A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells

Abstract

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.

Fig. 1

Expression of P2X7 mRNA transcripts and functional assays of P2X receptor expression. (A) RT-PCR was performed for P2X7 variants using the primers described in Table 1. Amplification products of the expected size were detected where E represents mouse embryonic stem cells, M represents J774.2 mouse macrophages and -T is the no template control. (B) Whole cell patch clamp recordings from undifferentiated mouse ES cells at the holding potential of − 60 mV. Histogram summarises the mean peak currents normalised to membrane capacitance detected at the ATP concentrations given. (C) Membrane permeabilisation measured by the uptake of ethidium and corresponding increase in fluorescent signal (544 nm excitation; 590 nm emission). An example kinetic trace of ethidium measurements from mouse ES cells with a saline vehicle (○) versus the addition of 1 mM ATP (●). (D) Concentration response relationship for ATP versus the rate of ethidium influx normalised to 1 mM ATP responses (n = 5) as described in methods. Data are plotted as the mean ± SEM.

Fig. 2

Pharmacological evidence for the expression of P2X7 receptors in mouse ES cells. Whole cell voltage clamp recordings from mouse ES cells with a holding potential of − 60 mV. Reversible inhibition of ATP-mediated current with a selective P2X7 antagonist A438079. Application of 5 mM ATP in the absence or presence of 10 μM A438079 as indicated.

Fig. 3

Evidence for P2X7 receptor expression by siRNA knockdown of receptor expression. (A) An example of ATP-mediated ethidium uptake in the absence or presence of 10 μM A438079 as indicated in the figure. Buffer control (ο) or addition of 1 mM ATP (●) as indicated. Representative of 3 independent experiments. (B) Knockdown of P2X7 expression by transfection of mouse ES cells with P2X7 targeting siRNA (P2X7 siRNA) versus a non-targeting control siRNA (NT siRNA) as indicated. P2X7 receptor expression was assessed by semi-quantitative RT-PCR using panP2X7 primers and β-actin as a positive control (primers Table 1). (C) Ethidium uptake is reduced in cells treated with 20 nM siRNA knockdown of P2X7 expression versus cells treated with the 20 nM control non-targeting (NT) siRNA as indicated in the figure. Representative of 3 independent experiments.

Fig. 4

Stimulation of mouse ES cells with exogenous ATP leads to necrosis. Release of lactate dehydrogenase (LDH) as a function of total cellular LDH (plotted as % total cellular LDH) for mouse ES cells transfected with 20 nM non-targeting siRNA (NT siRNA) or P2X7-targeted siRNA (P2X7 siRNA) as indicated (n = 3). Cells stimulated with ATP for 45 min at 37 °C. * P < 0.05 by unpaired two-tailed t test.

Fig. 5

Comparison of exogenous ATP hydrolysis between mES cells and human umbilical vein endothelial cells. (A–B) Measurement of extracellular ATP levels following addition of 100 nM ATP for HUVEC (A) or mES cells (B). (C) Histogram comparing the rate of ATP metabolism for HUVEC (n = 4) versus mouse ES cells (n = 5). Data are plotted as the mean ± SEM. *** P < 0.001 by unpaired two-tailed t test.

Fig. 6

The role of P2X7 receptors in mouse ES cell colony number and self-renewal. Mouse ES cells were plated at approximately 166 cells/cm² and cultured for 5 days (in the presence or absence of 10 μM A438079) prior to counting the percentage of colonies intensely staining for pure alkaline phosphatase activity (A–B) and total colony number normalised to control untreated cells (C). Cells cultured in GMEM or KO-DMEM (indicated in the figure) for 5 days as described in Materials and methods prior to analysis for colony number (C) and frequency of pure alkaline phosphatase positive colonies (B) where data are plotted as the mean ± SEM (n = 3). (A) A representative image of mES cells cultured in KO-DMEM in the presence or absence of 10 μM A438079. Scale bar = 400 μm. (D) Nanog-GFP expression in mES cells cultured in KO-DMEM for 48 and 72 h in the absence or presence of 10 μM A438079 as indicated in the figure. GFP fluorescence analysed by flow cytometry, representative images in figure with GFP^-ve control cells in insert. (E) Mean GFP fluorescence from flow cytometry experiments plotted as a histogram with n values indicated in the figure. Data plotted as mean ± SEM.