Effects of Conditioned Medium from Bone Marrow Cells on Human Umbilical Cord Perivascular Cells

Mesenchymal cells derived from human umbilical cord tissue are attracting increasing attention as a source for cell therapy. However, for applying the same in tissue engineering, it has been shown that the differentiation capacity of mesenchymal stromal cells (MSCs) is influenced by the tissue from which the cells are harvested. Thus, to explore the possibility of increasing the osteogenic capacity of MSCs derived from the perivascular tissue of the human umbilical cord (human umbilical cord perivascular cells, HUCPVCs), we cultured these cells using conditioned medium (CM) derived from cultures of human bone marrow-derived mesenchymal stromal cells (hBMMSCs). However, hBM-CM contains a wide variety of growth factors, the amounts and ratios of which are considered to vary with the cell culture stage. Thus, we aimed to evaluate the effects of hBM-CM derived from different stages of hBMMSC culture on the osteogenic capacity of HUCPVCs. The stages of hBMMSC culture were defined as follows: Stage 1 (mitogenic stage) represented the period from the start of hBMMSC culture to 70% cell confluence; Stage 2 (confluent stage) represented the period from 70% confluence to the initiation of calcified nodule formation; and Stage 3 (calcification stage) represented the period following the initiation of calcified nodule formation. An analysis of growth factors contained in the CM obtained at each stage by enzyme-linked immunosorbent assay showed that insulin-like growth factor 1 (IGF-1) was significantly elevated at Stage 2, whereas vascular endothelial growth factor (VEGF) was significantly elevated at Stage 3. HUCPVCs were cultured using the CM from each of the stages for 1, 2, or 3 weeks. RUNX2 expression was the most upregulated at week 1 and then downregulated in all the groups. The expression of collagen 1 was significantly elevated in Stage 2 HUCs at week 3. Alkaline phosphatase (ALP) activity, ALP, and alizarin staining were higher in Stage 2 HUCs and Stage 3 HUCs. The calcium content was the highest in Stage 2 HUCs. The calcium content of HUCPVC obtained by the method used in this study was six times higher than that reported in the previous study. Collectively, our results show that the CM obtained at Stage 2 was most effective in driving the osteogenic differentiation of HUCPVCs. Impact Statement Mesenchymal stromal cells (MSCs) derived from the perivascular tissue of umbilical cords are promising candidates for regenerative medicine. Because these are able to be differentiated into bone cells, cartilage cells, and adipocytes. The number of MSCs in perivascular tissue (HUCPVCs) is ∼1/300 but the number of HUCPVCs that differentiates into osteogenic cells is quite low. In order to promote osteogenic differentiation of HUCPVCs, we cultured HUCPVCs using conditioned medium collected from human bone marrow-derived mesenchymal stromal cells. Our study suggests that the use of conditioned medium can be effective on inducing osteogenic differentiation of HUCPVCs.