Calcium-Sensing Receptor-Mediated Osteogenic and Early-Stage Neurogenic Differentiation in Umbilical Cord Matrix Mesenchymal Stem Cells from a Large Animal Model

BACKGROUND: Umbilical 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 FINDINGS: Large (>8µm in diameter) and small (<8µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca(2+)) concentration ([Ca(2+)](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 [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) AMG641 (0.05, 0.1 or 1 µM) with high [Ca(2+)](o) and 4) the CaSR antagonist NPS2390 (10 mM for 30 min) followed by incubation with AMG641 in high [Ca(2+)](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/SIGNIFICANCE: Calcium- 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.