Vitamin D machinery and metabolism in porcine adipose-derived mesenchymal stem cells

Yovani Llamas Valle, Sami G. Almalki, Devendra K. Agrawal

Vitamin D, a hormone once thought to have a role limited to calcium homeostasis and bone mineralization, has pleiotropic effects on different types of cells. Vitamin D receptors are reported in vascular smooth muscle cells, endothelial cells, and cardiomyocytes. Adipose-derived MSCs (ADMSCs) are multipotent cells with the capacity to differentiate into cells of different lineages. To our knowledge, the presence of vitamin D machinery on porcine ADMSCs has not yet been examined. In this study, we investigated the presence of vitamin D machinery and metabolism in ADMSCs by analyzing the expression levels of vitamin D receptor (VDR), vitamin D metabolizing enzymes (CYP24A1 and CYP27B1) after in vitro stimulation with active vitamin D, calcitriol. ADMSCs isolated from porcine adipose tissue were characterized by positive staining for ADMSC markers, CD44, CD73, and CD90, and negative staining for macrophage marker CD11b and hematopoietic stem cell markers CD34 and CD45, and trilineage differentiation to osteocytes, chondrocytes, and adipocytes. No cytotoxicity was observed when MSCs were stimulated with 0.1-10 nM calcitriol. The ADMSCs were analyzed for mRNA and protein expression of CYP24A1, CYP27B1, and VDR by immunostaining, qPCR, and ELISA. A significant increase (p <0.01) in the mRNA expression of CYP24A1, CYP27B1, and VDR was observed after stimulation of ADMSCs with calcitriol (10 nM). The in vitro time-dependent effect of calcitriol (10 nM) on the components of vitamin D machinery in cultured MSCs was determined by qPCR. The VDR and CYP27B1 expression peaked at 3 h and CYP24A1 at 24 h, respectively. The in vitro biosynthesis of 1, 25(OH)2D3 by ADMSCs was analyzed by ELISA and Western blot. The levels of the active form of vitamin D were significantly decreased once the CYP enzymes were inhibited (p <0.01), demonstrating the ability of ADMSCs to convert inactive vitamin D into active vitamin D for cellular action. Porcine ADMSCs possess vitamin D hydrolases and VDR to metabolize and respond to vitamin D. Hence, in vivo circulating 25-hydroxy vitamin D levels may have a significant role in regulating the differentiation of ADMSCs into different lineages, which might assist in stem cell-based therapy.