 26: 63-72Crossref PubMed Scopus (90) Google Scholar), an important group of membrane lipids, the glycerophospholipids (GPL), composing the main part of membrane lipids has received less attention. The most prominent class, phosphatidylcholine (PC), makes up approximately one half of all membrane phospholipids. Phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) are the other common mammalian GPL classes. Each class consists of numerous different molecular species with different acyl, alkyl, or alkenyl chain assemblies (19van Meer G. Cellular lipidomics.EMBO J. 2005; 24: 3159-3165Crossref PubMed Scopus (404) Google Scholar). Consequently, a single cell contains more than a thousand different GPL molecular species (20Hicks A.M. DeLong C.J. Thomas M.J. Samuel M. Cui Z. Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry.Biochim. Biophys. Acta. 2006; 1761: 1022-1029Crossref PubMed Scopus (99) Google Scholar, 21Blom T.S. Koivusalo M. Kuismanen E. Kostiainen R. Somerharju P. Ikonen E. Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading.Biochemistry. 2001; 40: 14635-14644Crossref PubMed Scopus (63) Google Scholar). However, the ultimate reasons for the structural diversity of cellular lipids, apparently of great functional importance, are still largely unknown. Modern mass spectrometry and bioinformatics tools offer new ways to analyze the compositional changes in complex stem cell lipidomes and link those with the altered potency and quality of the cells (22Wenk M.R. Lipidomics: new tools and applications.Cell. 2010; 143: 888-895Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar–24Galle J. Bader A. Hepp P. Grill W. Fuchs B. Käs J.A. Krinner A. Marquaß B. Müller K. Schiller J. et al.Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.Curr. Med. Chem. 2010; 17: 2274-2291Crossref PubMed Scopus (41) Google Scholar). Membrane lipids not only form a protective layer around the cell but also mediate biological signals through several pathways employing G-protein activators, second messengers, and nuclear receptor activators (25Bensinger S.J. Tontonoz P. Integration of metabolism and inflammation by lipid-activated nuclear receptors.Nature. 2008; 454: 470-477Crossref PubMed Scopus (655) Google Scholar, 26Wymann M.P. Schneiter R. Lipid signalling in disease.Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (958) Google Scholar). The signaling pathways utilizing phosphorylated PIs (PIP), eicosanoids, or sphingolipids are well understood. Cellular mediators, such as inositol phosphates, diacylglycerols (DAG), lysophospholipids, ceramides, cleaved fatty acids (FA) [e.g., arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3)], or their derivatives, are released from membrane lipids and play an important role in many physiological processes including the regulation of inflammation. The essential polyunsaturated fatty acid (PUFA) 20:4n-6 is found primarily at the sn-2 position of most membrane phospholipids. It is a precursor for the synthesis of prostaglandins (PG), tromboxanes, and leukotrienes (27Funk C.D. Prostaglandins and leukotrienes: advances in eicosanoid biology.Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3039) Google Scholar), one of which, a specific PGE2, has been proposed as one of the soluble factors mediating the MSC immune function (7Aggarwal S. Pittenger M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105: 1815-1822Crossref PubMed Scopus (3648) Google Scholar, 28Cutler A.J. Limbani V. Girdlestone J. Navarrete C.V. Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation.J. Immunol. 2010; 185: 6617-6623Crossref PubMed Scopus (141) Google Scholar) The n-3 PUFAs, especially 22:6n-3, are precursors for resolvins, protectins, and maresins which, in contrast to 20:4n-6 and most n-6 PUFAs, exert their functions during the resolution phase of inflammation (29Stables M.J. Gilroy D.W. Old and new generation lipid mediators in acute inflammation and resolution.Prog. Lipid Res. 2011; 50: 35-51Crossref PubMed Scopus (250) Google Scholar) The aim of our study was to compare the GPL profiles of hBMSCs from young and old donors, study the effects of sequential expansion of the cells on these profiles, determine the expression of genes related to lipid metabolism and immunomodulation, and gain a better understanding of the possible relations of cell lipidome changes and functionality. Knowledge of the connections of lipid alterations during expansion and their effect on the immunosuppressive capacity of the therapeutic cells may have a profound influence on clinical cell expansion protocols. All patient protocols were approved by the Ethical Committee of Northern Ostrobothnia Hospital District or Ethical Committee of Hospital District of Helsinki and Uusimaa. The hBMSCs were obtained from bone marrow aspirates taken from the iliac crest or upper femur methaphysis of adult patients after written informed consent. The hBMSCs from donors of different ages (anonymous coding) were isolated as previously described (30Leskelä H.V. Risteli J. Niskanen S. Koivunen J. Ivaska K.K. Lehenkari P. Osteoblast recruitment from stem cells does not decrease by age at late adulthood.Biochem. Biophys. Res. Commun. 2003; 311: 1008-1013Crossref PubMed Scopus (111) Google Scholar, 31Peura M. Bizik J. Salmenperä P. Noro A. Korhonen M. Pätilä T. Vento A. Vaheri A. Alitalo R. Vuola J. et al.Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/PI3K pathway.Wound Repair Regen. 2009; 17: 569-577Crossref PubMed Scopus (34) Google Scholar). The cells were cultured in minimum essential α-medium (αMEM) supplemented with 20 mM HEPES, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Gibco, Invitrogen, Paisley, UK). The same serum lot was used throughout the study. The medium was renewed twice a week, the cells were harvested when 70–80% confluent and plated at a density of 1,000 cells/cm2. Population doublings for every passage were calculated using the formula NH = 2PD× N1, where NH is the number of cells harvested and N1 is the number of cells plated. For lipid analysis, the cells were washed with ice-cold PBS scraped on ice pelleted in silylated vials and stored at −70°C for later use. The expression of surface antigens CD13, CD14, CD19, CD29, CD34, CD44, CD45, CD49e, CD73, CD90, CD105, CD106, CD166, CD271, HLA-ABC, and HLA-DR were analyzed by flow cytometry (FACSAria, Becton Dickinson, San Jose, CA, and FlowJo 7.6.1 software, Treestar, Asland, OR) (supplementary Table I). Total lipids of the hBMSCs were extracted according to Folch et al. (32Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), spiked with internal standards, and dissolved in chloroform/methanol 1:2 for direct infusion experiments. Several internal standards were used to correct for the effects of the polar head group and acyl chain length on the instrument response according to previously reported procedures (33Koivusalo M. Haimi P. Heikinheimo L. Kostiainen R. Somerharju P. Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response.J. Lipid Res. 2001; 42: 663-672Abstract Full Text Full Text PDF PubMed Google Scholar–35Hermansson M. Uphoff A. Käkelä R. Somerharju P. Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry.Anal. Chem. 2005; 77: 2166-2175Crossref PubMed Scopus (170) Google Scholar). Just prior to the mass spectrometry analysis, 1% NH4OH was added, and the lipid extracts with the internal standards were infused to the electrospray source of a Quattro Micro triple quadrupole mass spectrometer (Micromass, Manchester, UK) at the flow rate of 8 μl/min. The collision energy of the instrument was set to 25–65 eV, and negative and positive ion modes were used. Argon was used as the collision gas. The PC and lysoPC (precursor of 184), PE (neutral loss of 141), PS (neutral loss of 87), and PI (precursor of 241) species were selectively detected using head group-specific MS/MS scanning modes (36Brügger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (733) Google Scholar, 37Sullards M.C. Merrill Jr, A.H. Analysis of sphingosine 1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.Sci. STKE. 2001; 2001: pl1PubMed Google Scholar). The acyl chain assembly of the major lipid species was confirmed by MS/MS techniques. The mass spectra were processed by MassLynx software (Micromass, Manchester, UK), and the individual lipid species were quantified by using the internal standards and LIMSA software (38Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (167) Google Scholar). The lipid species were abbreviated as follows: [total carbon number in the chains]:[total number of double bonds in the chains]. The relative concentrations of the lipid classes were obtained by summing the concentrations of the individual molecular species in a class. The FAs in the total lipids of the cells were determined as methyl ester derivatives by gas chromatography as detailed in Käkelä et al. (39Käkelä R. Käkelä A. Kahle S. Becker B.H. Kelly A. Furness R.W. Fatty acid signatures in plasma of captive herring gulls as indicators of demersal or pelagic fish diet.Mar. Ecol. Prog. Ser. 2005; 293: 191-200Crossref Scopus (58) Google Scholar). The FA composition was calculated as molar percentage, and the FAs were marked by using the following abbreviations: [carbon number]:[number of double bonds] n-[position of the first double bond calculated from the methyl end] (e.g., 22:6n-3). Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from healthy anonymous blood donors (Finnish Red Cross Blood Service) by density gradient centrifugation (Ficoll-Pague plus, GE Healthcare, Piscataway, NJ) and cryo-preserved for later use. Prior to use, the PBMCs were thawed gently and labeled with 5 µM 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) solution (Molecular probes, OR). The CFSE-labeled PBMCs were cultured in triplicates at 1.5 × 106 cells/well in a 48-well plate with hBMSCs in RPMI growth medium (RPMI, 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin). hBMSCs were allowed to attach for 2 h before PBMCs were added. To activate the T-cell proliferation, 100 ng/ml of antihuman CD3 antibody clone Hit3a (BioLegend, San Diego, CA) was added to the coculture. T-cell proliferation was recorded after four days of incubation as a dilution of fluorescent dye by flow cytometry. Telomere length were analyzed by the southern blot analysis of terminal restriction fragment (TRF) lengths (40Kimura M. Stone R.C. Hunt S.C. Skurnick J. Lu X. Cao X. Harley C.B. Aviv A. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths.Nat. Protocols. 2010; 5: 1596-1607Crossref PubMed Scopus (310) Google Scholar) Genomic DNA from snap-frozen cell pellets was purified using the Qiagen DNeasy Blood and Tissue Kit and extracted with ethanol. Quality of purified DNA was evaluated by 1% agarose gel electrophoresis. Telomere length analysis was performed using TeloTAGGG Telomere Length Assay Kit (Roche, Basel, Switzerland). DNA was digested using RsaI and HinfI enzymes and electrophoresed on a 0.8% agarose gel 5 V/cm. Southern blotting was performed using 20× salium sodium citrate (SSC) buffer. The blot was hybridized overnight using a digoxigenin (DIG)-labeled telomere-specific probe (TTAGGG) and incubated with alkaline phosphatase-labeled anti-DIG antibody. The blot was then incubated with CDP-Star chemiluminescent substrate and exposed to autoradiography film (GE Healthcare). The autoradiogram was scanned by densitometry, and TRF length was calculated using ImageJ analysis software (41Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat. Methods. 2012; 9: 671-675Crossref PubMed Scopus (35237) Google Scholar) according to TRF =ΣOD/Σ (ODi/Li), where ODi is optical density and Li is the length of the TRF at position i. TRF signals between 3 and 20 kb were used for telomere length measurements (40Kimura M. Stone R.C. Hunt S.C. Skurnick J. Lu X. Cao X. Harley C.B. Aviv A. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths.Nat. Protocols. 2010; 5: 1596-1607Crossref PubMed Scopus (310) Google Scholar). Snap-frozen cell pellets were lysed using RIPA buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentrations were determined using BCA protein assay kit (Pierce, Rockford, IL). Total protein (20 µg) was run on a 12% SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA) and electrotransferred to Hybond ECL Nitrocellulose membrane (GE Healthcare). The membrane was then blocked with 5% milk in PBS containing 0.1% Tween-20 and immunoblotted using anti-p16INK4A (1: 800, clone DCS-50, Sigma) and anti-p21 (1:250, Clone SXM30, BD Pharmingen). β-actin (1: 8000, monoclonal anti-β-actin, clone AC-74, Sigma) was used as a loading control. Polyclonal anti-mouse horseradish peroxidase (HRP)-conjugated antibody was used as a secondary antibody (1:1000, Dako Cytomation, Glostrup, Denmark). Detection was performed using enhanced chemiluminescent detection system (ECL, GE Healthcare). Quantification of band intensities were performed using GS 800 densitometer and Quantity One software (both from Bio-Rad Laboratories). RNA was extracted using Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, CA) and a Qiagen supplementary protocol, Purification of total RNA containing miRNA from animal cells using the RNAeasy Plus Mini Kit, according to the vendor's instructions. Extracted DNA was stored for later use and was not used in this study. Labeled RNAs (800 ng/sample) were hybridized onto Agilent SurePrint G3 Human GE 8 × 60 K, and then the slides were washed and scanned according to the manufacturer's recommendations. The raw data files (.txt files) were imported into R version 2.13 software (42R Development Core Team. 2011. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.Google Scholar) and preprocessed by the BioConductor package limma version 3.4.5 (43Smyth, G. K., 2005. Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions Using R and Bioconductor. R. Gentleman, V. Carey, S. Duboit, R. Irizarry, and W. Huber, editors. Springer, NY. 397–420.Google Scholar). After quality control of the data, the median probe intensities were log2-transformed and normalized according to the method of the quantiles (44Bolstad B.M. Irizarry R.A. Astrand M. Speed T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6415) Google Scholar). The probes for the same Entrez Genes or lincRNAs (as of 1 January 2012) were averaged. A linear model including the AGE*PASSAGE + SUBJECT + DYE terms followed by a moderated t-test was utilized for finding the differentially expressed genes (log2 fold change > 0.58) in the comparisons of interest (nominal P-value < 0.01). The differentially expressed genes were illustrated with violin plot by using R package vioplot with height argument 0.4 and analyzed for significant enrichments of GO-BP classes by using R library GOSim version 1.2.5 (45Fröhlich H. Speer N. Poustka A. Beissbarth T. GOSim–an R-package for computation of information theoretic GO similarities between terms and gene products.BMC Bioinformatics. 2007; 8: 166Crossref PubMed Scopus (149) Google Scholar). Enrichments with P-value < 0.01 were considered significant. Default parameters were used in the GO analysis. The data were analyzed in both univariate and multivariate ways. In the univariate approach, each GPL species or individual FA was analyzed separately and presented in figures as mean + SD. Statistical significances for the differences between hBMSCs from the young and old donors or early and late passages were calculated using Student paired t-test (*P < 0.05, **P < 0.01, ***P < 0.001). In addition, the changes in each lipid percentage were analyzed with linear mixed-effects models, fitting a model with fixed terms for age and passage and a random effect for cell line (a repeated measures type analysis). The effect of each term for every lipid was estimated using normal F-test P-values and visualized with interaction plots depicting the mean values in each passage and group. Software for statistical analysis was R version 2.12, using package "nlme" for a mixed-effects analysis. In the multivariate approach, the lipidome data were subjected to principal components analysis (PCA) to study differences between the samples in terms of the whole GPL profile and to find out which GPL species were mainly responsible for the variation in the data. PCA was computed using log10 normalized data, and the relative positions of the samples and variables were plotted using the first two principal components. In addition, quantitative multivariate measures of the differences among the sample groups were determined by soft independent modeling of class analogy (SIMCA) (46Svante, W., Sjöström, M., . 1977. SIMCA: a method for analyzing chemical data in terms of similarity and analogy. In Chemometrics: Theory and Application. B. Kowalski, editor. American Chemical Society, Washington, DC. 243–282.Google Scholar). hBMSCs isolated from five elderly donors (62–82 years of age, mean 74.6 years) and five young adult donors (20–24 years of age, mean 22.2 years) were characterized and shown to fulfill the established minimal criteria for MSCs (47Dominici M. Le Blanc K. Mueller I. Slaper-Cortenbach I. Marini F. Krause D. Deans R. Keating A. Prockop D. Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (12471) Google Scholar) (supplementary Fig. I). At the end of the cell culture (45–108 days, depending on the donor), the hBMSCs adopted typical senescent morphology ("fried egg") (Fig. 1A, right). The proliferation capacity of the primary hBMSCs was assessed by determining the cumulative population doublings as cells were passaged until senescence. The hBMSCs from old donors showed greater individual variation in their growth kinetics than those from young donors (Fig. 1B). It should be noted that the cell doublings before passage 4 were not included in the calculations. The replicative senescence was further studied from selected samples and passages. The replicative senescence enhanced the expression of cell-cycle components p16INK4A and p21CIP1/WAF1 (Fig. 1C). Additional proof was obtained from telomere length measurements. Telomere length in passage 11 cells was 1.0 ± 0.5 kbp shorter than that in passage 4 (Fig. 1D, supplementary Fig. II). The average values for GPL class totals for hBMSCs were PC, 41–46%; PE, 34–38%; PS, 5–8%; and PI, 4–8% (Table 1). In general, the hBMSCs from the young and old donors had very similar class profiles. The GPL species of the hBMSCs (Fig. 2) showed that the major PC species (in descending order) were 34:1, 36:2, 36:1, and 38:4 (Fig. 2A), whereas the most abundant PE species were 38:4, 36:1, 36:2, 40:5, 38:5, and 34:1 (Fig. 2B). The predominant PS species were 36:1, 40:5, and 40:6 (Fig. 2C), and a single species, 38:4, accounted for almost 50% of the total PI (Fig. 2D). In the early-
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