Abstract
BackgroundUmbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist.Methodology/Principal FindingsLarge (>8µm in diameter) and small (<8µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration ([Ca2+]o; 2.87 mM) and dose-response effects of AMG641 (0.01 to 3µM) on cell proliferation were evaluated. Both cell lines were then cultured in osteogenic or neurogenic differentiation medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) AMG641 (0.05, 0.1 or 1 µM) with high [Ca2+]o and 4) the CaSR antagonist NPS2390 (10 mM for 30 min) followed by incubation with AMG641 in high [Ca2+]o. Expression of osteogenic or neurogenic differentiation biomarkers was compared among groups. In both cell lines, AMG641 dose-dependently increased cell proliferation (up to P<0.001). Osteogenic molecular markers expression was differentially regulated by AMG641, with stimulatory (OPN up-regulation) in large or inhibitory (RUNX2 and OPN down-regulation) effects in small cells, respectively. AMG641 significantly increased alkaline phosphatase activity and calcium phosphate deposition in both cell lines. Following treatment with AMG641 during osteogenic differentiation, in both cell lines CaSR expression was inversely related to that of osteogenic markers and inhibition of CaSR by NPS2390 blocked AMG641-dependent responses. Early-stage neurogenic differentiation was promoted/triggered by AMG641 in both cell lines, as Nestin and CaSR mRNA transcription up-regulation were observed.Conclusions/SignificanceCalcium- and AMG641-induced CaSR stimulation promoted in vitro proliferation and osteogenic and early-stage neurogenic differentiation of UCM-MSCs. CaSR activation may play a fundamental role in selecting specific differentiation checkpoints of these two differentiation routes, as related to cell commitment status.
Highlights
Recent developments in stem cell biology research area have revealed that umbilical cord matrix (UCM, known as Wharton’s jelly) is a pivotal source of ‘‘young’’ mesenchymal stem cells (MSCs) considered as much more proliferative, immunosuppressive and even more therapeutically active than those from adult tissue sources [1]
In the large cell line, AMG641 significantly reduced DT values compared with controls (P,0.01 in cells treated with the two lowest AMG641 concentrations, i.e. 0.01 and 0.05mM AMG641; P,0.001 in cells treated with the four remaining higher AMG641 concentrations, i.e. 0.1, 0.5, 1 and 3 mM AMG641; Fig. 1, Panel A)
In the small cell line, AMG641 significantly reduced DT values compared with controls (P,0.05 in cells treated with the two lowest AMG641 concentrations; P,0.001 in cells treated with the remaining higher AMG641 concentrations; Fig. 1, Panel B)
Summary
Recent developments in stem cell biology research area have revealed that umbilical cord matrix (UCM, known as Wharton’s jelly) is a pivotal source of ‘‘young’’ mesenchymal stem cells (MSCs) considered as much more proliferative, immunosuppressive and even more therapeutically active than those from adult tissue sources [1]. The low immunogenicity makes eUCM-MSCs ideal for cell therapy and regenerative medicine applications, both for species-specific purposes in equine medicine and as a large animal model of pre-clinical trials. Enhancing research programs in this species could establish a suitable animal model useful for pre-clinical trials in humans. Stem cell therapy in equine orthopaedic diseases, a relatively new research area, has been based mainly on the use of bone marrow (BM-MSCs; [24,25]), adipose tissue (Ad-MSCs; [26]), amnionderived (AM-MSCs; [24]) and umbilical cord blood (UCB-MSCs; [27]). In a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist
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