Mechanism of granulocyte colony-stimulating factor for promoting cell viability of bone marrow mesenchymal stem cells / 生理学报

The present study was aimed to investigate the mechanism of the granulocyte colony-stimulating factor (G-CSF) on the viability of the bone marrow mesenchymal stem cells (MSCs). MSCs were cultured by classical whole bone marrow adhering method, and the MSCs were analyzed for the cell surface differentiation markers CD34, CD133, CD90 and CD105 by flow cytometry (FCM). The ability of the MSCs to differentiate into osteocytes and adipocytes was tested in osteogenic and adipogenic mediums, separately. The effect of G-CSF (20 mug/mL) on the passage 3 MSCs viability was evaluated by MTT method, and the molecular mechanism of the G-CSF mediated effects was assayed through the pretreatment of the signal pathway inhibitors including 50 nmol/L wortmannin (phosphatidylinoesitol 3 kinase inhibitor), 50 mumol/L PD98059 [extracellular signal-regulated-kinase1/2 (ERK1/2) inhibitor], 30 mumol/L SB203580 (p38 mitogen-activated protein kinase inhibitor), 10 mumol/L H89 (protein kinase A inhibitor), 20 mumol/L Y27632 (Rho kinase inhibitor), 1 mumol/L rapamycin [mammalian target of rapamycin (mTOR) inhibitor], 10 mmol/L straurosporine [protein kinase C (PKC) inhibitor], 6 nmol/L G0697 (PKCalpha inhibitor) and 50 mumol/L Pseudo Z (PKCzeta inhibitor). Cultured passage 3 MSCs expressed CD90 and CD105 strongly, and showed the ability of multi-differentiation into osteocytes and adipocytes. G-CSF promoted the viability of MSCs, and the promotion was completely inhibited by PKC inhibitor straurosporine and partially inhibited by wortmannin, rapamycin, PD98059, SB203580 or G0697. However, its effect was not inhibited by H89, Y27632 and Pseudo Z. It is thus suggested that the promoting effect of G-CSF on MSCs viability was closely related to AKT-mTOR-PKC signal pathway, and PKC maybe the central role in the signal pathway.

Erythropoietin promotes proliferation of human bone marrow mesenchymal stem cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the effects of recombinant human erythropoietin (rhEPO) on proliferation of human bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The aspirates of the bone marrow from healty volunteers were seeded in culture medium. Then MSCs were isolated according to characteristics adhering to the plastics. After three passages in culture, bone marrow-derived adherent cells were identified by growing morphological features, cell surface antigens and differentiation into multi-lineages. Then P3-MSCs which had been identified were incubated with different concentrations of rhEPO (0.5, 1, 5, 10 and 50 U/ml). Subsequently, proliferation of MSCs was measured by MTT assay, as well as cell counts. At the same time, cell cycle was detected by flow cytometry (FCM). The results indicated that the expressions of CD90 and CD105 in P3 bone marrow-derived adherent cells were positive, while the expressions of CD34 and CD45 were negative, and these cells could differentiate into adipocytes, osteocytes and chondrocytes in induction media. MTT assay showed that the optical density (OD) of group treated with EPO was significantly higher than that in the control group (p<0.05), and the group treated with 50 U/ml EPO achieved the most predominant effects. The results of cell count were coincident with that of MTT assay. Furthermore, the cell cycle analysis by FCM revealed that rhEPO could relatively decrease the cell ratio in G0/G1 phase, and increase the cell ratio in S and G2/M phases. As compared with the control group, all those differences were statistically significant (p<0.01). It is concluded that erythropoietin can promote proliferation of human bone marrow mesenchymal stem cells in vitro, which may be correlated with the increased entry into S and M phases of cell cycle of MSCs adjusted by EPO.