Differentiation character of adult mesenchymal stem cells and transfection of MSCs with lentiviral vectors

Abstract

This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lentiviral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and transfection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Aggracan was positive in chondrogenic lineages and the expression of aggracan and type collagen II was significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Keywords

mesenchymal stem cells / cell differentiation / lentiviral vectors

References

[1] FriedensteinA.J., CorskajaJ.F., KulaginaN.N.. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol, 1976, 4(5): 267-274

[2] HowlettC.R., CaveJ., WilliamsonM., et al.. Mineralization in in vitro cultures of rabbit marrow stromal cells. Clin Orthoped Rel Res, 1986, 213: 251-263

[3] MinguellJ.J., EricesA., CongetP.. Mesenchymal stem cells. Exp Biol Med, 2001, 226(6): 507-520

[4] WangL.F., GoodneadL., PratC., et al.. Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications. Hum Gene Ther, 2006, 17(1): 1-9

[5] AbdellatifA.A., PeltJ.L., BentonR.L., et al.. Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems. J Neurosci Res, 2006, 84(3): 553-567

[6] LefebvreV., HuangW., HarleyV.R., et al.. SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro- alpha1(II) collagen gene. Mol Cell Biol, 1997, 17(4): 2336-2346

[7] FosterJ.W., Dominguez-SteglichM.A., GuioliS., et al.. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature, 1994, 372(6506): 525-530

[8] FriedensteinA.J., ChailakhyanR.K., GerasimovU.V.. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet, 1987, 20(3): 263-272

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

[10] CongetP.A., MinguellJ.J.. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol, 1999, 181(1): 67-73

[11] Le BlancK., TammikC., RosendahlK., et al.. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol, 2003, 31(10): 890-896

[12] PeisterA., MelladJ.A., LarsonB.L., et al.. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood, 2004, 103(5): 1662-1668

[13] GronthosS., SimmonsP.J., GravesS.E., et al.. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone, 2001, 28(2): 174-181

[14] JavazonE.H., BeggsK.J., FlakeA.W.. Mesenchymal stem cells: Paradoxes of passaging. Exp Hematol, 2004, 32(5): 414-425

[15] BaddooM., HillK., WilkinsonR., et al.. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem, 2003, 89(6): 1235-1249

[16] WrightE., HargraveM.R., ChristiansenJ., et al.. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet, 1995, 9(1): 15-20

[17] KentJ., WheatleyS.C., AndrewsJ.E., et al.. A male-specific role for SOX9 in vertebrate sex determination. Development, 1996, 122(9): 2813-2822

[18] BellD.M., LeungK.K.H., WheatleyS.C., et al.. Sox9 directly regulates the type collagen gene. Nat Genet, 1997, 16(2): 174-178

[19] LefebvreV., HuangW., ZhouG., et al.. SOX9 is a potent activator of the chondrocyte-specific enhancer of the proα1(II) collagen gene. Mol Cell Bio, 1997, 17(4): 2336-2346

[20] AkiyamaH., ChaboissierM.C., MartinJ.F., et al.. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev, 2002, 16(21): 2813-2828

[21] SmitsP., LiP., MandelJ., et al.. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell, 2001, 1(2): 277-290

[22] LeL.P., EvertsM., DmitrievI.P., et al.. Fluorescently labeled adenovirus with pIX-EGFP for vector detection. Mol Imaging, 2004, 3(2): 105-116

[23] MagnessS.T., JijonH., Van Houten FisherN., et al.. In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse. J Immunol, 2004, 173(3): 1561-1570