Human mesenchymal stem cells (MSC) are popular candidates for tissue engineering. MSC are defined by their properties in two-dimensional (2D) culture systems. Cells in 2D are known to differ from their in vivo counterparts in cell shape, proliferation, and gene expression. Little is so far known about the phenotype and gene expression of cells in three-dimensional (3D) culture systems. To begin to unravel the impact of 3D versus 2D culture conditions on MSC, we have established MSC from adipose tissue and bone marrow in 3D cultures in alginate beads covalently modified with the tripeptide arginine-glycine-aspartic acid (RGD), the integrin-binding motif found in several molecules within the extracellular matrix. The MSC changed from their fibroblastoid shape (2D) to a small, compact shape when embedded in RGD alginate (3D). High viability was maintained throughout the experiment. The MSC retained expression of integrins known to bind RGD, and practically ceased to proliferate. Microarray analysis revealed that the gene expression in cells in RGD alginate was different both from the cells cultured in 2D and from prospectively isolated, uncultured MSC, but more similar to 2D cells. As alginate may be entirely dissolved, leaving the cells as single cell suspensions for various analyses, this represents a useful model for the study of cells in 3D cultures.
WoodburyD., ReynoldsK., BlackI.B.Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res, 69:908. 2002.
6.
OngS.Y., DaiH., LeongK.W.Hepatic differentiation potential of commercially available human mesenchymal stem cells. Tissue Eng, 12:3477. 2006.
7.
DominiciM., LeB.K., MuellerI., Slaper-CortenbachI., MariniF., KrauseD., DeansR., KeatingA., ProckopD., HorwitzE.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8:315. 2006.
8.
YamadaK.M., CukiermanE.Modeling tissue morphogenesis and cancer in 3D. Cell, 130:601. 2007.
9.
HansenL.K., MooneyD.J., VacantiJ.P., IngberD.E.Integrin binding and cell spreading on extracellular matrix act at different points in the cell cycle to promote hepatocyte growth. Mol Biol Cell, 5:967. 1994.
10.
GriffithL.G., SwartzM.A.Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol, 7:211. 2006.
BirgersdotterA., SandbergR., ErnbergI.Gene expression perturbation in vitro—a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol, 15:405. 2005.
13.
BoquestA.C., ShahdadfarA., FronsdalK., SigurjonssonO., TunheimS.H., CollasP., BrinchmannJ.E.Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture. Mol Biol Cell, 16:1131. 2005.
14.
AugstA.D., KongH.J., MooneyD.J.Alginate hydrogels as biomaterials. Macromol Biosci, 6:623. 2006.
15.
RuoslahtiE., PierschbacherM.D.Arg-Gly-Asp: a versatile cell recognition signal. Cell, 44:517. 1986.
16.
BoudreauN.J., JonesP.L.Extracellular matrix and integrin signalling: the shape of things to come. Biochem J, 339 (Pt 3):481. 1999.
17.
BoquestA.C., ShahdadfarA., BrinchmannJ.E., CollasP.Isolation of stromal stem cells from human adipose tissue. Methods Mol Biol, 325:35. 2006.
18.
KlokkT.I., MelvikJ.E.Controlling the size of alginate gel beads by use of a high electrostatic potential. J Microencapsul, 19:415. 2002.
19.
NaderiS., WangJ.Y., ChenT.T., GutzkowK.B., BlomhoffH.K.cAMP-mediated inhibition of DNA replication and S phase progression: involvement of Rb, p21Cip1, and PCNA. Mol Biol Cell, 16:1527. 2005.
20.
LivakK.J., SchmittgenT.D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods, 25:402. 2001.
21.
GentlemanR.C., CareyV.J., BatesD.M., BolstadB., DettlingM., DudoitS., EllisB., GautierL., GeY., GentryJ., HornikK., HothornT., HuberW., IacusS., IrizarryR., LeischF., LiC., MaechlerM., RossiniA.J., SawitzkiG., SmithC., SmythG., TierneyL., YangJ.Y., ZhangJ.Bioconductor: open software development for computational biology and bioinformatics. Genome Biol, 5:R80. 2004.
22.
WuZ., IrizarryR.A., GentlemanR., Martinez-MurilloF., SpencerF.A model-based background adjustment for oligonucleotide expression arrays. J Am Stat Assoc, 99:909. 2004.
23.
SmythG.K.Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol, 3. 2004.
24.
GautierL., CopeL., BolstadB.M., IrizarryR.Affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics, 20:307. 2004.
25.
BenjaminiY., HochbergY.Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc, 3:57. 1995.
26.
MarkusenJ.F., MasonC., HullD.A., TownM.A., TaborA.B., ClementsM., BoshoffC.H., DunnillP.Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. Tissue Eng, 12:821. 2006.
27.
OriY., HermanM., WeinsteinT., ChagnacA., ZevinD., MiloG., GafterU., MalachiT.Spontaneous DNA repair in human peripheral blood mononuclear cells. Biochem Biophys Res Commun, 320:578. 2004.
28.
BauerS., PattersonP.H.The cell cycle-apoptosis connection revisited in the adult brain. J Cell Biol, 171:641. 2005.
29.
DochevaD., PopovC., MutschlerW., SchiekerM.Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system. J Cell Mol Med, 11:21. 2007.
30.
KikuchiA., TairaH., TsurutaT., HayashiM., KataokaK.Adsorbed serum protein mediated adhesion and growth behavior of bovine aortic endothelial cells on polyamine graft copolymer surfaces. J Biomater Sci Polym Ed, 8:77. 1996.
31.
SacchettiB., FunariA., MichienziS., DiC.S., PiersantiS., SaggioI., TagliaficoE., FerrariS., RobeyP.G., RiminucciM., BiancoP.Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell, 131:324. 2007.
32.
RothS., Neuman-SilberbergF.S., BarceloG., SchupbachT.Cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell, 81:967. 1995.
33.
CastroC.P., PiscopoD., NakagawaT., DerynckR.Cornichon regulates transport and secretion of TGFalpha-related proteins in metazoan cells. J Cell Sci, 120:2454. 2007.
34.
MartelJ.A., MichaelD., Fejes-TothG., Naray-Fejes-TothA.Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells. Am J Physiol Renal Physiol, 293:F904. 2007.
35.
SmithS.A., HolikP.R., StevensJ., MelisR., WhiteR., AlbertsenH.Isolation and mapping of a gene encoding a novel human ADP-ribosylation factor on chromosome 17q12-q21. Genomics, 28:113. 1995.
36.
DanenE.H., SonnenbergA.Integrins in regulation of tissue development and function. J Pathol, 200:471. 2003.
37.
McCall-CulbreathK.D., ZutterM.M.Collagen receptor integrins: rising to the challenge. Curr Drug Targets, 9:139. 2008.
38.
TakagiJ.Structural basis for ligand recognition by RGD (Arg-Gly-Asp)-dependent integrins. Biochem Soc Trans, 32:403. 2004.
39.
PopovaS.N., Lundgren-AkerlundE., WiigH., GullbergD.Physiology and pathology of collagen receptors. Acta Physiol (Oxf), 190:179. 2007.
40.
WuY.N., YangZ., HuiJ.H., OuyangH.W., LeeE.H.Cartilaginous ECM component-modification of the micro-bead culture system for chondrogenic differentiation of mesenchymal stem cells. Biomaterials, 28:4056. 2007.
41.
TakikawaO.Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated L-tryptophan metabolism. Biochem Biophys Res Commun, 338:12. 2005.
NakayamaH., NakayamaN., ArisawaM., AokiY.In vitro and in vivo effects of 14alpha-demethylase (ERG11) depletion in Candida glabrata. Antimicrob Agents Chemother, 45:3037. 2001.
44.
MeuterS., SchaerliP., RoosR.S., BrandauO., BoslM.R., von AndrianU.H., MoserB.Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues. Mol Cell Biol, 27:983. 2007.
45.
TakahashiM., TakahashiY., TakahashiK., ZolotaryovF.N., HongK.S., IidaK., OkimuraY., KajiH., ChiharaK.CXCL14 enhances insulin-dependent glucose uptake in adipocytes and is related to high-fat diet-induced obesity. Biochem Biophys Res Commun, 364:1037. 2007.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
