Culturing of Human Mesenchymal Stem Cells as Three-dimensional Aggregates Induces Functional Expression of CXCR4 That Regulates Adhesion to Endothelial Cells*

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the α2 integrin subunit, and suppress the expression of CD49d, the α4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.

[1]  Paul E. Kennedy,et al.  HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor , 1996, Science.

[2]  R. Taichman,et al.  Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. , 2000, The Journal of clinical investigation.

[3]  P. Friedl,et al.  T lymphocyte locomotion in a three-dimensional collagen matrix. Expression and function of cell adhesion molecules. , 1995, Journal of immunology.

[4]  W. Rombouts,et al.  Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture , 2003, Leukemia.

[5]  M. Ratajczak,et al.  Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells In Vitro Is Regulated by Stromal‐Derived Factor‐1‐CXCR4 and Hepatocyte Growth Factor‐c‐met Axes and Involves Matrix Metalloproteinases , 2006, Stem cells.

[6]  W. Sierralta,et al.  Dynamic of Distribution of Human Bone Marrow-Derived Mesenchymal Stem Cells After Transplantation into Adult Unconditioned Mice , 2004, Transplantation.

[7]  J. Broach,et al.  A Point Mutation That Confers Constitutive Activity to CXCR4 Reveals That T140 Is an Inverse Agonist and That AMD3100 and ALX40-4C Are Weak Partial Agonists* , 2002, The Journal of Biological Chemistry.

[8]  Michael R Rosen,et al.  Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro , 2007, Stem cells.

[9]  O. Koç,et al.  The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. , 2006, Cardiovascular revascularization medicine : including molecular interventions.

[10]  P. Nelson,et al.  Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. , 2005, Stem cells and development.

[11]  S. Nishikawa,et al.  Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1 , 1996, Nature.

[12]  Andrea Bacigalupo,et al.  Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. , 2005, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[13]  J. Hoxie,et al.  CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons , 1997, Current Biology.

[14]  Fei Ye,et al.  Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. , 2004, The American journal of cardiology.

[15]  J. Chute.  Stem cell homing , 2006, Current opinion in hematology.

[16]  Franco Locatelli,et al.  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. , 2007, Blood.

[17]  J. Hows,et al.  Bone marrow-derived mesenchymal stem cells , 2005, Leukemia & lymphoma.

[18]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[19]  Gideon Rechavi,et al.  A Possible Role for CXCR4 and Its Ligand, the CXC Chemokine Stromal Cell-Derived Factor-1, in the Development of Bone Marrow Metastases in Neuroblastoma1 , 2001, The Journal of Immunology.

[20]  M. Sixt,et al.  Beta1 integrins: zip codes and signaling relay for blood cells. , 2006, Current opinion in cell biology.

[21]  J. Mosca,et al.  Characterization and functionality of cell surface molecules on human mesenchymal stem cells. , 2003, Journal of biomedical science.

[22]  Yi Li,et al.  Gliosis and brain remodeling after treatment of stroke in rats with marrow stromal cells , 2005, Glia.

[23]  L. Muul,et al.  Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Moore,et al.  AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor , 1998, Nature Medicine.

[25]  M. Endres,et al.  Towards in situ tissue repair: Human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2 , 2007, Journal of cellular biochemistry.

[26]  H. Augustin,et al.  Endothelial cells differentially express functional CXC-chemokine receptor-4 (CXCR-4/fusin) under the control of autocrine activity and exogenous cytokines. , 1998, Biochemical and biophysical research communications.

[27]  M. Jansson,et al.  Fetal Mesenchymal Stem-Cell Engraftment in Bone after In Utero Transplantation in a Patient with Severe Osteogenesis Imperfecta , 2005, Transplantation.

[28]  G. Rice,et al.  α2β1 integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis , 2007, Journal of Carcinogenesis.

[29]  J. Lieberman,et al.  Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells , 2007, Leukemia.

[30]  I. Ghiran,et al.  Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors , 2006, Stem cells.

[31]  E. Bonifacio,et al.  Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. , 2005, Blood.

[32]  Masahiko Kuroda,et al.  Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development , 1998, Nature.

[33]  R. Wong,et al.  Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4. , 2006, Biochemical pharmacology.

[34]  C. Voermans,et al.  Leukocyte-Endothelium Interaction Promotes SDF-1-dependent Polarization of CXCR4* , 2003, Journal of Biological Chemistry.

[35]  P. Conget,et al.  Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells , 1999, Journal of cellular physiology.

[36]  Yao‐Hua Song,et al.  Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells. , 2007, Biochemical and biophysical research communications.

[37]  A. Caplan,et al.  The Dynamic in vivo Distribution of Bone Marrow-Derived Mesenchymal Stem Cells after Infusion , 2001, Cells Tissues Organs.

[38]  L. Altucci,et al.  Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells , 2005, Journal of cellular biochemistry.

[39]  B. Brew,et al.  Human Mesenchymal Stem Cells Constitutively Express Chemokines and Chemokine Receptors That Can Be Upregulated by Cytokines, IFN-β, and Copaxone , 2007 .

[40]  I. Bellantuono,et al.  A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. , 2004, Blood.

[41]  S. T. Dheen,et al.  Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After Hypoglossal Nerve Injury , 2004, Stem cells.

[42]  Robert A. Kloner,et al.  Systemic Delivery of Bone Marrow–Derived Mesenchymal Stem Cells to the Infarcted Myocardium: Feasibility, Cell Migration, and Body Distribution , 2003, Circulation.

[43]  P. Ljungman,et al.  Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells , 2007, Leukemia.

[44]  R. Deans,et al.  Mesenchymal stem cells: biology and potential clinical uses. , 2000, Experimental hematology.

[45]  R. Alon,et al.  Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. , 1999, Science.

[46]  N. Fisk,et al.  Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. , 2001, Blood.

[47]  Carola Cavallo,et al.  Hyaluronan‐based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: Involvement of Cd44 and Cd54 , 2006, Journal of cellular physiology.

[48]  M. Chopp,et al.  MCP-1, MIP-1, IL-8 and Ischemic Cerebral Tissue Enhance Human Bone Marrow Stromal Cell Migration in Interface Culture , 2002, Hematology.

[49]  Geoffrey C Gurtner,et al.  Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1 , 2004, Nature Medicine.

[50]  Waldemar Kolanus,et al.  Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines , 2005, Nature Immunology.

[51]  A. Glodek,et al.  CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells1 , 2005, The Journal of Immunology.

[52]  M. Saito,et al.  Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. , 2002, Osteoarthritis and cartilage.