Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

Martino, Nicola Antonio; Consiglio, A. Lange; Crémonesi, F.; Valentini, Luisa; Caira, Michele; Guaricci, Antonio Ciro; Ambruosi, Barbara; Sciorsci, Raffaele Luigi; Lacalandra, Giovanni Michele; Reshkin, Stephan J.; Dell’Aquila, Maria Elena

doi:10.1371/journal.pone.0017714

Cited by 17 publications

(19 citation statements)

References 75 publications

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“…Although Barradas et al have recently shown that calcium-enhanced osteogenic differentiation in bone marrow-derived mesenchymal stromal cells is not mediated by CaSR activation [44], our results are in accordance with part of the literature in demonstrating that an elevation of Ca 2+ o concentration, as well as the use of calcimimetics, strongly activates functional CaSR in several cell lines [39], [42], [45].…”

Section: Discussionsupporting

confidence: 92%

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Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

et al. 2013

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Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

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Section: Discussionsupporting

confidence: 92%

“…In addition, recent works have suggested that this important receptor is expressed in stem cells such as size-sieved umbilical cord matrix equine stem cells [42], bone marrow hematopoietic stem cells [43] and mouse embryo stem cells [18].…”

Section: Discussionmentioning

confidence: 99%

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

et al. 2013

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“…However, we suspect that the calcium sensing receptor (CaSR) may play a role in inhibiting chondrogenesis in response to increased extracellular calcium, as it has been shown to be expressed and functionally active in osteoblast-like cell lines 40 and several types of stem cells, including human and ovine amniotic fluid stem cells, 41 equine umbilical cord matrix, 42 and bone marrow mesenchymal stem cells (MSCs). 43 More recently, the calcium-sensing receptor was reported to play a potential role in the osteogenic differentiation of human amniotic fluid MSCs.…”

Section: Camentioning

confidence: 99%

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

Mellor

Mohiti-Asli

Williams

et al. 2015

Tissue Engineering Part A

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We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factorb1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold.

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“…Calcium concentration was selected on the basis of a previous study from our unit [13] in which 2.5 mM CaCl 2 was considered as CaSR EC 50 in both cell lines. External Ca 2+ concentration ([Ca 2+ ] o ) in the present study, as in our previous study by Martino et al, [13] was actually either 0.37 (low [Ca 2+ ] o ) or 2.37 mMCa 2+ (high [Ca 2+ ] o ).…”

Section: Experimental Designmentioning

confidence: 99%

“…Indeed, a recent study from our unit reported the CaSR is expressed in eUCM-MSCs and is functionally active since calcium and the selective CaSR agonist NPS R-467 stimulate cell growth/proliferation in these cell lines, an effect which is reversed by the CaSR antagonist NPS2390 [13]. On the other hand, limited information is available on the role of CaSR in cell differentiation.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

Cited by 17 publications

References 75 publications

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

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

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