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

Abstract

Spontaneous cytosolic calcium transients and oscillations have been reported in various tissues of nonhuman and human origin but not in human midbrain-derived stem cells. Using confocal microfluorimetry, we studied spontaneous calcium transients and calcium-regulating mechanisms in a human ventral mesencephalic stem cell line undergoing proliferation and neuronal differentiation. Spontaneous calcium transients were detected in a large fraction of both proliferating (>50%) and differentiating (>55%) cells. We provide evidence for the existence of intracellular calcium stores that respond to muscarinic activation of the cells, having sensitivity for ryanodine and thapsigargin possibly reflecting IP₃ receptor activity and the presence of ryanodine receptors and calcium ATPase pumps. The observed calcium transient activity potentially supports the existence of a sodium-calcium antiporter and the existence of calcium influx induced by depletion of calcium stores. We conclude that the cells have developed the most important mechanisms governing cytosolic calcium homeostasis. This is the first comparative report of spontaneous calcium transients in proliferating and differentiating human midbrain-derived stem cells that provides evidence for the mechanisms that are likely to be involved. We propose that the observed spontaneous calcium transients may contribute to mechanisms involved in cell proliferation, phenotypic differentiation, and general cell maturation.

Figure 1

Immunocytochemical characterization of proliferating (a, b, c) and differentiating human midbrain-derived stem cells (d, e, f, g, h, i). Immunofluorescence staining of cultures propagated for 4 days by exposure to epidermal growth factor and basic fibroblast growth factor was performed. Almost all cells expressed Ki67, a marker of dividing cells (a). Moreover, a large proportion of the cells expressed the intermediate filament protein nestin, a marker of neural progenitor cells (b). Only very few cells had spontaneously differentiated into β-tubulin III- (β-tub III-) positive neurons (c). Cells were differentiated for 10 days by exposure to fibroblast growth factor 8 for three days followed by exposure to forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor for seven days and immunostained for neuronal and astroglial markers. At this time point, some cells expressed the early marker of migrating neuronal cell doublecortin (DCX) (d), whereas extensive staining for another early neuronal marker, β-tub III (e), and a mature neuronal marker, microtubule-associated protein 2ab (Map2) (f), was seen. A large proportion of cells expressed tyrosine hydroxylase (TH), a marker of catecholaminergic neurons (i), whereas only few cells were found to express γ-aminobutyric acid (GABA), a marker for GABAergic neurons (h). Very few cells were found to express glial fibrillary acidic protein (GFAP), a marker of astroglial cells (g). Scale bars = 50 μm.

Figure 2

Spontaneous calcium transients in cytosol of differentiating stem cells are dependent on intracellular and extracellular calcium supply. Fura-2-loaded cells were measured for 20 min at room temperature. The pattern of calcium transients from five representative cells is shown (a). The fraction of cells with calcium transients (cytosolic calcium increase followed by decrease) was calculated in each experiment (b), as was the number of transients per cell (c). The cells were incubated in the presence or absence of extracellular calcium as indicated on the figure. Pretreatment with thapsigargin in the absence of calcium was used to deplete the intracellular calcium stores (third bars in the two histograms). Ryanodine was used to study the role of ryanodine receptors. The mean values and SEM from 6–15 independent experiments (b) and 40–378 cells (c) are shown. ∗ indicate significant difference from the control value. Consecutive recordings (still images) of Fluo-4-loaded cells with a 60 sec interval ((d); corresponding video available, see Supplemental Figure 1).

Figure 3

Effects of thapsigargin on cytosolic calcium (a) and calcium addition to store-depleted differentiating stem cells (b). Cells were incubated in a calcium-free medium with 10 μM EGTA and then 2 μM thapsigargin was added (arrow). The cells were dye loaded in Krebs-Ringer solution (containing 2 mM calcium). The results from five independent experiments are shown, each represents the mean value from 29 to 65 cells, all of which with a calcium transient in response to thapsigargin (a). The cells were dye loaded in the presence of calcium and thapsigargin (2 μM). The dye was removed and the cells were primed with 2 μM thapsigargin for 12 min in calcium-free solution. Then 2 mM calcium was added, and the incubation lasted for 8–11 min. The results from four independent experiments are shown, each representing the mean value obtained from analysis of 23 to 39 cells, all of which displayed calcium transients in response to the addition of calcium (b).

Figure 4

Calcium-induced increase of cytosolic Ca²⁺ in differentiating cells incubated in the absence or presence of Na⁺. After dye loading in the presence of Ca²⁺, cytosolic Ca²⁺ was monitored in cells incubated in a calcium-free medium with or without Na⁺ and then 2 mM Ca²⁺ was added (arrow) (a). The area under the curve (AUC) of the effect of Ca²⁺ addition is shown (b). The fraction of cells that respond to the addition of 2 mM Ca²⁺ with increased cytosolic Ca²⁺ is shown (c). The cells were incubated in the absence and presence of Na⁺ and then exposed to 2 mM Ca²⁺ ((c), columns A, B). Columns C and D show the fraction of cells with spontaneous Ca²⁺ increase in the absence and presence of Na⁺. AUC of the spontaneous Ca²⁺ increase is shown in columns C and D in Figure 4(d). GraphPad Prism4 was used to calculate AUC, expressed as fluorescence intensity (excitation ratio 360 nm/380 nm) per cell per 12 min (b) and 20 min (d), respectively. Mean value and SEM from 7 and 9 independent experiments (Figure 4(a)). Mean values and SEM from 296 cells and 270 cells incubated in the absence or presence of sodium, respectively (Figure 4(b)). Mean values and SEM from 7–14 experiments (Figure 4(c)) and 226 cells and 384 cells in Figure 4(d) (columns C and D, resp.). Column D in Figure 4(c) represents the data shown in Figure 2.

Figure 5

Immunocytochemical characterization of proliferating human midbrain-derived stem cells. Immunostaining of cells during, (1) standard propagation (upper panel) in a medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and (2) after removal of EGF/bFGF and one-day exposure to fibroblast growth factor 8 (FGF8) (lower panel), which represents the initial step of the induced neuronal differentiation (sequential addition of various factors, see Materials and Methods for details). To visualize all cells, cultures were immunostained using an antibody against human nuclei (HN). Almost all cells in both experimental groups expressed Ki67, a marker of dividing cells. Moreover, a very large proportion of the cells expressed the intermediate filament protein nestin, a marker of neural progenitor cells. Only very few cells had spontaneously differentiated into β-tubulin III- (β-tub III-) positive or doublecortin- (DCX-) positive immature neurons in the EGF/bFGF group, whereas some β-tub III-positive and DCX-positive neuronal cells were seen after short-term FGF8 treatment. Scale bar = 20 μm.

Figure 6

Effect of thapsigargin and calcium on proliferating stem cells incubated in a calcium-free medium. The cells were dye loaded and washed in a calcium-containing medium and then incubated in a calcium-free medium with 10 μM EGTA. Thapsigargin (2 μM, final concentration) was added after initiation of the incubation. After incubation for 300 sec, 2 mM calcium (final concentration) was added to the cells. The results from one of three independent experiments are shown (a). 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 SEM from 31 cells. All the cells responded to the addition of thapsigargin and calcium. Peak values from all three experiments, after addition of thapsigargin (open column) and calcium (closed column) can be seen (b). Mean values and SEM from 31–64 cells. Taken together, 149 cells were studied in three independent experiments, and 98% of the cells responded to the addition of both thapsigargin and calcium.

Figure 7

The fraction of proliferating cells with calcium transients during 15 min of incubation. Representative calcium traces from cells kept under six different experimental conditions (trace from five cells in each group) (a). The cells were loaded with 5 μM Fluo-4. Calcium was present during the loading and the incubation procedures as indicated on the figure. Thapsigargin was used for depletion of calcium stores. Thapsigargin was present during the loading procedure. Carbachol and pirenzepin were present during the incubation. Sodium-free solution was used only for the incubation. Mean values and SEM, 6–14 independent experiments (b).