**2.2. Preparation of cells for Ca2+ imaging**

Before assaying with Ca2+ imaging, cells were maintained in a 12-socket plate for 12 h in the medium described above but without antibiotics. For isolation, cells cultured in a 1-ml socket were rinsed twice with the Versene solution (Sigma-Aldrich) that was then substituted for 200 μl HyQTase solution (HyClone) for 3–5 min. The enzymatic treatment was terminated by the addition of a 0.8 ml culture medium to a socket. Next, cells were resuspended, put into a tube, and centrifuged at 50 g for 45 s for moderate sedimentation. Isolated cells were collected by a plastic pipette and plated onto a photometric chamber of nearly 150 μl volume. The last was a disposable coverslip (Menzel-Glaser) with attached ellipsoidal resin wall. The chamber bottom was coated with Cell-Tak (BD Biosciences), enabling strong cell adhesion. Attached cells were then loaded with dyes for 20 min at room temperature (23–25°С) by adding Fluo-4 AM (4 μM) and Pluronic (0.02%; all from Molecular Probes) to a bath solution. Loaded cells were rinsed with the bath solution for several times and kept at 4°C for 1 h prior to recordings. Generally, incubation of MSCs at low temperature stabilized intracellular Ca2+ and decreased a fraction of spontaneously oscillating cells.

stanford.edu). In this low Ca2+ bath solution, the glucose concentration was increased to 13 mM to keep osmolarity. All chemicals used in experiments described below were applied by the complete replacement of the bath solution in a 150-μl photometric chamber for nearly 2 s using a perfusion system driven by gravity. The used salts and buffers were from Sigma-

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

In a typical experiment, nearly a hundred of MSCs loaded with Fluo-4 resided in a photometric camera, and their responsiveness to different ligands was assayed with Ca2+ imaging. Consistently with observations of others [3], functional heterogeneity was characteristic of a MSC population derived from each particular donor. Although a variety of GPCR agonists were found to stimulate Ca2+ signaling in MSCs, including ATP, ADP, noradrenaline or adrenaline, acetylcholine or its analog carbachol, GABA, glutamate, serotonin, and UTP, only a relatively small group of cells in a given MSC population was specifically responsive to a par-

agonists applied at different combinations, and a particular cell was either irresponsive to all stimuli or responded to one, rarely two, particular compound (**Figure 1A**–**C**). ATP-sensitive cells composed the most abundant subgroup of 9–15% (12% on average), depending on MSC preparation (**Figure 1B**). The percentage of cells responsive to other agonists was on average: ADP—7.1, adenosine—8.7, carbachol—3.4, GABA—5, glutamate—1.2, noradrenaline—6.7, serotonin—6.6, and UTP—6 (**Figure 1B**). The more or less accurate analysis of distribution of MSC responsivity was performed for nucleotides. In designated experiments, wherein cells were sequentially stimulated by ATP, ADP, and UTP, 125 purinergic MSCs were assayed overall, and only 13 cells (10%) were found to respond to all three agonists at the indicated concentrations (**Figure 1C**). Both ATP and ADP stimulated Ca2+ signaling in 40 cells (32%) that did not respond to UTP; 33 cells (26%) preferred the ATP-UTP pair. In addition, 20, 9, and 7 cells (16, 7, and 6%) responded exclusively to ATP, ADP, or UTP, respectively (**Figure 1C**).

Thus, the results presented above (**Figure 1**) clearly demonstrated that responsiveness to a given agonist varied from cell to cell. Note that GPCRs from most subfamilies, e.g. P2Y receptors, can couple to several signaling pathways, depending on cellular context [26–29]. Hence, in cells nonresponsive in terms of Ca2+ signaling to a particular agonist, appropriate GPCRs

In the present study, we focused on transduction of adrenergic and purinergic agonists capable of stimulating Ca2+ signaling in the MSC cytoplasm. We first aimed at evaluating dose dependencies of cellular responses to tested agonists. The analysis, which initially involved adrenergic transduction, revealed that Ca2+ responses varied with noradrenaline concentration in an "all-or-nothing" fashion. In other words, noradrenaline never caused detectable effects, when applied below 100 nM, but above the threshold of 100–200 nM, it elicited marked Ca2+ transients that were similarly shaped irrespective of agonist concentration (**Figure 2A**).

might be either not expressed or not coupled to Ca2+ mobilization.

**3.1. Dose dependence of MSC responses to adrenergic and purinergic agonists**

MSCs were sequentially stimulated by multiple

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

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Aldrich, and agonists and inhibitors were from Tocris.

ticular agonist (**Figure 1**). Overall, nearly 10<sup>3</sup>

**3. Results**
