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.
Highlights
Calcium is a versatile intracellular messenger controlling a wide range of cellular processes [1,2,3] including cell proliferation, cell differentiation, and general gene transcription [4,5,6,7]
Spontaneous calcium oscillations have been observed in early postnatal cerebellar Purkinje neurons [20], embryonic mouse cortical brain slices [21], mouse spinal cord neurons [22], slice cultures of the spinal cord and dorsal root ganglia prepared from mouse embryos [23], and undifferentiated cells and neural progenitor cells derived from a mouse bone marrow [24]
Prior to analysis of calcium transients, cells were differentiated for 10 days in vitro according to our neuronal differentiation protocol (CK4 protocol) in which fibroblast growth factor 8 (50 ng/ml), forskolin (25 μM), glial cell line-derived neurotrophic factor (5 ng/ml), and sonic hedgehog (25 ng/ml) are added in a sequential manner [34, 35]
Summary
Calcium is a versatile intracellular messenger controlling a wide range of cellular processes [1,2,3] including cell proliferation, cell differentiation, and general gene transcription [4,5,6,7]. Spontaneous calcium transients and oscillations have been reported in a number of tissues of nonhuman origin [19]. Spontaneous calcium oscillations have been observed in early postnatal cerebellar Purkinje neurons [20], embryonic mouse cortical brain slices [21], mouse spinal cord neurons [22], slice cultures of the spinal cord and dorsal root ganglia prepared from mouse embryos [23], and undifferentiated cells and neural progenitor cells derived from a mouse bone marrow [24]. There have been reports on spontaneous calcium oscillations in human mesenchymal stem cells [25,26,27], human embryonic stem cellderived neurons [28], and human cardiac progenitor cells [29]. It appeared that calcium supply to cytosol was derived from intracellular calcium stores by IP3-dependent release
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