Mayuri Sinha Prasad

Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K+) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K+ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K+ currents composed by Ca2+-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K+ channels.

Growth of Human Embryonic Stem Cells in Long-Term Hypoxia

Human embryonic stem cells (hESCs) hold a great promise for regenerative medicine and tissue engineering. In order to obtain uniform hESC cultures without spontaneous differentiation, which is of interest for basic investigations as well as the development of future therapeutic protocols, it is important that specific culture conditions are adhered to. Here, we describe in detail a procedure for propagation of hESCs that by virtue of exposure of the cultures to low atmospheric oxygen (5%) enables the maintenance of their undifferentiated phenotype in long term. The critical steps and impact of possible modifications on the final outcome are discussed, and useful hints are provided to streamline the troubleshooting.