Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

Abstract

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H2O2) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100μM H2O2 dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs.

Keywords: Antioxidative enzymes; Extracellular matrix; Mesenchymal stem cells; Osteogenesis; Reactive oxygen species.

Fig.1.

Evaluation of decellularized extracellular matrix (ECM) deposited by UC-MSCs. (A) Decellularized ECM showed a fibrous structure in a representative bright field image. Scale bar = 100 μm. (B) Scanning electron microscopy analysis revealed that decellularized ECM was composed of net-like lattices with small bundles of collagen fibers. Scale bar = 3 μm. (C) Representative images of immunofluorescence staining identified four matrix proteins (type I and III collagens, fibronectin, and laminin) in decellularized ECM. Scale bar = 100 μm.

Fig.2.

Effects of culturing on ECM on cell proliferation and cell cycle phase distribution in UC-MSCs. (A) UC-MSCs were seeded on TCPS and ECM-coated surfaces at the cell density of 1,000 cells/cm². The DNA content, representing cell proliferation, was assessed on days 3, 5, and 7. (B) UC-MSCs were seeded on TCPS and ECM at the cell density of 100, 1,000, and 5,000 cells/cm². The DNA content was assessed on day 5. (C) Cell morphology and density were observed in representative fluorescence images labeled by fluorescein diacetate (FDA) when UC-MSCs were initially cultured at the cell density of 1,000 cells/cm². Scale bar = 100 μm. (D) The cell cycle phase distribution of UC-MSCs cultured on TCPS and ECM was measured by flow cytometry analysis. Values are the mean ± S.E. of five independent experiments (n = 5) in the DNA content assay and of three independent experiments (n = 3) in the cell cycle phase distribution analysis. Statistically significant differences are indicated by ** (p < 0.01). TCPS: tissue culture polystyrene; ECM: extracellular matrix.

Fig.3.

Culturing on ECM attenuated accumulation of intracellular reactive oxygen species (ROS). (A) Intracellular ROS of UC-MSCs cultured on TCPS and ECM was labeled by DCF-DA and fluorescence intensity was measured by flow cytometry. (B) Intracellular hydrogen peroxide of UC-MSCs cultured on TCPS and ECM was labeled by Amplex^® Red dye. (C) The mRNA level of SIRT1 was measured by real-time RT-PCR. (D) Culturing on ECM upregulated the protein level of SIRT1, determined by western blot analysis. The α-tubulin lane served as a loading control. (E) Culturing on ECM downregulated phosphorylation (p-) of p38 MAPK. The level of p-p38 was normalized to total p38 protein. The level of p38 was normalized to the α-tubulin protein. Values are the mean ± S.E. of four independent experiments (n = 4) in ROS analysis, hydrogen peroxide analysis, real-time RT-PCR, and western blot analysis. Statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01). P: TCPS/ tissue culture polystyrene; E: ECM/extracellular matrix.

Fig.4.

Effects of culturing on ECM on superoxide dismutase (SOD) in UC-MSCs. (A) Culturing on ECM upregulated SOD1 mRNA expression in UC-MSCs, determined by real-time RT-PCR. (B) The protein levels of SOD1, measured by western blot analysis, did not change in cells cultured on TCPS and ECM. (C) Culturing on ECM increased SOD2 mRNA expression in UC-MSCs. (D) The protein level of SOD2 was enhanced in ECM-cultured UC-MSCs. (E) Culturing on ECM improved SOD activity in UC-MSCs. Values are the mean ± S.E. of four independent experiments (n = 4) in real-time RT-PCR, western blot analysis, and SOD activity analysis. Statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01). P: TCPS/ tissue culture polystyrene; E: ECM/extracellular matrix.

Fig.5.

Effects of culturing on ECM on catalase and GPx1 in UC-MSCs. (A) Culturing on ECM upregulated CAT mRNA expression in UC-MSCs, determined by real-time RT-PCR. (B) The protein levels of catalase, measured by western blot analysis, did not change in cells cultured on TCPS and ECM. (C) Culturing on ECM improved catalase activity in UC-MSCs. (D) The mRNA level of GPX1 did not change in ECM-cultured cells. (E) The protein level of GPx1 did not change in ECM-cultured cells. Values are the mean ± S.E. of four independent experiments (n = 4) in real-time RT-PCR, western blot analysis, and catalase activity analysis. Statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01). P: TCPS/ tissue culture polystyrene; E: ECM/extracellular matrix.

Fig.6.

Culturing on ECM improved resistance of UC-MSCs to exogenous H₂O₂ and restored the osteogenic differentiation capacity. UC-MSCs were cultured on TCPS and ECM, and continuously treated with 100 μM H₂O₂ during osteogenic differentiation. (A) Representative images of Alizarin Red S staining, indicating mineralization of the matrix, showed that treatment with H₂O₂ suppressed matrix calcification in TCPS-cultured cells, but culturing on ECM retained the capacity of UC-MSCs for calcified matrix deposition. (B) The stained mineral layers were dissolved in 1% hydrochloric acid and were quantified via a spectrophotometer. (C)-(H) The mRNA levels of osteoblast-specific marker genes, including COL1A1 (C), SPP1 (D), ALP (E), RUNX2 (F), BGLAP (G), and SP7 (H) were measured by real-time RT-PCR. Values are the mean ± S.E. of four independent experiments (n = 4) in Alizarin Red S staining and real-time RT-PCR. Statistically significant differences are indicated by * (p < 0.05) or ** (p < 0.01). TCPS: tissue culture polystyrene; ECM: extracellular matrix; NEG: negative control; OS: osteogenic induction; H₂O₂: hydrogen peroxide.