**1. Introduction**

Mesenchymal stromal cells (MSCs) are described as a heterogeneous cellular pool that includes immature cells responsible for the replenishment of supportive and connective tissues due to their capability of maintaining self-renewal and multipotent differentiation [1–3]. By unique biologic properties, cultured MSCs from different sources attract sufficient interest in the fields of regenerative medicine and immunotherapy [4–6]. Despite evident progress in MSC biology spurred by the therapeutic potential of these cells, current knowledge on their receptor and signaling systems remains scarce. Evidence exists that MSCs are capable of sensing complex extracellular cues, including hormones, cytokines, and nucleotides [7, 8]. This implies that MSCs employ multiple surface receptors and signaling pathways to adjust their physiological functions to specific tissue microenvironment.

are ubiquitously involved in Ca2+ signaling [21]. Although α<sup>2</sup>

been documented [22]. All three β-subtypes are linked to adenylyl cyclase by G<sup>s</sup>

isoforms of adrenergic and purinergic receptors are coupled to Ca2+ mobilization in diverse cell types, we considered Ca2+ imaging as an adequate approach to detail purinergic and

MSCs of the first passage were obtained from the Faculty of Basic Medicine at Lomonosov Moscow State University. All procedures that involved human participants were performed in accordance with the ethical standards approved by the Bioethical Committee of the Faculty based on the 1964 Helsinki declaration and its later amendments. The study involved 21 healthy (not suffered from infectious or systemic diseases and malignancies) individuals from

Cells were isolated from subcutaneous fat tissue of healthy donors using enzymatic digestion as previously described [24]. Briefly, the adipose tissue was extensively washed with two volumes of Hank's Balanced Salt Solution (HBSS) containing 5% antibiotic/antimycotic solution (10,000 units of penicillin, 10,000 μg of streptomycin, and 25 μg of Amphotericin B per mL; HyClone), fragmented, and then digested at 37°C for 1 h in the presence of collagenase (200 U/ml, Sigma-Aldrich) and dispase (10 U/ml, BD Biosciences). Enzymatic activity was neutralized by adding an equal volume of culture medium (HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Basal Medium for human undifferentiated mesenchymal stem cells containing 10% of HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Growth Supplement (CGS), 1% antibiotic/antimycotic solution (HyClone) and centrifuged at 200 g for 10 min. This led to the sedimentation of diverse cells, including MSCs, macrophages, lymphocytes, and erythrocytes, unlike adipocytes that remained floating. After removal of supernatant, a

let to lyse erythrocytes, and cell suspension was centrifuged at 200 g for 10 min. Sedimented cells were resuspended in the MSC culture medium and filtered through a 100-μm nylon cell strainer (BD Biosciences). As indicated by flow [24], after isolation and overnight preplating, the obtained cell population contained not only MSC cells that basically represented the most abundant subgroup but also admixed macrophages and lymphocytes. The two last cell subgroups were dramatically depleted by culturing for a week in the MSC culture medium

at a subconfluent level (~80% confluency) and passaged using HyQTase (HyClone). By using the methodology described previously [25], cultured cells were demonstrated to differentiate into the osteogenic, chondrogenic, and adipogenic directions, the finding confirming their multipotency. In experiments, MSCs of the second to fourth passages were usually used.

21 to 55 years old, and informed consent was obtained from each participant.

Cl, 10 mM KHCO3

lyl cyclase via G<sup>i</sup>

also couple to G<sup>i</sup>

adrenergic transduction in MSCs.

**2. Materials and methods**

**2.1. Cell isolation and culturing**

lysis solution (154 mM NH4

and humidified atmosphere (5% CO<sup>2</sup>

and β<sup>3</sup>

isoforms widely regulate adeny-

http://dx.doi.org/10.5772/intechopen.79097

, and 0.1 mM EDTA) was added to a cell pel-

) at 37°C. The obtained MSC population was maintained

, although β<sup>2</sup>

141

, their coupling to phospholipase C (PLC) and Ca2+ mobilization has also

Calcium Signaling Initiated by Agonists in Mesenchymal Stromal Cells from the Human Adipose…

, and directly do not control intracellular Ca2+ [23]. Given that certain

Here, we studied MSCs derived from the human adipose tissue and examined Ca2+ signaling initiated by a variety of agonists of G-protein coupled receptors (GPCRs). We specifically focused on adrenergic and purinergic signaling systems that attracted us for the following reasons. It has been known for a long time that noradrenaline released by sympathetic nerves regulates distinct physiological processes in the adipose tissue such as lipid and glucose metabolism and secretion of distinct signaling molecules, including adipocytokines and cytokines [9]. Hence, MSCs that reside in the adipose tissue can be subjected to the action of noradrenaline and factors released by adipocytes on adrenergic stimulation. Purinergic agonists have been documented as an important factor determining MSC fate [7, 8, 10–12]. Reportedly, ATP serves both as an adipogenic regulator and an osteogenic factor, while its downstream product adenosine switches off adipogenic differentiation and promotes osteogenesis [13, 14]. Damaged tissues are an abundant source of extracellular ATP that may be converted by extracellular nucleotidases to ADP and eventually to adenosine [15]. It therefore might be expected that MSCs are exposed to and regulated by nucleotides and adenosine when these cells migrate *in vivo* or are transplanted *ex vivo* into an injured tissue.

The responsiveness to purines and pyrimidines is widespread among eukaryotic cells, which express numerous purinoreceptors from the P1 and P2 families. The P1 subgroup includes four G-protein-coupled receptors (A<sup>1</sup> , A2A, A2B, A<sup>3</sup> ) recognizing adenosine as an endogenous agonist [16]. The more diverse P2 family is composed of ionotropic P2X and metabotropic P2Y receptors. P2X receptors are cationic channels specifically gated by ATP, while P2Y receptors are activated by multiple purine and pyrimidine nucleotides or by sugar nucleotides and couple to intracellular second messenger pathways by heteromeric G proteins [17, 18]. In mammals, seven genes encode P2X subunits (P2X1–7) that can form homo- and heterotrimeric cation channels with noticeable Ca2+ permeability [19, 20]. The P2Y subfamily includes eight members (P2Y1,2,4,6,11,12,13,14), which are distinct by ligand specificity and coupling to downstream signaling pathways, including the ubiquitous phosphoinositide cascade [17, 18].

Nine genes encode human adrenoreceptors, which all belong to the GPCR superfamily and compose three distinctive subgroups, including three α<sup>1</sup> (α1A, α1B, α1D), three α<sup>2</sup> (α2A, α2B, α2C), and three β (β<sup>1</sup> , β<sup>2</sup> , β<sup>3</sup> ) receptor subtypes. Canonically, α<sup>1</sup> -adrenoreceptors couple to Gq and are ubiquitously involved in Ca2+ signaling [21]. Although α<sup>2</sup> isoforms widely regulate adenylyl cyclase via G<sup>i</sup> , their coupling to phospholipase C (PLC) and Ca2+ mobilization has also been documented [22]. All three β-subtypes are linked to adenylyl cyclase by G<sup>s</sup> , although β<sup>2</sup> and β<sup>3</sup> also couple to G<sup>i</sup> , and directly do not control intracellular Ca2+ [23]. Given that certain isoforms of adrenergic and purinergic receptors are coupled to Ca2+ mobilization in diverse cell types, we considered Ca2+ imaging as an adequate approach to detail purinergic and adrenergic transduction in MSCs.
