Potassium: a fifth “element” for the regulation of pluripotency and the cellular state in human pluripotent stem cells

Many inorganic elements are critically involved in the modulation of biochemical reactions and cell signaling pathways, suggesting that cells in unique states may display distinct elemental profiles and have specific requirements for different elements. Using X-ray fluorescence (XRF) spectrometry and inductively coupled plasma mass spectrometry (ICPMS) techniques, we measured the amounts of 56 major and trace inorganic elements in undifferentiated human pluripotent stem cells (hPSCs), their isogenic differentiated derivatives, and somatic cells used for cell reprogramming. While the amounts of most elements analyzed did not appear correlated with the pluripotent state of cells, the amount of potassium cation in undifferentiated hPSCs was significantly lower than that in multiple types of non-pluripotent cells. This phenomenon was reproducibly and consistently shown by both XRF spectrometry and ICPMS analyses in multiple hPSC lines and differentiated cells. Flow cytometry analysis using a cell-permeable fluorescence indicator for potassium, APG2-AM, also suggested that higher percentages of cells in pluripotent populations have a low level of intracellular potassium than those in non-pluripotent populations. To test whether the cellular pluripotency could be influenced by the manipulation of intracellular potassium, we used pharmacological tools to alter the permeability and intracellular concentration of potassium in hPSCs. The treatment with two potassium channel blockers, tetraethylammonium and 4-aminopyridine, increased intracellular potassium in human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), accompanied by the dose- and time-dependent downregulation of pluripotency markers POU5F1 and NANOG. In contrast, treatment with two types of potassium channel activators led to a decrease in intracellular potassium and the upregulation of POU5F1 and NANOG. Via 4-aminopyridine, we further exploited the link between cellular states and potassium thresholds, selectively eliminating hPSCs from differentiated derivatives within a dose window. Collectively, our data indicates that the amount of intracellular potassium is associated with the cellular states of hPSCs, and that the manipulation of intracellular potassium with pharmacological tools has functional impact on the regulation of pluripotency signaling in hPSCs. Potassium-altering agents may therefore be utilized in regenerative medicine for one of several purposes, including cellular purification and changing cellular identity. We demonstrate the first evidence that at the most basic level, a periodic element can be manipulated and have physiological, and potentially, therapeutic consequences.