Human umbilical cord perivascular cells (HUCPVCs) derived from regions surrounding the umbilical cord vessels represent an attractive cell source for cellular therapies, given their proliferative potential and the accessibility of donor material compared with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). However, these cells remain poorly characterised. Using flow cytometry, HUCPVCs were shown to express conventional MSC markers CD29, CD44, CD73, CD90, CD105, CD146, CD166 and integrins alpha1 to -5, alphaV, alphaVβ3, alphaVβ5, β1 and β3, but not CD14, CD34, CD45, STRO-1 or integrin alphaVβ6. HUCPVC marker profiles were consistent between three donors and at different passage numbers. Immunostaining for smooth muscle cell (SMC) markers; alpha-SMA, SM22alpha and smoothelin revealed that HUCPVCs shared expression of these markers with SMCs. However, in comparison with SMCs, HUCPVCs deposited more extensive fibronectin-rich matrices. When compared with hBM-MSCs, HUCPVCs differentiated along adipogenic and osteogenic lineages more slowly and did not progress to terminal phenotypes. mRNA expression of recently identified mesenchymal progenitor cell markers, ROR2, EPHA2, PLXNA2, CDH13 and CD9, was confirmed in HUCPVCs from two donors. In addition, all these markers (except EPHA2) were detected in the umbilical cord vessel wall cells of three donors, confirming their expression in both cultured HUCPVCs and cells of the primary tissue. To determine the roles of these markers in HUCPVCs, they were depleted individually using siRNA. Knockdown (KD) efficiencies of 90-97% were achieved. CD9 KD cells appeared elongated compared to cells treated with control siRNA, and these cells along with ROR2, EPHA2 and PLXNA2 KD cells exhibited larger cell areas than controls. All KD cells also showed decreased proliferative potential by day 6 compared with control siRNA or lipofectamine treated cells. A decrease in total β1 integrins was detected in the CD9 KD cells. Up-regulation of ROR2 and PLXNA2 mRNA expression was detected in HUCPVCs from two donors, when they underwent osteogenic differentiation. ROR2 and PLXNA2 knockdown resulted in increases in PLXNA2 and ROR2 mRNAs respectively, when cells were cultured in osteogenic medium compared with basal conditions. In addition, each individual knockdown revealed that the KD cells showed trends in increasing RUNX2 mRNA expression after 13-16 days in osteogenic medium. These data suggest that ROR2 and PLXNA2 may co-operate in promoting an osteogenic phenotype. Culturing HUCPVCs on non-mineralised BVSMC-derived matrices had very little impact on their differentiation status. In contrast, when HUCPVCs were cultured on mineralised BVSMC-derived matrices in osteogenic medium, their ability to further deposit mineralised matrix was enhanced by 7 days; no accompanying changes in RUNX2, ROR2 or PLXNA2 mRNA expression were detected. Taken together, early up-regulation of RUNX2, ROR2 and PLXNA2 appears to be important in driving osteogenic differentiation in HUPCVCs, whilst subsequent down-regulation of these markers may be required for mineralisation to occur. HUCPVCs express ROR2, PLXNA2, CDH13 and CD9 in vitro and in situ; these markers have distinct roles in regulating cell proliferation, shape and differentiation which may be regulated via changes in β1 integrins. It is not known why HUCPVCs might differentiate along adipogenic and osteogenic lineages more incompletely than hBM-MSCs. Further comparative characterisation of HUCPVCs and hBM-MSCs is a prerequisite for exploiting their vast clinical potential.