Abstract
BackgroundThe present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.Methodology/Principal FindingsA large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca2+]o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.Conclusions/SignificanceIn conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.
Highlights
Mesenchymal stem cells (MSCs) are undifferentiated cells able to self-renew, that have a high proliferative capacity and grow as adherent cells [1]
Experiment 1: Effects of high external calcium concentration and the calcimimetic NPS R-467 on growth/proliferation of equine size-sieved UC matrix (UCM)-MSCs Increasing external Ca2+ to 2.5 mM did not affect cell growth, in the large (Fig. 1A’) cell line, whereas in the small cell line (Fig. 1B’), it significantly increased cell growth compared with controls (P,0.05)
The addition of NPS R-467, in presence of 2.5 mM exsternal Ca2+, in both cell lines significantly increased cell growth compared with controls (P,0.001 for both the large cell line and P,0.05 for the small cell line)
Summary
Mesenchymal stem cells (MSCs) are undifferentiated cells able to self-renew, that have a high proliferative capacity and grow as adherent cells [1]. Immunophenotyping by flow cytometry revealed that these cells are positive for specific MSC markers such as CD73, CD90, CD105 [3,19,20,21] and they are negative for the hematopoietic line markers such as CD34 and CD45 [1,3,19,20,21,22] These cells are negative for the human leukocyte antigen HLA-DR [1,3,19,20,22,23] suggesting a potential role as a human allogenic cell source for cell-based therapies. The present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine sizesieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level
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