Effects Of Bioactive Factors From Medium Conditioned By A Murine Bone Marrow Endothelial Cell Line On Proliferation Of MSC In Vitro

The regulation of proliferation and differentiation in stem cells is an important aspect in biomedical researches at the present time. The mechanisms underlying proliferation and differentiation of stem cells need to be elucidated for potential application of them, so that they can be manipulate in vitro and treated in vivo. The bone marrow mesenchymal stem cell (MSC)is believed as multipotential stem cell. The biological behaviors of hematopoietic and nonhematopoietic lineage cells can be controlled in various ways within the bone marrow microenvironment. The bone marrow endothelial cells greatly contribute that which are main components of comprising of bone marrow microenvironment. There is a close relationship between bone marrow endothelial cells and MSCs during the marrow tissue development and in the tissue location, and the cytokines they secrete may play an important role in the regulation of biological behavior of bone marrow MSCs. Presently, little is known about the bioactive products from bone marrow endothelial cells regulating the proliferation and differentiation of MSCs.This study was aimed to investigate in vitro effects of bioactive factors, which are secreted by a murine bone marrow endothelial cell line, on the proliferation of bone marrow MSCs, so as to provide experimental evidences for clarifying the role of bone marrow endothelial cells in the regulation of bone marrow MSC growth.In this study, the murine bone marrow endothelial cell line was characterized once mre since it has been established for 10 years. The serum-free conditioned medium of murine bone marrow endothelial cell line (mBMEC-CM) was collected, and then ultrafiltered using a 3000-relative molecular mass (RMM) cut-off membrane. The retained and filtrated fractions of mBMEC-CM were obtained, which were designated as RMM＞3000 fraction and RMM＜3000 one respectively. By using primary bone marrow MSC colony formation test and MSC subculture with or without the addition of mBMEC-CM, RMM＞3000 fraction and RMM＜3000 one respectively, the proliferative responses of MSC-lineage cells to them were evaluated with the colony yield and the absorbance value for MTT assay. The transforming growth factorβ1 (TGF-β1) was detected from the RMM＞3000 fraction of mBMEC-CM by Western blot analysis. The effects of bioactive products from bone marrow endothelial cells on the in vitro proliferation of MSCs were examined further by the anti-TGF-β1 antibody neutralization test using the recombinant murine TGF-β1 as the positive control.All mBMEC-CM and its RMM＞3000 and RMM＜3000 fractions suppressed the colony-forming capacity of primary murine bone marrow MSCs in vitro. The inhibitory effect of RMM＞3000 fraction was most significant compared with mBMEC-CM and RMM＜3000 fraction, and the maximal inhibition was observed at 20% of final volume fraction in cultures. But mBMEC-CM and its two ultrafiltered fractions had no effects on the proliferation of the third subculture cells from primary murine bone marrow MSCs. TGF-β1 occurred in RMM＞3000 fraction. The inhibitory effects of RMM＞3000 fraction on the primary MSC colony formation were neutralized in the cultures with an addition of anti-TGF-β1 antibody, evidenced by the increased colony numbers. RMM＞3000 fraction significantly reduced the mitotic index of the primary MSC-lineage cells. Cells in G₀/G₁ phases were markedly increased, while cells in S + G₂/M phases were obviously decreased in the cultures with an addition of RMM＞3000 component. These effects of RMM＞3000 fraction were not, however, observed in the third subculture cells from primary murine bone marrow MSCs.Together, these results suggest that murine bone marrow endothelial cells can secrete more than one bioactive factor which have inhibitory effects on the proliferation of primary bone marrow MSCs in vitro. TGF-β1 is one of those factors. The inhibitory effects of them are dose-dependent. These inhibitory factors block or retard the switch of quiescent MSCs into cell cycle. Therefore, the present study has given experimental evidences for getting an insight into the mechanisms underlying MSC growth regulation.