Figure 4. Effects of gain of function of Notch ligand杕ediated signals on EPC differentiation. A, Scheme of the insert culture system with 0.4-祄 pores, in which receptors on the surface of the target stem cells in the upper chamber are capable of directly interacting with ligands on the cells in the bottom chamber. In the present study, BM-Lin?/SUP> cells were seeded in the upper chamber, whereas 3T3 stromal cells overexpressing a specific Notch ligand, Jag-1 or Dll-1, were placed in the bottom chamber. B, Reverse-transcription polymerase chain reaction revealed enhanced expression of the specific Notch ligand in the stromal cells stimulated by Jag-1 or Dll-1 signal compared with those transduced with empty vector. C, Reverse-transcription polymerase chain reaction revealed expression of Hes-1 and Hes-5, target effector genes of active Notch, in BM-Lin?/SUP> cells, which were cocultured with 3T3 stromal cells specifically expressing the target Notch ligand gene. D, Fluorescence-activated cell sorting analysis revealed more frequent expression of Flk-1 and CD31 in BM-Lin?/SUP> cells stimulated by Jag-1杕ediated signals, but not Dll-1杕ediated signals, than in those stimulated by empty vector. E, Reverse-transcription polymerase chain reaction to detect expression of typical EPC surface markers and vascular endothelial growth factor in BM-Lin?/SUP> cells stimulated by specific Notch ligand or empty vector. The cellular mRNA level of CD31, Flk-1, and vascular endothelial cadherin (VE-cadherin) was elevated in the Jag-1 group compared with the Dll-1 and empty-vector groups. In contrast, the cellular mRNA level of vascular endothelial growth factor and Flt-1 was similar in all groups. PECAM indicates platelet and endothelial cell adhesion molecule. F, EPC colony-forming assay using BM-KSLs stimulated by specific Notch ligand杕ediated signals revealed significant augmentation of vasculogenic capacity in the Jag-1 group, but not the Dll-1 group, compared with the empty-vector group. CFU indicates colony-forming units. **P<0.01 (n=3 in each group). G, Frequency of apoptotic cells (TUNEL-positive cells) in BM Sca-1+/Lin?/SUP> cells in vitro was significantly lower in EPC-enriched cells stimulated by Jag-1 signal than in those stimulated by Dll-1 signal or empty vector. *P<0.05 (n=3 in each group).