Conditioned media from human adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells efficiently induced the apoptosis and differentiation in human glioma cell lines in vitro

Abstract

Human mesenchymal stem cells (MSCs) have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs). We found (1) MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2) MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3) real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4) furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

Figure 1

Growth inhibition of U251 glioma cells in MSCs conditioned media. The proliferation of U251 cells was inhibited by conditioned media from both ASC-CM and UC-MSC-CM. The number of cells in control cultures of U251 glioma cells was taken as 100%. The effect on proliferation of U251 under experimental conditions was calculated in relation to single cultures of U251 cells. Numbers on the X-axis indicated plated cell number per well.

Figure 2

Growth inhibition of HT 29, A549, and MCF-7 cell lines in MSCs conditioned media.

Figure 3

Annexin and PI flow cytometric assay. (a) Representative annexin and PI flow cytometric picture of U251 cells under ASC- and UC-MSC-conditioned media coculture conditions. (b) Statistical results of three independent flow experiments.

Figure 4

Quantitative real-time PCR of caspase 3, caspase 9, survivin, XIAP, and GFAP after MSCs conditioned medium induction. MSCs conditioned medium treatment leads to significant overexpression of caspase 3 and caspase 9 and significant downregulation of survivin and XIAP. GFAP expression is also significantly increased after induction.

Figure 5

Cell cycle analysis of U251 cells after ASCs and UC-MSCs conditioned medium induction. MSCs conditioned medium treatment led to G0/G1 growth arrest in U251 cells.

Figure 6

MSCs conditioned media coculture leads to significant differentiation of U251 cells towards a normal glial cell phenotype. Phase contrast image of U251 cells before and after MSCs conditioned media coculture. U251 glioma cells are usually with short processes. Much more and longer processes were seen after MSCs conditioned media induction and these processes connected with neighboring cells, forming an interconnected network.

Figure 7

Scratch assay. U251 cells were induced with ASC-CM and UC-MSC-CM for two days. The cells quickly grew over the scratch for U251 cells cultured alone. After 48 hours, the scratch was invisible. For ASC-CM and UC-MSC-CM culture, although there are still some cells growing over the scratch line, the scratch is still visible. Fewer cells are seen grown over the line.

Figure 8

Matrigel invasion assay. U251 cells or U87 cells (induced or noninduced) were placed in transwells with 8 μm pore size polycarbonate filters, precoated with Matrigel. The lower wells were filled with DMEM with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours. Nonmigratory cells on the upper surface of the transwells were removed and the migratory cells from the lower surface of the transwell were fixed and stained with 1% crystal violet. Cells were photographed and average cells per field were calculated. Experiments were done in triplicates on three times independently.

Figure 9

Cellomics high content screening (HCS) system analysis indicated much more prominent cell process outgrowth after MSCs conditioned media induction. (a) Representative fluorescent picture of U251 glioma cells before and after conditioned media induction. GFAP positive cells are shown in red. Much longer cell processes were seen after conditioned media induction. (b) Statistical analysis of cell process outgrowth before and after MSCs conditioned media induction. Both ASC- and UC-MSC-conditioned media led to more significant outgrowths of cell processes.