Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death

Abstract

Schwann cells (SCs) are fundamental for development, myelination and regeneration in the peripheral nervous system. Slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine. Several molecules, including receptors for neurosteroids and neurotransmitters, have been suggested to be implicated in regulating physiology and regenerative potential of SCs. Adipose-derived stem cells (ASCs) can be differentiated into SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid receptors, which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitters in ASC. In this study, we investigated the expression of purinergic receptors in dASC. Using reverse transriptase (RT)-PCR, western blot analyses and immunocytochemistry, we have demonstrated that ASCs express P2X3, P2X4 and P2X7 purinoceptors. Differentiation of ASCs towards glial phenotype was accompanied by upregulation of P2X4 and P2X7 receptors. Using Ca2+-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5′-triphosphate (ATP) triggers intracellular Ca2+ signals, indicating functional activity of these receptors. Whole-cell voltage clamp recordings showed that ATP and BzATP induced ion currents that can be fully inhibited with specific P2X7 antagonists. Finally, using cytotoxicity assays we have shown that the increase of intracellular Ca2+ leads to dASC death, an effect that can be prevented using a specific P2X7 antagonist. Altogether, these results show, for the first time, the presence of functional P2X7 receptors in dASC and their link with critical physiological processes such as cell death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for nerve repair.

Purinergic and Store-Operated Ca2+ Signaling Mechanisms in Mesenchymal Stem Cells and Their Roles in ATP-Induced Stimulation of Cell Migration

“…This study examined hDP-MSCs from several donors and obtained consistent evidence to demonstrate that P2X7, P2Y 1 , and P2Y 11 are the purinergic P2 receptors responsible for ATP-induced Ca 21 signaling in hDP-MSCs ( Figs. 1 and 2), confirming expression of these P2 receptors in MSCs reported by some previous studies using BM-MSCs and AT-MSCs [19][20][21][22]28]. Nonetheless, this study observed noticeable variations in the results obtained in cells from different donors, for example, at both mRNA and functional expression levels (Figs.…”

“…Accumulating evidence shows that MSCs release ATP constitutively or in response to mechanical stimulation [17][18][19][20]. Previous studies consistently demonstrated that ATP induced robust Ca 21 responses but reported expression of a bewildering variety of P2X and P2Y receptors in MSCs from different tissues and species [17][18][19][20][21][22][23][24][25][26][27][28] and, as a result, the cognate intrinsic mechanisms remains contentious. There is also evidence for occurrence of storeoperated Ca 21 entry in BM-MSCs [17], but the molecular identity of the Ca 21 channels is still elusive.…”

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…In the eye, subretinal hemorrhage increases the [ATP] o , leading to a P2X7R-dependent Ca 2ϩ influx that activates caspase-8 and initiates photoreceptor cell apoptosis (38). In the central nervous system, P2X7Rs are found on microglia, oligodendrocytes, and astrocytes, where they contribute to the genesis of neuropathic and chronic inflammatory pain (25) and influence cell survival (39). Although the presence of P2X7Rs on central neu-rons is controversial (6,40), recent data suggest they play a role in modulation of release of transmitters and hormones (41,42).…”

Baicalin promotes the viability of Schwann cells in vitro by regulating neurotrophic factors

“…Furthermore, SCs create a suitable axonal growth environment by producing neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and neurotrophic factors-3, -4/5 and -6 (NT-3, NT-4/5, and NT-6, respectively) (8)(9)(10)(11)(12). However, the slow growth rate of SCs is reported to be one of the major limitations of SC application in regenerative medicine (13). In addition, elevating the proliferation ability of SCs is important for constructing tissue-engineered nerves (6).…”