Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro

Abstract

Mesenchymal stem cells (MSCs) were induced into a nucleus pulposus-like phenotype utilizing simulated microgravity in vitro in order to establish a new cell-based tissue engineering treatment for intervertebral disc degeneration. For induction of a nucleus pulposus-like phenotype, MSCs were cultured in simulated microgravity in a chemically defined medium supplemented with 0 (experimental group) and 10 ng/mL (positive control group) of transforming growth factor β1 (TGF-β1). MSCs cultured under conventional condition without TGF-β1 served as blank control group. On the day 3 of culture, cellular proliferation was determined by WST-8 assay. Differentiation markers were evaluated by histology and reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-β1 slightly promoted the proliferation of MSCs. The collagen and proteoglycans were detected in both groups after culture for 7 days. The accumulation of proteoglycans was markedly increased. The RT-PCR revealed that the gene expression of Sox-9, aggrecan and type II collagen, which were chondrocyte specific, was increased in MSCs cultured under simulated microgravity for 3 days. The ratio of proteoglycans/collagen in blank control group was 3.4-fold higher than positive control group, which denoted a nucleus pulposus-like phenotype differentiation. Independent, spontaneous differentiation of MSCs towards a nucleus pulposus-like phenotype in simulated microgravity occurred without addition of any external bioactive stimulators, namely factors from TGF-β family, which were previously considered necessary.

Keywords

mesenchymal stem cells / simulated microgravity / cell differentiation / transforming growth factor β1

References

[1] MacfarlaneG.J., ThomasE., CroftP.R., et al.. Predictors of early improvement in low back pain amongst consulters to general practice: the influence of pre-morbid and episode-related factors. Pain, 1999, 80(1–2): 113-119

[2] LuomaK., RiihimäkiH., LuukkonenR., et al.. Low back pain in relation to lumbar disc degeneration. Spine, 2000, 25(4): 487-492

[3] GruberH.E., LeslieK., IngramJ., et al.. Cell based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers. Spine J, 2004, 4(1): 44-55

[4] MaroudasA., StockwellR.A., NachemsonA., et al.. Factors involved in the nutrition of the human lumbar intervertebral disc: Cellularity and diffusion of glucose in vitro. J Anat, 1975, 120(Pt1): 113-130

[5] PittengerM.F., MackayA.M., BeckS.C., et al.. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143-147

[6] MastrogiacomoM., CanceddaR., QuartoR.. Effect of different growth factors on the chondrogenic potential of human bone marrow stromal cells. Osteoarthritis Cartilage, 2001, 9(SupplA): S36-40

[7] SunJ., ZhengQ.. Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro. J Huazhong Univ Sci Technol Med Sci, 2009, 29(4): 512-516

[8] YamamotoY., MochidaJ., SakaiD., et al.. Upregulation of the viability of nucleus pulposus cells by bone marrow-derived stromal cells: Significance of direct cell-to-cell contact in coculture system. Spine, 2004, 29(14): 1508-1514

[9] BosnakovskiD., MizunoM., KimG., et al.. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system. Exp Hematol, 2004, 32(5): 502-509

[10] TaoF., LiF., LiG., et al.. Differentiation of mesenchymal stem cells into nucleus pulposus cells in vitro. J Huazhong Univ Sci Technol Med Sci, 2008, 28(2): 156-158

[11] LucaG., CalvittiM., NastruzziC., et al.. Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells. Cell Transplant, 2006, 15(1): 55-65

[12] UnsworthB.R., LelkesP.I.. Growing tissues in microgravity. Nat Med, 1998, 4(8): 901-907

[13] LeeJ.Y., HallR., PelinkovicD., et al.. New use of a three-dimensional pellet culture system for human intervertebral disc cells. Spine, 2001, 26(21): 2316-2322

[14] GruberH.E., IngramJ., HanleyE.N.Jr.. An improved staining method for intervertebral disc tissue. Biotech Histochem, 2002, 77(2): 81-83

[15] FarndaleR.W., ButtleD.J., BarrettA.J.. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta, 1986, 883(2): 173-177

[16] MaldonadoB.A., OegemaT.R.Jr.. Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. J Orthop Res, 1992, 10(5): 677-690

[17] LuZ.F., Zandieh DoulabiB., WuismanP.I., et al.. Differentiation of adipose stem cells by nucleus pulposus cells: configuration effect. Biochem Biophys Res Commun, 2007, 359(4): 991-996

[18] VadalàG., SobajimaS., LeeJ.Y., et al.. In vitro interaction between muscle-derived stem cells and nucleus pulposus cells. Spine J, 2008, 8(5): 804-809

[19] Le VisageC., KimS.W., TatenoK., et al.. Interaction of human mesenchymal stem cells with disc cells: Changes in extracellular matrix biosynthesis. Spine, 2006, 31(18): 2036-2042

[20] VadalàG., StuderR.K., SowaG., et al.. Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion. Spine, 2008, 33(8): 870-876

[21] ChenS., EmeryS.E., PeiM.. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-β1. Spine, 2009, 34(12): 1272-1280

[22] MwaleF., RoughleyP., AntoniouJ.. Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: A requisite for tissue engineering of intervertebral disc. Eur Cell Mater, 2004, 8: 58-64