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Stem Cells International
Volume 2017, Article ID 9605432, 14 pages
Research Article

Comparative Analysis of Spontaneous and Stimulus-Evoked Calcium Transients in Proliferating and Differentiating Human Midbrain-Derived Stem Cells

1Department of Cardiovascular and Renal Research, University of Southern Denmark, J.B.Winsløws Vej 21, 5000 Odense C, Denmark
2Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, J.B.Winsløws Vej 21, 5000 Odense C, Denmark
3Department of Molecular Biology and Center of Molecular Biology Severo Ochoa, UAM-CSIC, Campus Cantoblanco, 28049 Madrid, Spain

Correspondence should be addressed to Morten Meyer; kd.uds.htlaeh@reyemm

Received 30 June 2017; Accepted 10 September 2017; Published 22 October 2017

Academic Editor: Luca Vanella

Copyright © 2017 Torben Johansen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Figure 1 Video of differentiating stem cells. Recordings through 5 min with a frequency of 0.33Hz of fluo-4-loaded differentiating human midbrain-derived stem cells using a laser-scanning microscope (Olympus FluoView FV1000MPE multiphoton laser scanning microscope). Supplementary Figure 2 Calcium transients induced by carbachol in differentiating stem cells. The differentiating cells were loaded with Fura-2 and then exposed to various concentrations of carbachol for 15 min in presence or absence of extracellular calcium and pirenzepine. (A) shows the dose-dependent increase of the fraction of cells with calcium transients in response to carbachol. Mean values and SEM are shown for cells incubated in presence (square) or absence (triangle) of calcium and in the presence of pirenzepine and calcium (circle). The values from two (carbachol 100 μM) and 3-8 independent experiments are shown. (B) shows the effect of increasing concentrations of carbachol on the total calcium increase (AUC) during the 15 min of exposure to carbachol. Mean values and SEM from 43-194 cells. Supplementary Figure 3 Thapsigargin induced calcium transient in proliferating stem cells suspended in calcium-free medium. Cells were loaded in a calcium containing medium, and then washed in a medium containing calcium. Incubation was performed in a calcium-free medium with 10 μM EGTA. Thapsigargin (2 μM, final concentration) was added to the cells during the incubation. The figure shows the results from two independent experiments. Abscissa: Time of incubation (sec). Ordinate: Intracellular calcium content. The fluorescence intensity was normalized to the level of the fluorescence intensity of untreated cells (100 %). Mean values and S.E.M. from 50 and 39 cells from two independent experiments, responding to the addition of thapsigargin. The cells represent 76% and 60% of the observed cell population, respectively. Supplementary Figure 4 Calcium-induced calcium increase in calcium-depleted proliferating stem cells. The cells were calcium-depleted by preincubation with 2μM thapsigargin for 30 min in calcium-free medium containing 10μM EGTA, washed and incubated in a calcium-free medium with EGTA but without thapsigargin. Calcium (2mM, final concentration) was added to the cells after 200 sec incubation (arrow). Time course of calcium increase from a representative experiment (A) and the mean value of the peak levels from 7 independent experiments (B). Mean and S.E.M. from 81 cells (A) and from seven independent experiments (B). Taken together 332 cells were studied in seven experiments, and all the cells responded to the addition of calcium.

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