Intimal medial thickening (IMT) and vascular regeneration are hallmarks of arteriosclerosis disease. The source of lesional vascular smooth muscle (vSMC)-like cells contributing to the neointima thickening and the molecular mechanisms that dictate their fate and function have been the topic of much debate. Our lab along with other research groups have previously shown a putative role for re-capitulation of Notch and Hedgehog signalling components in promoting neointima thickening and progression of IMT in murine models of arteriosclerosis. In recent years, a population of smooth muscle myosin heavy chain (SM-SMHC)-negative multipotent vascular stem cells (MVSCs) have been isolated in the tunica media and shown to proliferate and differentiate into vSMCs and further invade the intima in animal models of vascular injury. The risk of vascular disease may depend, in part, on the embryological origin of vSMC progenitors; with vSMCs of neuroectodermal (NE) origin (ascending aorta, aorta arch and carotid artery) exhibiting a higher risk compared to their paraxial mesodermal (PM) counterpart (descending aorta). Herein, we demonstrate that NE (S100β+) stem cell-derived progeny substantially contribute to neointima thickening within carotid arteries of S100β-eEGP-CreERT2- Rosa26-tdT transgenic mice following ligation-induced injury and regeneration in vivo. S100β+ vascular stem cells (vSCs) of NE origin were successfully isolated and characterised from the two discrete embryological regions of arterial vessels (neuroectoderm versus mesoderm) in vitro and responded in the same manner to different myogenic stimuli including transforming growth factor-β1 (TGF-β1), the Notch ligand Jagged1 and the morphogens wnt4 and sonic hedgehog (Shh). The primary inductive stimulus for myogenic differentiation was the Jagged1/Notch signalling pathway, which was shown to enrich the H3K4me2 epigenetic mark at the SM-MHC locus. Furthermore, enhanced calponin1 (Cnn1) and Sm-mhc gene expression along with an increase in the number of CNN1 and SM-MHC positive cells was attenuated using γ-secretase inhibitor DAPT. In conclusion, neuroectodermal- derived (S100β+) vSCs may be responsible for the progression of pathological vascular remodelling and ultimately cardiovascular disease through re-capitulation of the Notch signalling pathway by the ligand, Jagged1.