Among different pathways coordinating intracellular signaling, the most prominent is intracellular calcium signaling (ICS), controlling various cellular processes including proliferation, motility, apoptosis and differentiation . ICS is impressively diverse and consists of mechanisms that differ in frequency, amplitude and spatio-temporal patterning depending on an extensive molecular repertoire of signaling components. The free intracellular calcium concentration ([Ca2+
i) of a resting cell is in the range of 10–100 nM. Following physiological stimulation, [Ca2+
i levels can rise up to 1-2 μM concentrations. ICS is codified by the peak amplitude and frequency of [Ca2+
i transients, promoted by the entry of external Ca2+ through Ca2+ channels or the release of Ca2+ from internal stores. These internal stores are deposited within internal membrane structures such as the endoplasmic reticulum (ER). Following activation of G-protein-coupled receptors, phospholipase C-β (PLC-β) cleaves phosphatidylinositol 4,5-bisphosphate, releasing diacylglycerol and inositol-1,4,5-trisphosphate (IP3) which diffuses into the cell for activation of IP3 receptors (IP3R) and releasing Ca2+ from the ER. Moreover, Ca2+ enters the cytosol and activates ryanodine receptor (RYR) channels following activation of voltage-operated channels (VOCs), or receptor-operated channels (ROCs); this process is called Ca2+-induced Ca2+ release [2–4].
There are mainly two types of spontaneous intracellular Ca2+ transients: waves and spikes. The first one is mediated by IP3R and/or RYR activation and involves sensitivity to [Ca2+
i levels. This Ca2+ entry pathway is active at resting potential and is amplified by Ca2+ release from intracellular stores, and increases [Ca2+
i to a lower level than that attained by spikes. In the presence of gap junctions connecting cells, these intracellular waves can spread to neighboring cells, thereby coordinating neural activity and physiological processes of many cells [5, 6]. Compared to Ca2+ spikes, waves reveal a lower frequency with a mean duration of more than 30s, as observed in growth cones; their generation does not depend on action potentials. Waves occur locally and decay with distance from the site of initiation.
Calcium spikes depend on Ca2+ influx through VOCs or ROCs and Ca2+ release from intracellular stores (Ca2+ induced Ca2+ release via RYR), and achieve mean [Ca2+]i levels of 500 nM. They are characterized by their frequency (mean duration approximately 10s), and occur throughout an excitable cell, since they involve Ca2+-dependent action potentials.
Spontaneous Ca2+ spike frequency in cultured neurons initially varies from 1-10/h and then declines. Similar patterns of spike activity were observed in neural tube stages in vivo[2, 7] and during neuronal differentiation of embryonal carcinoma (CSC, a model for pluripotent embryonic stem cells) and adult bone marrow mesenchymal (hMSC) stem cells [8, 9]. Notably, these low frequencies of calcium transients regulate gene transcription and are suggested to be essential for the progress of neural differentiation and phenotype specification [2, 10–12].
Chemical and electrical signals, mediated by metabotropic and ionotropic receptors and VOCs promote intracellular calcium signaling and subsequent induction of differentiation. Adenosine 5’-triphosphate (ATP)-activated metabotropic and ionotropic receptors, also denominated as purinergic receptors, have drawn a lot of attention, due to their wide expression in almost every cell including stem cells. These receptors belong to the first neurotransmitter receptors expressed during development [13, 14]. ATP is the mainly purinergic messenger molecule and is released from cells in physiological conditions by exocytosis, transporters or even lysosomes. When the release occurs by damaged cells in an uncontrolled manner, ATP contributes to cell death and disease states. Once released into the extracellular space, ATP is degraded by ectonucleotidases producing the signaling molecules adenosine diphosphate (ADP), adenosine monophosphate  and adenosine . Based on pharmacological and structural properties, purinergic receptors are divided into metabotropic P1 and P2Y receptors as well as P2X ionotropic receptors.
P1 receptors subtypes are selective for adenosine and are classical seven-transmembrane metabotropic receptors coupled to several families of Gi, Go and Gs proteins. There are four types of adenosine receptors (A1, A2A, A2B and A3) differing by pharmacological and functional properties . A1 and A3 receptors exert inhibitory effects on adenylyl cyclase activity (mediated through Gi/o proteins) and also regulate PLC-β activity and thus IP3 synthesis . P2 receptor subtypes are activated by ATP, ADP, uridine-5'-triphosphate (UTP), uridine-5'-diphosphate (UDP) or UDP-glucose. P2 receptors are further divided into P2X and P2Y subtypes based on their structural characteristics .
P2X receptors as ATP-gated cationic (Na+/K+/Ca2+) channels [13, 16], are assembled in trimeric form as homomeric or heteromeric receptors from seven subunits (P2X1-P2X7). Recombinant P2X1, P2X2, P2X3, P2X4, P2X2/3, P2X2/6, P2X4/6, P2X1/5 as well as P2X7 receptors, when activated in a silent environment of cells not expressing any endogenous purinergic receptor, were all shown to be permeable for Ca2+. P2X receptors are mostly expressed by excitable cells, and Ca2+ entry through P2X receptor channels provides an important regulation mechanism of physiological responses in vivo, while aberrant Ca2+ entry, mostly mediated by P2X7 receptors, is suggested to be involved in pathophysiological conditions such as cell death, neuroinflammation and excitotoxic brain damage during epilepsy [18–22].
Metabotropic P2Y purinergic receptors activated by ATP, ADP, UTP, UDP or UDP glucose are composed by P2Y1,2,4,6,11,12,13,14 subtypes based on phylogenetic similarity . P2Y1,2,4,6,11 subtypes are coupled to Gq/G11 proteins activating PLC-β, thus inducing IP3-mediated Ca2+ release from the ER [13, 14, 23]. P2Y12,13,14 receptors inhibit adenylyl cyclase activity via Gi/o proteins. These latter-mentioned receptors also participate in regulating [Ca2+
i levels. For instance, P2Y receptor subtypes acting via Gi/o proteins can regulate N-type Ca2+ channel activity [24–26]. P2Y receptors are expressed in the central and autonomic nervous systems as well as by most non-excitatory cells where they exert long-term effects by regulating crucial cellular functions including proliferation and differentiation .
Recent studies have focused on changes of expression patterns and functions of this receptor family during differentiation from embryonic into neuronal cells [8, 27]. The developmental fate of differentiating stem cells depends on the ‘niche’, in which the cells exist, and several associated signaling systems have been pinpointed [28, 29]. In this review, we discuss the importance of ICS for the differentiation process of stem cells into neural cells with special emphasis on purinergic receptor function during Ca2+ signaling.