Condition medium of HepG-2 cells induces the transdifferentiation of human umbilical cord mesenchymal stem cells into cancerous mesenchymal stem cells

Abstract

This study aimed to investigate the transdifferentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into cancer-associated mesenchymal stem cells (CA-MSCs) after incubation with condition medium (CM) from liver cancer HepG-2 cells, and the biobehaviors (proliferation and migration) of these CA-MSCs were further evaluated. The supernatant of HepG-2 cells was collected and mixed with equal volume of low glucose DMEM. The resultant medium was used to treat hUCMSCs for 48 h. The expression of CA-MSCs related proteins and miR-221 was detected in cells. The supernatant of induced hUCMSCs was mixed with equal volume of high glucose DMEM, and the resultant medium was used treat HepG-2 cells for 48 h and the proliferation and migration of HepG-2 cells were evaluated. Moreover, HepG-2 cells were co-cultured with hUCMSCs and then the proliferation and migration of HepG-2 cells were assessed. After incubation with the supernatant from HepG-2 cells, hUCMSCs showed significantly elevated expression of vimentin, fibroblast activation protein (FAP) and miR-221. The supernatant of induced hUCMSCs was able to significantly increase the proliferation and migration of HepG-2 cells. Following co-culture, the proliferation and migration of HepG-2 cells increased dramatically. These findings suggest that the supernatant of HepG-2 cells is able to induce the phenotype of CA-MSCs and the supernatant of CA-MSCs may promote the proliferation and migration of HepG-2 cells. These findings provide experimental evidence for the cellular remodeling in tumor microenvironment and the safety of clinical use of hUCMSCs.

Keywords: Stem cells; cell migration; hepatocellular carcinoma; transdifferentiation; umbilical cord mesenchymal stem cells.

Figure 1

A. Expression of vimentin and FAP in hUCMSCs after treatment with the supernatant from HepG-2 cells (Western blotting); B. Quantification of protein expression of vimentin and FAP. In hUCMSCs, treatment with the supernatant from HepG-2 cells significantly increased the protein expression of vimentin and FAP (*P<0.05 vs control group); C. Protein expression of vimentin and FAP. Treatment with the supernatant from HepG-2 cells markedly increased the protein expression of vimentin and FAP in hUCMSCs (immunofluorescence staining, scale bar =150 μm).

Figure 2

miR-221 expression in hUCMSCs. Treatment with the supernatant from HepG-2 cells significantly increased miR-221 expression in hUCMSCs (quantitative RT-PCR) (*P<0.05 vs control group).

Figure 3

A. Colony formation assay of HepG-2 cells. B. Quantification of colonies formed by HepG-2 cells in different groups. C. Number of HepG-2 cells from the fifth day to the tenth day in different groups. Supernatant from treated hUCMSCs significantly increased the colony formation capability of HepG-2 cells (*P<0.05 vs control group).

Figure 4

A. Migration of HepG-2 cells (scale bar =200 μm); B. Number of HepG-2 cells crossing the membrane in different groups. Treatment with the supernatant from treated hUCMSCs markedly increased the number of HepG-2 cells crossing the membrane (*P<0.05 vs control group).

Figure 5

A. Colony formation assay of HepG-2 cells co-cultured with hUCMSCs; B. Number of colonies formed by HepG-2 cells after co-culture with hUCMSCs (*P<0.05); C. Quantification of HepG-2 cells crossing the membrane from the fifth day to the tenth day. The number of colonies formed HepG-2 cells increased significantly after treatment with supernatant from treated hUCMSCs (*P<0.05 vs control group).

Figure 6

A. Migration of HepG-2 cells (scale bar =200 μm); B. Number of migrated HepG-2 cells in two groups. More HepG-2 cells migrates across the membrane after treatment with supernatant from treated hUCMSCs (*P<0.05 vs control group).