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.
Highlights
Amniotic fluid stem (AFS) cells, isolated during pregnancy for prenatal genetic tests, have been recognized as an efficient source of cells with therapeutic potential [1]
Confirming our previous data [8], the results indicated that ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) did not display any hematopoietic molecular markers (CD14, 31 and 45), while they expressed several surface adhesion molecules (CD29, CD58, CD166 and CD90) and intracellular stemness markers (TERT, SOX2 and Nanog)
To better assess the potential calcimimetic Calcium Sensing Receptor (CaSR)-mediated pathway in osteogenic differentiation, we showed in Figure 4 that 1 mM R-568 increased Alkaline Phosphatase (ALP) activity significantly greater than both 1.8 or 2.5 mM calcium, which is a principal agonist of CaSR receptor and considered a current gold standard in osteogenic process
Summary
Amniotic fluid stem (AFS) cells, isolated during pregnancy for prenatal genetic tests, have been recognized as an efficient source of cells with therapeutic potential [1]. AFS cells are widely multipotent, express some pluripotency markers and can be differentiated within the tissues of the three germ layers [1,2]. Their properties such as low immunogenicity, the inability to form tumors, easy accessibility, and the absence of ethical problems associated with their use, make them ideal candidates for regenerative medicine [3,4,5]. Large animals form an optimal preclinical model on which to study various diseases, such as bone disease In this context, oAFMSCs used in allotransplantation of injured Achilles tendon led to matrix organization and tissue regeneration [8]. OAFMSCs have been used in tissue renovation such as the repair of diaphragmatic tendon [9] and prenatal tracheal reconstruction [10]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
