Question: 

ES-cell derived embryoid bodies show a maximum of Flk-1 expression at day 4 and 5 of spontaneous differentiation. It is postulated that the Flk-1⁺ subpopulation represents endothelial, hematopoetic and cardiac progenitor cells. It has to be evaluated if the Flk- subpopulation is able to generate new Flk+ cells and if both subpopulationes influence eatch other by paracrine effects.

Methods: 

We subcultivated Flk-1⁺ and Flk-1^- cells derived from 4-day-old ES cells after enzymatic dissociation and magnetic beat separation to study their cell fate at distinct time points of differentation using immunohistochemistry, PCR and si-RNA techniques.

Results: 

At day 4 Flk-1⁺ cells express the transcription factors Runx1, Fli-1 and Tbx5 at a higher level compared to Flk-1^- cells. The day 4 Flk-1⁺ cell fraction contains 83% Flk-1⁺ cells, decreasing to 20% at day 4+6 of subculture. The Flk-1^- cell fraction contains 13% Flk-1⁺ cells at day 4+1 of subculture which slightly increases to 30% at day 4+5. Following 6 days of Flk-1⁺ cell subculture immunocytochemical analysis of the pan leukocyte marker CD45 revealed 18% positive cells. The percentage of CD31⁺ cells reached a maximum of 31% at day 4+2 in subcultured Flk-1⁺ cells; 13% of Flk-1⁺ cells were positive for the cardiac marker alpha actinin. In contrast no sign of endothelial differentiation and no expression of CD45 was observed in subcultures of Flk-1^- cells. Both isolated Flk-1 subpopulations differed in their senescence capacity which was characterized by their proliferation potential, size of cell nuclei and beta-galactosidase activity.

Treatment of Flk-1⁺ cells with conditioned medium from the Flk-1^- subculture did not significantly change gene expression. In contrast treatment of Flk-1^- cells with medium from the Flk-1⁺ subculture increased Flk-1⁺ cells from 13 to 27% at day 4+2. Conditioned medium from Flk-1⁺ cells initiated differentiation of 12% CD45⁺, 13% CD31⁺ and 8% alpha actinin⁺ cells at day 4+6. No significant influence on proliferation capacity was observed in the former Flk-1^- fraction under these conditions.

Conclusions: 

Paracrine factors from Flk-1⁺ cells stimulate the recruitment of new Flk-1⁺ progenitor cells from the former Flk-1^- subpopulation. This further directs their differentiation into leukocytes, endothelial cells and cardiomyocytes.