Effects of CD4+ T lymphocytes from ovariectomized mice on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation.

Abstract

The present study was designed to investigate the effects of T cells on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs were co-cultured with CD4+ T cells that had been pretreated with anti-TNF-α or controls and were derived from ovariectomized (OVX) mice or sham control mice. MTT was used to assess the proliferative ability of BMMSCs and flow cytometry was used to analyze the BMMSC cell cycle. Following the induction of osteogenic differentiation in BMMSCs, calcium nodules were observed using alizarin red staining and alkaline phosphatase (ALP) staining. The expression levels of the osteogenesis-associated genes, runt related transcription factor 2 (Runx2) and osteocalcin (OCN) in BMMSCs were quantified using reverse transcription-quantitative PCR and western blotting. Osteogenesis-related signaling pathways, including ERK, JNK and p38 MAPK were also examined by western blotting. BMMSCs co-cultured with CD4+ T cells from OVX mice exhibited reduced proliferative ability compared with sham mice and the cell cycle was arrested at the G2/M phase. Additionally, BMMSCs co-cultured with CD4+ T cells from OVX mice presented with reduced levels of osteogenic differentiation and lower ALP activity, less calcium deposition and reduced expression of Runx2 and OCN compared with sham mice. The reduced levels of proliferation and osteogenic differentiation of BMMSCs induced by CD4+ T cells were not seen when the T cells were had been pretreated with anti-TNF-α. The results indicated that CD4+ T cells from OVX mice inhibited the proliferation and osteogenic differentiation of BMMSCs by producing high levels of TNF-α and may provide a novel insight into the dysfunction of BMMSCs caused by estrogen deficiency.