Figure 1
Figure 1. Existence of a hierarchy among BM progenitors in their ability to differentiate and function as PACs. (A) BM-derived progenitors were isolated and grown in EGM-2 or IMDM medium for 7 days. VEGFR2 (KDR) expression was most induced in CMP- and GMP-derived PACs by flow cytometry. *P < .01 versus MEP. (B) CMP- and GMP-derived PACs exhibited more efficient uptake of DiI-AcLDL and Ulex lectin than MEPs and HSCs. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse TE-2000-u microscope at original magnification ×10 and Spot Version 5.0 software (Diagnostics Instruments Inc). (C) PACs derived from CMPs and GMPs expressed higher levels of PAC markers CD31, CD133, CD34, CD45, Tie2, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin by flow cytometry. *P < .05 versus HSCs. **P < .01 versus HSCs. (D-E) PACs derived from CMPs and GMPs more readily adhered to fibronectin-coated plates (D) and migrated faster than the MEPs and HSCs or the progenitors grown in IMDM (E). *P < .01 versus IMDM. **P < .01 versus MEPs. For adhesion assays, PACs (CMPs, GMPs, MEPs, and HSCs grown in EGM-2 for 7 days) were plated onto fibronectin-coated 24-well plates at a density of 20 000 cells per well. After 15 minutes of incubation at 37°C, unattached cells were removed by washing with phosphate-buffered saline, and the number of attached cells was quantitated. Migration assay was performed using a transwell system. (F) PACs derived from CMPs and GMPs promote network formation, as evidenced by Matrigel studies. The indicated PACs were incubated with human umbilical vein endothelial cells, and the number of “tubes” per high powered field was quantitated. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse 80i microscope at original magnification ×4 and Spot Version 5.0 software (Diagnostics Instruments Inc). (G) PACs derived from CMPs and GMPs incorporated more efficiently into networks compared with MEPs and HSCs. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse TE-2000-u microscope at original magnification ×10 and Spot Version 5.0 software (Diagnostics Instruments Inc). (H) PACs derived from CMPs and GMPs exhibited higher levels of VEGF by ELISA (left). *P < .01 versus MEPs. IL-1β (right) was not significantly different among the groups.

Existence of a hierarchy among BM progenitors in their ability to differentiate and function as PACs. (A) BM-derived progenitors were isolated and grown in EGM-2 or IMDM medium for 7 days. VEGFR2 (KDR) expression was most induced in CMP- and GMP-derived PACs by flow cytometry. *P < .01 versus MEP. (B) CMP- and GMP-derived PACs exhibited more efficient uptake of DiI-AcLDL and Ulex lectin than MEPs and HSCs. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse TE-2000-u microscope at original magnification ×10 and Spot Version 5.0 software (Diagnostics Instruments Inc). (C) PACs derived from CMPs and GMPs expressed higher levels of PAC markers CD31, CD133, CD34, CD45, Tie2, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin by flow cytometry. *P < .05 versus HSCs. **P < .01 versus HSCs. (D-E) PACs derived from CMPs and GMPs more readily adhered to fibronectin-coated plates (D) and migrated faster than the MEPs and HSCs or the progenitors grown in IMDM (E). *P < .01 versus IMDM. **P < .01 versus MEPs. For adhesion assays, PACs (CMPs, GMPs, MEPs, and HSCs grown in EGM-2 for 7 days) were plated onto fibronectin-coated 24-well plates at a density of 20 000 cells per well. After 15 minutes of incubation at 37°C, unattached cells were removed by washing with phosphate-buffered saline, and the number of attached cells was quantitated. Migration assay was performed using a transwell system. (F) PACs derived from CMPs and GMPs promote network formation, as evidenced by Matrigel studies. The indicated PACs were incubated with human umbilical vein endothelial cells, and the number of “tubes” per high powered field was quantitated. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse 80i microscope at original magnification ×4 and Spot Version 5.0 software (Diagnostics Instruments Inc). (G) PACs derived from CMPs and GMPs incorporated more efficiently into networks compared with MEPs and HSCs. *P < .01 versus MEPs. Images were visualized using a Nikon eclipse TE-2000-u microscope at original magnification ×10 and Spot Version 5.0 software (Diagnostics Instruments Inc). (H) PACs derived from CMPs and GMPs exhibited higher levels of VEGF by ELISA (left). *P < .01 versus MEPs. IL-1β (right) was not significantly different among the groups.

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