Control of leucocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue

Embryonic stem (ES) cells spontaneously differentiate capillary-like structures as well as leucocytes such as monocytes/macrophages, neutrophils, natural killer (NK) cells and cytototoxic T lymphocytes. The interplay between vasculogenesis and leucocyte differentiation as well as the population of tumour tissues with ES cell-derived leucocytes and endothelial cells is, however, not sufficiently specified. In the present study, gene expression of the cell surface markers CD68 and CD14 (expressed on monocytes and macrophages), Mac-1 (CD11b) (expressed on granulocytes, monocytes and NK cells) and CD16 (expressed on neutrophils) was investigated in murine CGR8 ES cells in relation to the endothelial cell markers CD31 and vascular endothelial (VE)-cadherin. Expression of leucocyte markers increased from day 7-8 of cell culture on. Furthermore, addition of macrophage colony-stimulating factor to the cell culture medium resulted in a threefold increase in the number of CD68+ monocytes/macrophages. Treatment of embryoid bodies with lipopolysaccharide (LPS) up-regulated CD14 thus suggesting functionality of the CD14 LPS receptor. Differentiation of vascular structures positive for CD31 and VE-cadherin preceded leucocyte differentiation by 2 days (i.e. from day 5-6 on) suggesting that vasculogenesis may be a determinant of leucocyte differentiation. Consequently the Flk-1 antagonist SU5416 which inhibits vasculogenesis of ES cells significantly blunted leucocyte differentiation. Confrontation culture of embryoid bodies with multicellular breast tumour spheroids initiated significant increase of leucocyte cell numbers and invasion of leucocytes into the tumour tissue. In summary our data demonstrate that during ES cell differentiation vasculogenesis precedes leucocyte differentiation, and point towards the direction that leucocyte cell invasion into tumour tissue may initiate the pro-inflammatory microenvironment necessary for tumour vascularization.