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

Abstract

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.

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

“…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.…”

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

“…Wnt signaling is also involved in AFMSC commitment toward osteogenesis [13] . Recently, we identified for the first time, the presence of Calcium Sensing Receptor (CaSR) in ovine and human AFMSCs [44,68] . CaSR, originally cloned from parathyroid glands, acts by controlling the secretion of parathyroid hormone in response to changes in extracellular calcium levels [75] .…”

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry

“…We have analyzed the fresh supernatant of MSC cultures, containing EVs displaying the phenotype of their parental cells. In order to identify the best marker to study MSC-derived EVs, we tested different MSC specific antibodies, as listed in Table S1 and we confirmed that, given that CD90 represents a well-recognized pan-MSC- marker, it is also well expressed on MSC-derived EVs [ 42 , 51 , 53 ]. Therefore, for each sample, 100 μL of MSC supernatant were stained using 3 μL (0.006 μg/μL) of an anti-human CD90 conjugated to R-phycoerythrin (PE) (BD Biosciences, San Jose, CA, USA; Clone 5E10, Cat.…”