Poultry is vulnerable to bone problems throughout their lives or production period due to rapid growth in broilers and the active laying cycle in layers. The calcium-sensing receptor (CaSR) is important in calcium and bone metabolism. The objective of this study was to investigate the effect of the CaSR ligand (l-Trp) and 1,25-dihydroxycholecalciferol (1,25OHD3) on the regulation of proliferation and osteogenic differentiation of chicken mesenchymal stem cells (MSCs) isolated from the compact bones of 14-day-old Ross 308 chickens and Dekalb pullets, which can provide cell-based evidence for the prevention or alleviation of skeletal disorders in the poultry industry. First, the dose- (0, 0.5, 1, 2, 5, 10, and 15 mM) and time-effects (0, 7, and 14 days) of l-Trp on the proliferation and osteogenic differentiation in chicken MSCs were investigated. The 5 mM l-Trp had a balanced effect between proliferation and osteogenic differentiation in broiler and layer MSCs when differentiated for 7 days. The broiler and layer MSCs were then treated with (1) osteogenic medium, osteogenic medium supplemented with (2) 1 nM 1,25OHD3, (3) 2.5 mM Ca2+, (4) 2.5 mM Ca2+ + 5 mM l-Trp and (5) 2.5 mM Ca2+ + 5 mM l-Trp + 1 μM NPS-2143, separately for 7 days. Results showed that the 5 mM l-Trp significantly inhibited the proliferation of broiler and layer MSCs on day 7 (P < 0.05), but 1 nM 1,25OHD3 significantly promoted the proliferation of layer MSCs (P < 0.05). Only the 2.5 mM Ca2+ + 5 mM l-Trp group significantly increased the mineralization process during osteogenic differentiation (P < 0.05), and this treatment also significantly upregulated the mRNA expression of the vitamin D receptor (VDR), β-catenin, and osteogenesis genes in broiler MSCs (P < 0.05). The osteogenic differentiation process in layer MSCs was faster than that in broiler MSCs. In layer MSCs, Ca2+ alone significantly facilitated mineralization and ALP activity after 7-day osteogenic differentiation (P < 0.05). However, the 5 mM l-Trp significantly inhibited the differentiation and mineralization process by downregulating the mRNA expression of CaSR, VDR, β-catenin, and osteogenic genes (P < 0.05) in layer MSCs. Taken together, l-Trp and 1,25OHD3 can regulate proliferation and osteogenic differentiation in both broiler and layer MSCs depending on the dose, treatment time, and cell proliferation and differentiation stages.
Keywords: 1,25-dihydroxycholecalciferol; L-tryptophan; calcium-sensing receptor; chicken mesenchymal stem cells; osteogenic differentiation.