Characterisation of serum‐induced intracellular Ca²⁺ oscillations in primary bone marrow stromal cells

Abstract

Intracellular Ca2+ signalling is pivotal to cell function and [Ca2+]i oscillations permit precise and prolonged modulation of an array of Ca2+-sensitive processes without the need for extended, global elevations in [Ca2+]i. We have studied [Ca2+]i signalling in primary rat marrow stromal cells exposed to foetal calf serum (FCS) constituents at concentrations up to those required to promote growth and differentiation in culture. Spontaneous [Ca2+]i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca2+ oscillations in 41 +/- 3% of cells. Incidence of FCS-induced oscillations was dose-dependent, saturating at 0.5%. These oscillations were arrested by disruption of Ca2+ stores with 100 nM-1 microM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP3-receptors by 50 microM 2-amino-ethoxydiphenyl borate (2-APB) and inhibition of phospholipase C with 5 microM U73122. The oscillations decreased in frequency and amplitude following inhibition of Ca2+ influx with EGTA or La3+ but were poorly sensitive to nifedipine (1-10 microM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca2+]i oscillations are heat stable, insensitive to disulphide bond reduction with 20 mM dithioerythritol and retained by a 30 kDa molecular weight filter. Serum is routinely present in culture medium at 10%-15% [v/v] and marrow stromal cells maintained under culture conditions exhibited sustained oscillations. This is the first demonstration of agonist-induced complex Ca2+ signals in marrow stromal cells. We conclude that Ca2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation.

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration

“…Importantly, it has long been known that repetitive Ca 2þ spikes may be exploited by growth factors to drive proliferation, migration, and differentiation [29,[41][42][43][44], which are the main steps underlying the formation of a novel vascular network by circulating ECFCs. This study provides, for the first time, the evidence that VEGF stimulates ECFCs to proliferate and form tubule-like structures by inducing oscillations in [Ca 2þ ] i and the consequent nuclear translocation of the Ca 2þ -sensitive transcription factor, NF-jB.…”

“…[Ca 2þ ] i oscillations have been recorded in a variety of progenitor and stem cells, such as serum-stimulated primary rat marrow stromal cells [43], human preadipocytes [44], and human cardiac fibroblasts [57]. Moreover, spontaneous repetitive Ca 2þ spikes may arise in human mesenchymal stem cells (HMSCs) [30,58], adult mice neural stem cells [59], and human cardiac progenitor cells (HCPCs) [60].…”

“…Moreover, spontaneous repetitive Ca 2þ spikes may arise in human mesenchymal stem cells (HMSCs) [30,58], adult mice neural stem cells [59], and human cardiac progenitor cells (HCPCs) [60]. Similar to VEGF-induced Ca 2þ waves in ECFCs, the oscillatory response to serum as well as the spontaneous Ca 2þ transients in HMSCs [30] were shaped by the interplay between InsP 3 -dependent Ca 2þ release and SOCE [43,44,57]. Conversely, HCPCs exploit the rhythmic activation of InsP 3 Rs, but not SOCE, to generate repetitive Ca 2þ spikes [60].…”

Human AB Serum and Thrombin-Activated Platelet-Rich Plasma Are Suitable Alternatives to Fetal Calf Serum for the Expansion of Mesenchymal Stem Cells from Adipose Tissue

“…Most studies have concluded that serum-free media cannot promote MSC growth without the addition of cytokines [35,36]. This is possibly due to the fact that serum induces intracellular Ca 2ϩ oscillations which are vital to MSC proliferation and differentiation [37]. Recently, several studies have concentrated on the use of autologous serum [11,16,23,36].…”

From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera?

“…Serum-free culture experiments on animal models demonstrate the maintenance in culture of MSCs, but only after two passages in FCS-supplemented media (Lemnon et al, 1995), and due to MSC species-specific differences, even maintenance in serum-free conditions may not be applicable in the human system. Serum-free media can not promote MSC growth without the addition of cytokines or growth factors, possibly because serum induces intracellular calcium oscillations which are vital to stem cell proliferation and differentiation (Kuznetsov et al, 1997;Foreman, et al, 2006).…”