Loading Human Mesenchymal Stem Cells with Trehalose by Fluid-Phase Endocytosis

Abstract

Human mesenchymal stem cells (MSCs) have shown great promise in the area of tissue engineering. Regardless of their regenerative potential, however, they will not be useful on a large scale unless an improved and more stable form of cellular storage is developed. An ideal storage condition would be dehydrated cells, as this would allow room temperature storage and would not require refrigeration or freezing equipment. As a first step toward developing a method for storing MSCs in a desiccated state, we have characterized the ability of these cells to take up solutes from the extracellular milieu, as the introduction of protective solutes into the cytosol is a critical step in the dehydration process. Lucifer yellow (LYCH), a well-known probe in the study of fluid-phase endocytosis, indicated the uptake process was inhibited below 20°C. Fourier transfer infrared spectroscopy studies suggested that this inhibition is associated with the membrane physical state. In addition, fluorescence microscopy revealed endosomes stained with LYCH accumulated in the MSCs, and suggested that the dye entered the cytosol over time. Trehalose, a protective disaccharide, was accumulated by the MSCs as well. Uptake was proportional to the length of incubation and showed a nonsaturating dependence on extracellular concentration, characteristic of fluid-phase endocytosis. Endocytosis inhibitors were used to investigate further the mechanism of uptake. Colchicine and nocodazole, both of which depolymerize microtubules, blocked trehalose uptake. Dimethyl sulfoxide, which affects microtubules differently, by forming shorter and more abundant microtubules, also inhibited trehalose uptake. However, cytochalasin B, which depolymerizes actin filaments, and thus blocks both macropinocytosis and caveolae-dependent pinocytosis, did not cause a decrease in trehalose uptake. Amiloride, which blocks sodium channels and inhibits clathrin-independent pinocytosis, also did not inhibit trehalose uptake. Taken together, these results suggest that human MSCs are capable of loading trehalose from the extracellular space by a clathrin-dependent fluid-phase endocytotic mechanism that is microtubule-dependent but actinindependent.