Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro.

Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1(+)) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G(2)-M phases of the cell cycle than Flk-1(+) ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle alpha-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1(+) ES cells into the endothelial cell lineage. The shear stressed Flk-1(+) ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1(+) ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1(+) ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1(+) ES cells.

[1]  Gabriel Acevedo-Bolton,et al.  Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis , 2003, Nature.

[2]  J. Ando,et al.  Fluid shear stress increases the production of granulocyte-macrophage colony-stimulating factor by endothelial cells via mRNA stabilization. , 1998, Circulation research.

[3]  J. Ando,et al.  Flow-induced Endothelial Gene Regulation , 1999 .

[4]  J. Ando,et al.  P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells , 2000 .

[5]  B. Berk,et al.  Ligand-Independent Activation of Vascular Endothelial Growth Factor Receptor 2 by Fluid Shear Stress Regulates Activation of Endothelial Nitric Oxide Synthase , 2003, Circulation research.

[6]  D. Rifkin,et al.  The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium. , 2000, Molecular biology of the cell.

[7]  M. Shibuya,et al.  VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells , 1999, Oncogene.

[8]  M. Gimbrone,et al.  Mechanical Forces and the Endothelium , 1999 .

[9]  S. Chien,et al.  Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway. , 2004, American journal of physiology. Heart and circulatory physiology.

[10]  Jun Yamashita,et al.  Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage. , 2003, Blood.

[11]  M. Kaufman,et al.  Establishment in culture of pluripotential cells from mouse embryos , 1981, Nature.

[12]  平島 正則 Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis , 1999 .

[13]  E. Dejana,et al.  Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. , 1996, Blood.

[14]  A M Wobus,et al.  Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. , 1994, Developmental biology.

[15]  D. Gottlieb,et al.  Embryonic stem cells express neuronal properties in vitro. , 1995, Developmental biology.

[16]  Y. Saijoh,et al.  Determination of left–right patterning of the mouse embryo by artificial nodal flow , 2002, Nature.

[17]  S. Nishikawa,et al.  TGF-β receptor kinase inhibitor enhances growth and integrity of embryonic stem cell–derived endothelial cells , 2003, The Journal of cell biology.

[18]  Kimiko Yamamoto,et al.  Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. , 2003, Journal of applied physiology.

[19]  S. Nishikawa,et al.  A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells. , 2003, Blood.

[20]  Jun Yamashita,et al.  Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors , 2000, Nature.

[21]  G. Martin,et al.  Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[22]  P. Davies,et al.  Flow-mediated endothelial mechanotransduction. , 1995, Physiological reviews.