Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Angiogenesis. 2022 Feb;25(1):113-128. doi: 10.1007/s10456-021-09815-4. Epub 2021 Sep 3.


Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Keywords: BMP4; ES cells; Endothelial differentiation; FGF2; PA6 cells; TGFβ1; WNT6.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Morphogenetic Protein 4
  • Cell Differentiation
  • Embryonic Stem Cells
  • Endothelial Cells*
  • Fibroblast Growth Factor 2* / pharmacology
  • Mice
  • Proto-Oncogene Proteins
  • Stromal Cells
  • Transforming Growth Factor beta1 / genetics
  • Transforming Growth Factor beta1 / physiology*
  • Wnt Proteins


  • Bmp4 protein, mouse
  • Bone Morphogenetic Protein 4
  • Proto-Oncogene Proteins
  • Tgfb1 protein, mouse
  • Transforming Growth Factor beta1
  • Wnt Proteins
  • Wnt6 protein, mouse
  • Fibroblast Growth Factor 2