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

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.

The Incorporation of Marine Coral Microparticles into Collagen-Based Scaffolds Promotes Osteogenesis of Human Mesenchymal Stromal Cells via Calcium Ion Signalling

“…Interestingly, however, MSC-seeded collagen/coral scaffolds both demonstrated positive staining for CaSR. CaSR is G protein-coupled receptor which binds to extracellular calcium ions [37,38] and has been demonstrated to promote osteogenic differentiation of MSCs in vitro [39,40] and to determine the route through which bone formation occurs in vivo, with hyperstimulation of CaSR being shown to inhibit chondrogenic differentiation thus directing MSCs down an intramembranous ossification pathway [41]. Interestingly, previous studies have shown that extracellular calcium ions, delivered from calcium phosphate glass/polylactic acid scaffolds and acting through CaSR, also elicits a pro-angiogenic effect in endothelial progenitor cells [42].…”

Equine adipose tissue derived stem cells and their multilineage differentiation

“…Researchers had reported similar results from different sources of various species such as human (Lee et al 2011), bovine (Ozden-Akkaya et al 2019), canine (Altunbaş et al 2016) in the context of neurosphere generation under the influence of EGF and FGF, supporting our findings. Although equine stem cells of different sources like equine umbilical cord (Martino et al 2014), equine amniotic membrane (Lange-Consiglio et al 2012) known to be able to differentiate into neurogenic cells; there were no reports found on neurospheres generation for equine. This study shows that EASCs are able to form neurospheres.…”

Regenerative Potential of Human Schneiderian Membrane: Progenitor Cells and Epithelial‐Mesenchymal Transition

“…Although that was not an in situ evaluation, such as is the case for our current study, the osteogenic potential of MSCs in the SM was strongly supported. The extracellular calcium‐sensing receptor (CaSR) plays a role in osteogenic differentiation of MSCs (Martino et al, ) and additional calcium up‐regulates the expression of CaSR and nestin (Martino et al, ) and stimulates overexpression of osteogenic markers in MSCs (Gonzalez‐Vazquez et al, ). It is thus reasonable to speculate here a direct relation of the innate osteogenic potential of the SM and the active vitamin D production which was demonstrated in sinonasal mucosa (Sultan et al, ), being known the role of vitamin D in calcium homeostasis.…”