Extracellular matrix degradation products and low-oxygen conditions enhance the regenerative potential of perivascular stem cells

Abstract

Tissue and organ injury results in alterations of the local microenvironment, including the reduction in oxygen concentration and degradation of the extracellular matrix (ECM). The response of perivascular stem cells to these microenvironment changes are of particular interest because of their wide distribution throughout the body and their potential involvement in tissue and organ response to injury. The chemotactic, mitogenic, and phenotypic responses of this stem cell population were evaluated in response to a combination of decreased oxygen concentration and the presence of ECM degradation products. Culture in low-oxygen conditions resulted in increased proliferation and migration of the cells and increased activation of the ERK signaling pathway and associated integrins without a change in cell surface marker phenotype. The addition of ECM degradation products were additive to these processes. Reactive oxygen species within the cells were increased in association with the mitogenic and chemotactic responses. The increased proliferation and chemotactic properties of this stem cell population without any changes in phenotype and differentiation potential has important implications for both in vitro cell expansion and for in vivo behavior of these cells at the site of injury.

FIG. 1.

Perivascular stem cells exhibit increased growth in 6% compared with 21% oxygen. Growth of perivascular stem cells plated at 6% (filled circles) or 21% (open circles) oxygen. Cells were plated at an initial seeding density of 1 × 10⁴ per well and duplicate wells harvested every 24 h with total cells per well counted. All of the data are means of triplicate determinations with SD (some values are too small to be visible). Equivalent trends have been observed on two further occasions. Inset: the doubling rate of perivascular stem cells per 24 h period under 6% (filled bars) or 21% (open bars) oxygen. SD, standard deviation.

FIG. 2.

Cells have elevated DNA synthesis and metabolism in 6% compared with 21% oxygen, which addition of UBM further elevates. (A) Cells were plated at 6% (filled columns) or 21% (open columns) oxygen at 5 × 10⁴ cells per well containing 0, 2, 5, or 15 μg/mL UBM and incubated for 18 h. New DNA synthesis was monitored via 5-bromo-2′-deoxyuridine incorporation. The presence of 5-bromo-2′-deoxyuridine in DNA was determined via immunoassay and quantified colormetrically at 370 and 492 nm. All of the data are means of triplicate determinations with SD. Proliferation of cells in UBM was compared with cells in UBM-free media at the same oxygen concentration. Statistical analysis was performed using the Student's t-test where *p < 0.05. Equivalent trends have been observed on two further occasions. (B) About 1 × 10⁵ cells per well were plated at 6% (filled columns) or 21% (open columns) oxygen in media containing 10% alamarBlue and 0, 10, 25, 50, or 100 μg/mL UBM. The metabolism of alamarBlue was quantified colormetrically at 600 nm at time 0 and after 6 h incubation. All of the data are means of triplicate determinations with SD. Metabolism of cells in UBM was compared with cells in UBM-free media at the same oxygen concentration, where *p < 0.05. Equivalent trends have been observed on two further occasions. UBM, urinary bladder matrix.

FIG. 3.

Perivascular stem cells in 6% oxygen have elevated reactive oxidative species and greater superoxide dismutase activity. (A) Cells grown in 6% (filled symbols) or 21% (open symbols) oxygen were loaded with dichlorofluorescine diacetate before plating into media containing 0 (circles) or 20 μg/mL (triangles) UBM digest material. Fluorescence intensity was quantified at excitation wavelength (Ex) 485 nm and emission wavelength (Em) 538 nm at 5 min intervals. (B) Cells grown in 6% (filled columns) or 21% (open columns) oxygen were exposed to 0 or 20 μg/mL UBM digest material for 6 h before quantification of cellular superoxide dismutase activity against a superoxide dismutase standard curve.

FIG. 4.

Cells cultured in 6% oxygen maintain mesenchymal stem cell markers and mutilineage potential. (A) Cells cultured at 21% (left panels) or 6% oxygen (right panels) were analyzed for mesenchymal stem cell markers CD105 coexpressed with CD44 (top) and CD90 coexpressed with CD 73 (bottom). (B) Perivascular stem cells were cultured in 6% (left) or 21% (right) oxygen and tested for the ability to differentiate. Cells were maintained in adipogenic medium, fixed, and stained for lipids with oil red O (Top). Stem cells were centrifuged into a pellet and cultured in the chondrogenic medium containing TGF-β1. Pellets were then sectioned, and stained with Alcian blue and nuclear fast red for detection of sulfated glycosaminoglycans and nuclei, respectively (middle). Cells cultivated in the osteogenic medium were stained with alizarin red at pH 4.2, staining calcium deposits red (bottom). The high degree of differentiation is shown by the almost solid blue (sulfated glycosaminoglycans, middle image) and solid red (calcium, bottom image) observed. Images at 40 ×.

FIG. 5.

Low oxygen and the presence of UBM digest material increase phosphorylation of ERK1/2. The phosphorylation of ERK1/2 after exposure of perivascular stem cells to 0, 50, or 100 μg/mL UBM digest material for 16 h at 6% or 21% oxygen was monitored via immunoblot. The equivalent intensity of the bands for β-actin and ERK across loading conditions indicates equal loading of protein per well.

FIG. 6.

Migration of perivascular stem cells toward UBM digest material is elevated at 6% compared with 21% oxygen. The migration of cells toward 0, 25, 50, 100, and 250 μg/mL UBM was monitored at 6% (filled columns) or 21% (open columns) oxygen. Chemotaxis chambers containing 3 × 10⁴ cells per well were incubated for 3 h at their respective oxygen concentration, and migrated cells counted. All of the data are means of quadruplicate determinations with SD. Migration of cells in UBM was compared with cells in UBM-free media at the same oxygen concentration. Statistical analysis was performed using the Student's t-test, where *p < 0.05. Equivalent trends have been observed on two further occasions.