FIG. 1.
Mesenchymal stem cells (MSCs) in contact with endothelial cells (ECs) inhibit EC proliferation and S-phase cell cycle progression in a dose-dependent fashion. (A) Antibody-based separation of MSCs from ECs using an antibody against CD44 (which is present on the surface of MSCs). After magnetic activated cell sorting (MACs) separation with negative selection, a pure population of CD44-negative cells (ECs) is generated for use in our subsequent assays. (B) Two representative histograms generated by flow cytometry from BrDU stained ECs alone and ECs+MSCs (with contact). ECs were costained with CD31 antibodies and the histograms are representative of proliferation phase of the ECs. Red indicates S-phase cells, green indicates G2/M phase cells, and blue indicates G1 phase cells. Quantitative representation of these findings is indicated in the bar graphs. ECs cocultured in contact with MSCs demonstrate a significant decrease (*P<0.05) in S-phase cells, which is not the case for MSCs cultured in transwells (without contact) with ECs (ECs+MSC [TW]). MSCs in contact with ECs demonstrate a slight increase in G1 phase cells, which is significant P<0.05 indicated by(+) between ECs and ECs+MSCs. MSCs cultured in transwells with ECS (ECS+MSC [TW]) result in a significant increase (*P<0.05) in G2/M phase cells. The ratio of coculture in all of these experiments was 1:5 (MSCs: ECs). (C) MSCs in contact with ECs exhibit a dose-dependent inhibition of EC proliferation. ECs were seeded at a fixed density into wells with increasing numbers (0.25–1.0×105) MSCs added to the well. Decreases in S-phase were dose dependent and significant between treatment groups (*P<0.05). Color images available online at www.liebertpub.com/scd