The AML microenvironment suppresses GMP and CD34⁺ progenitors. (A) plasma from patients with AML suppresses murine GMP proliferation. CFSE-labeled murine GMPs were isolated and cultured in the presence of plasma from patients with AML and in the presence of L-NMMA (0.5 mM) and NOHA (0.5 mM). A representative histogram plot of 5 patient AML plasma experiments is shown. Independent experiments were performed on 2 separate occasions. (B) AML-induced GMP suppression is overcome by the addition of L-NMMA and NOHA. GMPs cultured in the presence of AML plasma (50% of final volume) have significantly reduced proliferation compared with those cultured in the presence of healthy donor plasma (P = .0001) or AML plasma with L-NMMA (0.5 mM) and NOHA (0.5 mM) (P = .0001). (C) Plasma from patients with AML suppresses human CD34⁺ HSC proliferation. CFSE-labeled human CD34⁺ HSCs were isolated and cultured in the presence of plasma from AML patients and in the presence of L-NMMA (0.5 mM) and NOHA (0.5 mM). Data are representative of 5 experiments with plasma from patients with AML. Independent experiments were performed on 2 separate occasions. (D) AML-induced human CD34⁺ HSC suppression is overcome by the addition of L-NMMA and NOHA. CD34⁺ HSCs cultured in the presence of AML plasma have significantly reduced proliferation compared with those cultured in the presence of healthy donor plasma (P = .0002) or AML plasma with L-NMMA and NOHA (P = .003). Independent experiments were performed on 2 separate occasions.