ACE and KDR expression in d7EB cells was monitored because these markers are associated with differentiation of hematopoietic and endothelial progenitor cells. 12, 28 Neither ACE nor KDR were expressed in undifferentiated stem cells; however, increased cell surface expression of these markers was observed after onset of mesodermal differentiation, starting at day 3 and reaching maximum at day 7 to 8 when 23% of d7EB cells were ACEϩKDRϩ and 23% were ACEϩKDRϪ ( Figure 6A ). To determine whether these two markers identified distinct ) in supernatants of directly co-cultured human d7EB and mouse CD11bϩ cells (ratio, 1:5) isolated from septic mice. The co-cultured cells were stimulated with LPS, 1 g/ml, for either 6 or 12 hours. At both times, NO production was significantly lower in the co-cultured wells compared with CD11bϩ cells alone. N ϭ 3 for each experiment. *P Ͻ 0.05; error bars represent SEM. D: Decreased iNOS expression in CD11bϩ cells co-cultured with human d7EB cells. After 12 hours of co-culture, CD11bϩ cells were selected using CD11b magnetic beads, and lysed. Western blot for iNOS protein was performed. Blot is representative of 3 separate experiments. E: Decreased intracellular iNOS expression in CD11bϩ cells isolated from lungs of mice transplanted or not with d7EB cells 12 hours after CLP. CD11bϩ cells were selected using magnetic beads, and permeabilized for intracellular staining for iNOS. Plot representative of 3 separate experiments. Open lines represent isotype-matched control. F: Exposure of CD11bϩ cells to NO alters subsequent inflammatory profile. CD11bϩ cells isolated from septic mice were stimulated with LPS, 1 mg/ml, and the indicated amounts of the NO donor diethylenetriamine NONOate (DETA NO) for 12 hours. Supernatants were collected, and TNF-␣ and IFN-␥ concentrations were measured using an ELISA. lumen-containing tubes. The ACEϩKDRϪ cells gave rise to colonies of hematopoietic precursors that stained positive for hematopoietic markers such as CD43 ( Figure  6B ). The endothelial and hematopoietic colony-forming capacity of the FACS-sorted cells was quantified by counting the number and types of colonies formed per 10,000 ACEϩ/KDRϩ, ACEϩ/KDRϪ, ACEϪ/KDRϪ, and ACEϪ/KDRϩ cells. Endothelial colonies were formed almost exclusively by the ACEϩ/KDRϩ fraction, with a small contribution from the ACEϪ/KDRϩ cells, whereas the ACEϩ/KDRϪ fraction showed great efficiency in production of hematopoietic colonies ( Figure 6C ). To determine the importance of ACE and KDR markers in the observed protective phenotype against sepsis, fractionated d7EB cells were co-cultured according to their expression of ACE and KDR with the CD11bϩ cells from septic mice as described above. The co-cultured cell fractions were stimulated in vitro with LPS for 12 hours, and mouse TNF-␣ and IFN-␥ production was monitored. Both the ACEϩ/KDRϪ fraction and the ACEϩ/KDRϩ fraction significantly reduced TNF-␣ and IFN-␥ production ( Figure 6D ), indicating the critical role of the progenitor cells expressing only ACE in reducing TNF-␣ and IFN-␥ production by CD11bϩ cells. In addition, a strong cell surface co-expression of ACE and toll-like receptor 4 (TLR4) was observed in d7EB cells, suggesting an association between ACE expression and the capability to respond to LPS ( Figure 6E ). To further validate these observations, the ACEϩ cells were fractionated and used for survival studies comparing them with the ACEϪ fraction. Only ACEϩ d7EB cells reproduced the protective effect of the mixed d7EB population in survival against sepsis, whereas ACEϪ cells were not protective ( Figure 6F ). To examine whether the reduced production of cytokines was associated with differential NO production by the ACEϩ progenitor cells, flow cytometry was used to determine co-expression of ACE and KDR with NO production. NO was produced only by the ACE-expressing cells, both ACEϩ/KDRϩ and ACEϩ/KDRϪ, whereas ACEϪ cells did not produce NO ( Figure 6G ).