Co-culture of mesenchymal stem cells with umbilical vein endothelial cells under hypoxic condition

Abstract

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdM^NOR, CdM-CdM^HYP and HUVEC-CdM^HYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdM^NOR and HUVEC-CdM^HYP groups, the survival rate, migration and angiogenesis of HUVECs in MSC-CdM^HYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdM^HYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Keywords

hypoxia / mesenchymal stem cells / umbilical vein endothelial cells / co-culture / femoral head necrosis

References

[1] NReisN.D., SchwartzO., MilitianuD., et al.. Hyperbaric oxygen therapy as a treatment for stage-I avascular necrosis of the femoral head. J Bone Joint Surg Br, 2003, 85(3): 371-375

[2] AlvesE.M., AngrisaniA.T., SantiagoM.B.. The use of extracorporeal shock waves in the treatment of osteonecrosis of the femoral head: a systematic review. Clin Rheumatol, 2009, 28(11): 1247-1251

[3] LaiK.A., ShenW.J., YangC.Y., et al.. The use of alendronate to prevent early collapse of the femoral head in patients with nontraumatic osteonecrosis. A randomized clinical study. J Bone Joint Surg Am, 2005, 87(10): 2155-2159

[4] CastroF.P.Jr, BarrackR.L.. Core decompression and conservative treatment for avascular necrosis of the femoral head: a meta-analysis. Am J Orthop (Belle Mead NJ), 2000, 29(3): 187-194

[5] NakamuraY., KumazawaY., MitsuiH., et al.. Combined rotational osteotomy and vascularized iliac bone graft for advanced osteonecrosis of the femoral head. J Reconstr Microsurg, 2005, 21(2): 101-105

[6] Lieberman JR, Conduah A, Urist MR. Treatment of osteonecrosis of the femoral head with core decompression and human bone morphogenetic protein. Clin Orthop Relat Res, 2004, (429):139–145

[7] Granero-MoltoF., WeisJ.A., LongobardiL., et al.. Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair. Expert Opin Biol Ther, 2008, 8(3): 255-268

[8] Granero-MoltoF., WeisJ.A., MigaM.I., et al.. Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cells, 2009, 27(8): 1887-1898

[9] HorwitzE.M., ProckopD.J., FitzpatrickL.A., et al.. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med, 1999, 5(3): 309-313

[10] CuiQ., XiaoZ., LiX., et al.. Use of genetically engineered bone-marrow stem cells to treat femoral defects: an experimental study. J Bone Joint Surg Am, 2006, 88(Suppl3): 167-172

[11] YanZ., HangD., GuoC., et al.. Fate of mesenchymal stem cells transplanted to osteonecrosis of femoral head. J Orthop Res, 2009, 27(4): 442-6

[12] HungS.C., PochampallyR.R., ChenS.C., et al.. Angiogenic effects of human multipotent stromal cell conditioned medium activate the PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis. Stem Cells, 2007, 25(9): 2363-2370

[13] IkenagaS., HamanoK., NishidaM., et al.. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res, 2001, 96(2): 277-83

[14] Tateishi-YuyamaE., MatsubaraH., MuroharaT., et al.. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet, 2002, 360(9331): 427-435

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

[16] OwenM.. Marrow stromal stem cells. J Cell Sci Suppl, 1988, 10: 63-76

[17] PhinneyD.G., ProckopD.J.. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells, 2007, 25(11): 2896-2902

[18] GangjiV., ToungouzM., HauzeurJ.P.. Stem cell therapy for osteonecrosis of the femoral head. Expert Opin Biol Ther, 2005, 5(4): 437-442

[19] GangjiV., HauzeurJ.P.. Treatment of osteonecrosis of the femoral head with implantation of autologous bone-marrow cells. Surgical technique. J Bone Joint Surg Am, 2005, 87(Suppl1Pt1): 106-112

[20] Hernigou P, Beaujean F. Treatment of osteonecrosis with autologous bone marrow grafting. Clin Orthop Relat Res, 2002, (405):14–23

[21] CuiQ., XiaoZ., LiX., et al.. Use of genetically engineered bone-marrow stem cells to treat femoral defects: an experimental study. J Bone Joint Surg Am, 2006, 88(Suppl3): 167-172

[22] JiW.F., TongP.J., ZhengW.B., et al.. Experimental study on treatment of femoral head necrosis with arterial perfusion of marrow stem cells]. Zhongguo Zhong Xi Yi Jie He Za Zhi (Chinese), 2004, 24(11): 999-1002

[23] LiZ.H., LiaoW., CuiX.L., et al.. Intravenous transplantation of allogeneic bone marrow mesenchymal stem cells and its directional migration to the necrotic femoral head. Int J Med Sci, 2011, 8(1): 74-83

[24] HorwitzE.M., GordonP.L., KooW.K., et al.. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci USA, 2002, 99(13): 8932-8937

[25] TangY.L., ZhaoQ., QinX., et al.. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann Thorac Surg, 2005, 80(1): 229-236

[26] GnecchiM., HeH., LiangO.D., et al.. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med, 2005, 11(4): 367-368

[27] RaffaghelloL., BianchiG., BertolottoM., et al.. Human mesenchymal stem cells inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche. Stem Cells, 2008, 26(1): 151-162

[28] CeradiniD.J., KulkarniA.R., CallaghanM.J., et al.. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med, 2004, 10(8): 858-864

[29] PeledA., PetitI., KolletO., et al.. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science, 1999, 283(5403): 845-848

[30] LiuH., PanZ., LiA., et al.. Roles of chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) in metastasis of hepatocellular carcinoma cells. Cell Mol Immunol, 2008, 5(5): 373-378

[31] PotierE., FerreiraE., AndriamanalijaonaR., et al.. Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. Bone, 2007, 40(4): 1078-1087

[32] MullerI., VaeglerM., HolzwarthC., et al.. Secretion of angiogenic proteins by human multipotent mesenchymal stromal cells and their clinical potential in the treatment of avascular osteonecrosis. Leukemia, 2008, 22(11): 2054-2061