Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells

The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pHi), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pHi and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pHi took place in this population. In contrast, depolarization caused an increase in pHi (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pHi (pH 6.9–7.1) also responded to acute hypoxia. During hypoxic treatment the resting pHi decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

► pHi measurements on BCECF loaded differentiating neural stem cells showed that two main populations were present.
► These populations responded differently when they were exposed to acute hypoxia, extracellular acidosis or NH4+.
► Neural stem cells tolerated pH values down to pHo 6.5 while pHo 6.2 totally inhibited neural stem cell development.
► Depolarization caused an increase in pHi in the GLAST positive cell population located closer to the neurosphere body.
► Live pH measurements can be used to select specific cell populations in the neurosphere culture.