Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

Background
Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.
Principal Findings
P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.
Conclusions
In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.

背景
P2X7受体（P2X7 receptor, P2X7R）已被证实具有新的发育生物学功能，包括增殖刺激与神经分化。经视黄酸（retinoic acid）诱导神经分化的小鼠胚胎干细胞（embryonic stem cell, ESC），能够模拟早期胚胎神经外胚层发育过程中的相关事件。
主要研究结果
未分化状态的小鼠胚胎干细胞中，P2X7R与多能性标志物Oct-4的表达水平最高。在未分化细胞中，P2X7R激动剂Bz-ATP（benzoyl-ATP, Bz-ATP）可加速细胞周期进程，该效应可被特异性P2X7R抑制剂KN-62阻断。经视黄酸诱导神经分化的小鼠胚胎干细胞，其Oct-4与P2X7R的表达水平均显著下调。与既往相关研究相比，未分化及神经分化的细胞中，仅需更低浓度的Bz-ATP配体即可触发P2X7R介导的细胞内钙流反应。
KN-62的存在可使表达SSEA-1（阶段特异性胚胎抗原1, stage-specific embryonic antigen 1, SSEA-1）、Dcx（双皮质素, doublecortin, Dcx）及β3-微管蛋白（β3-tubulin）的细胞数量增加，同时使SSEA-1与β3-微管蛋白双阳性细胞的数量增多，这证实神经外胚层分化的启动与神经元命运决定依赖于P2X7R活性的抑制。此外，在P2X7R被抑制的条件下，Ki-67（增殖标志物Ki-67）阳性细胞数量增加，这表明干细胞祖细胞得以重新进入细胞周期，进而增加成神经细胞的数量，最终促进神经发生。
结论
在胚胎干细胞中，P2X7R的表达与活性上调可维持细胞增殖；而当胚胎干细胞被诱导进行神经分化时，P2X7受体的表达与活性需要被抑制。