Figure 6.
Cell surface marker ACE defines the subpopulation of protective d7EB progenitor cells responsible for the anti-inflammatory effect. A: FACS analysis of d7EB cells for the cell surface markers ACE and KDR identified the following cell populations: ACE+/KDR+ (23% of total) and ACE+/KDR− (23%). These cells were subsequently characterized and used for transplantation. Numbers indicate percentages. Undifferentiated human ES cells did not express either ACE or KDR. Upper small panel, gate used for cell analysis is depicted in red inset. Lower small panel, Isotype controls for ACE and KDR antibodies. B: ACE+/KDR− cell fraction gave rise to hematopoietic and endothelial colonies. Upper panels from left to right, Characteristic architecture of hematopoietic colony arising from ACE+/KDR− cells; characteristic architecture of endothelial colony arising from ACE+/KDR+ cells; ACE+/KDR+ cells are capable of forming lumen-containing tubes in 2-dimensional Matrigel. Lower panels from left to right, Hematopoietic colony staining positive for CD43; endothelial colony staining positive for VE-cadherin; endothelial colony staining positive for von Willebrand factor. Arrows indicate lumen. Scale bar = 150 mm. C: Quantification of colony efficiency of human d7EB cells. d7EB cells were fractionated as described, based on ACE and KDR expression, and subcultured. The number of endothelial and hematopoietic colonies with the previously shown architecture and phenotype was quantified. The ACE+/KDR+ fraction gave rise to 450 ± 50 endothelial colonies per 105 d7EB cells and 40 ± 10 hematopoietic colonies, whereas the ACE+/KDR− fraction gave rise exclusively to 730 ± 60.8 hematopoietic colonies. Error bars represent SEM. D: ACE+ and ACE+/KDR+ cells decreased TNF-α and IFN-γ production by mouse CD11b+ cells. d7EB cells were fractionated by FACS according to their expression of ACE and KDR, and placed in direct co-cultures with mouse CD11b+ cells (ratio, 1:5). The co-cultured cells were stimulated with LPS, 1 μg/ml, for 12 hours, and culture supernatants were collected for cytokine measurement using an ELISA. N = 3 for each experiment. *P < 0.05; error bars represent SEM. E: ACE is co-expressed withTLR4 in d7EB cells. Cell surface co-expression of ACE withTLR4 was determined via FACS using human-specific anti-TLR4 antibody. Fifty percent of ACE+ cells co-expressed TLR4. Experiments were repeated at least 3 times. F: ACE+ d7EB fraction significantly improved survival after CLP. ACE+ cells were injected i.v. (300,000 cells in 200 μL of PBS) 1 hour after CLP. At the end of 120 hours of observation, survival in the control group receiving an equal number of ACE− d7EB cells was 10% versus 50% in the ACE+ d7EB recipient group. N = 20 in each group. P < 0.0001 using a log-rank test. G: Only ACE+ cells produce NO. d7EB cells were stimulated in vitro with LPS, 1 μg/ml, for 1 hour, and subsequently stained for ACE and /KDR and intracellular NO. Of the four ACE- and KDR-based fractions of d7EB cells, only the ACE+/KDR+ and the ACE+/KDR− fractions produced NO, with more than 90% of the total cells in each fraction being positive, whereas no ACE− fractions exhibited any significant NO production. Experiments were repeated at least 3 times. FSc. forward scatter.