Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocytes

Abstract

Adipose stem cell-conditioned medium (ASC-CM) has been successfully used to treat multiple types of tissue and organ defects, including skin wounds both in vitro and in vivo. However, the mechanisms through which ASC-CM promotes wound healing remain unclear. We hypothesized that the wound healing effect of ASC-CM is mediated in part by the promotion of the migration of vascular endothelial cells, fibroblasts and keratinocytes, the three cell types essential for wound healing. We reported that ASC-CM stimulated the migration of these cells sequentially, and endothelial cells were the first cell type to respond to ASC-CM stimulation (4 h), followed by fibroblasts (12 h) and then keratinocytes (24 h). We also determined the optimal concentration of ASC-CM in stimulating these cells (50% dilution) in addition to the optimal time to intervene in order to maximize the wound healing activity of ASC-CM. Our data suggest an important role for ASC-CM in wound healing, possibly through the synthetic action of multiple adipose stem cell-derived cytokines that in turn promote cell migration. Thus, ASC-CM appears to have significant potential in wound healing applications.

Keywords: adipose stem cell-conditioned medium; fibroblasts; keratinocytes; migration; vascular endothelial cells.

Figure 1.

(A) Human umbilical vein endothelial cells (HUVECs), (B) keratinocytes and (C) fibroblasts from skin, ×100 magnification. Cells stained with crystal violet after migration, (D) HUVECs, (E) keratinocytes and (F) fibroblasts, ×200 magnification.

Figure 2.

Isolation and culture of human adipose-derived stem cells (hASCs) in vitro [(A) P0 (B) P1 (C) P2 (D) P3, ×100 magnification]. (Ea-d) Flow cytometry performed on the hASCs in the 3rd passage showed that isolated hASCs positively expressed CD13, CD44, CD90, CD105 and negatively expressed CD14, CD34. (F) Adipogenic differentiated ASCs were positively stained by Oil Red O. (G) Osteogenic differentiated adipose-derived stem cells (ASCs) were positively stained by Alizarin red. (H) Protein microarray analysis of ASC-CM. ASC-CM, ASC-conditioned medium. P0, primary ASCs; P1, passage 1; P2, passage 2; P3, passage 3.

Figure 3.

The effect of different concentrations of adipose stem cell-conditioned medium (ASC-CM) on migration of (A) human umbilical vein endothelial cells (HUVECs), (B) fibroblasts and (C) keratinocytes. The migratory effects of 50% ASC-CM were significantly greater than those of either lower concentrations (0, 10 and 25%) or higher concentrations (75 and 100%). Migration assay of (D) HUVECs, (E) fibroblasts and (F) keratinocytes stimulated by 50% ASC-CM at different time periods. Results showed that HUVEC migration started at 4 h, and peaked at 12 h. Fibroblasts started to migrate at 12 h and reached a maximum in 24 h. Keratinocytes appeared to be the slowest to respond to ASC-CM stimulation. Control, 0% ASC-CM; ^*P<0.05.

Figure 4.

Comparison of the migration of human umbilical vein endothelial cells (HUVECs), fibroblasts and keratinocytes stimulated by 50% adipose stem cell-conditioned medium (ASC-CM). Results showed 50% ASC-CM had a clear effect on cell migration, particularly on HUVEC and fibroblast migration, but the effect on keratinocyte migration was less marked, with 0% ASC-CM as the control.