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.

Effects of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells in mice / 中国实验血液学杂志

To explore the effect of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells (MSCs) in mice, 20 Kunming mice (35 +/- 5 g) were divided randomly into 4 groups: low-dose TPO group, moderate-dose TPO group, high-dose TPO group and normal control group (n = 5). The experimental groups were subjected to intraperitoneal injections of TPO at a dose of 25, 50, 100 microg/kg, respectively, and normal control group were treated with saline at a dose of 0.1 ml/g per day for 5 days. The bone marrow was harvested on 12 hours after the final administration. The bone marrow nucleated cells (BMNCs) were counted and seeded at a density of 10(6) cells/cm(2). The colony-forming unit-fibroblast (CFU-F) of MSCs was cultured and evaluated. The CFU-F of MSCs underwent osteo-genic induction and adipogenic induction, and cytochemical and immunocytochemical staining were performed to verify their multipotential. CFU-F and the cell percentage of CD90(+), CD105(+), CD34(+) in BMNCs were analyzed by flow cytometry. The results showed that the number of BMNCs and the cell percentage of CD90(+), CD105(+), CD34(+) and CFU-F increased obviously in TPO groups as compared with the normal control group (p < 0.05). The number of BMNCs increased most obviously in the 50 microg/kg TPO group. However, there was no significant difference in number of CFU-F between 50 microg/kg and 100 microg/kg TPO group (p > 0.05). The CFU-F of MSCs in bone marrow had their osteogenic and adipogenic differentiation potentials in vitro. It is concluded that the number of BMNCs and the cell percentage of CD90(+), CD105(+) and CFU-F increased after administration with TPO. It means that TPO can enhance MSCs to proliferate in bone marrow. However, the number of BMNCs and CFU-F can not increase with the increase of TPO dose.

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.

The effect of recombinant human erythropoietin on the migration of bone marrow derived mesenchymal stem cells in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of rhEPO on the migration of bone marrow(BM) derived mesenchymal stem cells(MSCs) and its probable signal transduction mechanism.</p><p><b>METHODS</b>MSC was cultured by classical whole BM adherence method; MSC characteristics was identified by multi-differentiation and surface marker (CD90, CD133, CD34, CD105). The effect of different concentrations EPO (1, 10, 100, 1000 IU/ml) on MSCs migration were observed. Then 30 minutes later, MSC were treated with signal transduction pathway inhibitors, 50 nmol/L wortmannin, 50 micromol/L PD98059, 10 micromol/L U73122, 4 microg/ml Anti EPO-R IgG, 30 micromol/L SB203580, 10 mmol/L Staurosporine, 6 nmol/L G06976 and 50 micromol/L Pseudo Z, respectively. The efficacy of MSC migration was analyzed by Transwell in vitro migration assay.</p><p><b>RESULTS</b>Cultured MSCs had the capacities for osteogenic and adipogenic differentiation and highly expressed CD105, CD90 and EPO-R. The efficiency of MSC in vitro migration increased gradually in a concentration-dependent manner with increasing concentration of rhEPO, and the ability peaked at a concentration of 100 IU/ml. Furthermore, the migration ability was decreased on treated with U73122, Anti EPO-R IgG, Staurosporine, Pseudo Z treatment.</p><p><b>CONCLUSION</b>EPO/EPO-R-mediated MSCs migration is related with MAPK, PI-PLC/PKC-zeta signal pathways, PKC-zeta signal pathway may be of central role for MSCs migration.</p>

Effects of rhG-CSF on mobilization of mouse mesenchymal stem cells / 中国实验血液学杂志

To evaluate the effects of rhG-CSF on mobilization of mesenchymal stem cells (MSCs) of mouse bone marrow at different time point, thirty mice were randomly divided into rhG-CSF treatment group and control group. The mice were subcutaneously injected with rhG-CSF in a dose of 80 microg/kg or saline for 5 days. The bone marrow and peripheral blood were obtained at time points of 6, 12, 168 hours after final injection of rhG-CSF or saline. Bone marrow mononuclear cells (BMMNCs) were seeded at density of 1 x 10(6) MNCs onto 12-well plate for culture expansion in DMEM supplemented with 10% FBS, and the number of colony forming unit - fibroblast (CFU-F) was counted after 14 days. The cells were collected by trypsinization and the surface antigens CD34, CD133, CD90 and CD105 were analyzed by flow cytometry. The multi-differentiation of MSCs were done in the culture condition of induced-adipocyte and osteocyte. Peripheral blood MNCs examination was same as the bone marrow. The results indicated that the number of CFU-F of bone marrow in rhG-CSF group was more than that in control group (p < 0.01), the number of CFU-F in rhG-CSF group at 6 hours was more than that at 12 hours and 168 hours, respectively (p < 0.01). There was no obvious difference between CFU-F at 12 hours and at 168 hours (p > 0.05). MSCs were positive for CD90, CD105 and negative for CD34 and CD133. MSCs were found to differentiate into adipocyte and osteocyte in vitro. The CFU-F of PBMNCs obtained and cultured in vitro in the same culture conditions could be observed after the rhG-CSF injection at 6 hours, but cloning efficiency was (0.50 +/- 0.11) x 10(-6) MNCs and showed statistical difference as compared with control. It is concluded that rhG-CSF to mobilize hemopoietic stem cells can be used to induce mouse MSCs in vivo expansion, which showed the peak value within 6 hours after final injection of rhG-CSF. rhG-CSF have the mini-mobilization effect on murine MSCs derived from bone marrow.

Effects of HGF on GVHD and Th1/Th2-related cytokines in ALL mice after allo-BMT / 中国实验血液学杂志

To observe the effects of hepatocyte growth factor (HGF) on graft-versus-host disease (GVHD) and Th1/Th2 related cytokines in mice with acute lymphoblastic leukemia (ALL) after allogenic bone marrow transplantation (allo-BMT), BALB/c mice were conditioned by total body irradiation with 11 Gy and then were transplanted with allogeneic bone marrow after establishing ALL model. BALB/c mice were divided into groups A and B. The mice of group A were injected subcutaneously with HGF from day 0 to 7 after allo-BMT, and the mice of group B were injected subcutaneously with PBS from day 0 to 7 after allo-BMT. The symptoms of GVHD and the GVHD pathological changes of liver and small intestine and skin were observed. The serum levels of both IFN-gamma and IL-4 were determined by ELISA. The results showed that the score of GVHD in group A was lower than that in group B (P < 0.05). The levels of IFN-gamma in both groups A and B were all higher than that in normal group (P < 0.05 and P < 0.001, respectively), However, the level of IFN-gamma in group A was lower than that in group B (P < 0.01). The levels of IL-4 in both group A and B were all lower than that in normal group (P < 0.05), but the level of IL-4 in group A was higher than that in group B (P < 0.05). It is concluded that HGF can alleviates the severity of GVHD, because of its balancing the Th1/Th2-related cytokines after allo-BMT.

The effect of HGF on graft-versus-host disease and graft-versus-leukemia after allogeneic bone marrow transplantation in acute lymphoblastic leukemia mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of hepatocyte growth factor (HGF) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) after allogeneic bone marrow transplantation (allo-BMT) and related mechanism in acute lymphoblastic leukemia (ALL) mice.</p><p><b>METHODS</b>Twenty nude mice were randomly divided into control (group A) and test (group B) groups for monitoring relapse, and 20 BALB/c mice into control (group C) and test (group D) groups for GVHD. HGF as injected from day 0 to day 7 after BMT for groups B and D, while PBS for A and C. CD4(+) and CD8(+) T cell were evaluated by flow cytometry. The survival of mice after BMT was recorded. The level of tumor necrosis factor-alpha (TNF-alpha) was evaluated by ELISA.</p><p><b>RESULTS</b>The median past-BMT survival were 7.00 +/- 1.58, 9.00 +/- 1.58, 11.00 +/- 3.95 and 24.00 +/- 13.44 days for groups A, B, C, D, respectively, being prolonged in group D. HGF could decrease the quantity of CD4(+) T cells [group D (10.39 +/- 1.15)% vs group C (13.50 +/- 1.80)%, P < 0.01] and increase CD8(+) T cell [group D (12.25 +/- 2.85)% vs group C (6.12 +/- 1.99)%, P < 0.01], decrease the level of TNF-alpha in transplanted ALL mice [group D (112.10 +/- 18.99) pg/ml vs group C (143.90 +/- 25.35) pg/ml, P < 0.01] and reduce the degree of GVHD.</p><p><b>CONCLUSION</b>HGF could alleviate post-allo-BMT GVHD but retain GVL effect.</p>

Expansion of marrow hematopoietic progenitor cells ex vivo supported by culture system from mouse marrow embryonic fibroblasts and leukemia inhibitory factor / 中国实验血液学杂志

The objective of this study was to explore the effect of culture system from embryonic fibroblasts and leukemia inhibitory factor (LIF) on expansion of mouse bone marrow hematopoietic progenitor cells ex vivo, and to observe its effect on the expression of homing-related cell adhesion molecules among VLA-4 (CD49e), VLA-5 (CD49e), LFA-1 (CD11a), HCAM (CD44) and L-selectin (CD62L). The culture system from the mouse embryonic fibroblasts inactivatd by mitomycin C and contained LIF was used to culture with mouse BMMNC for 7 days. The total number of BMMNC, CFC, Sca-1(+) cells, cell apoptosis rate and the expression of above cell adhesion molecules were counted. The results showed that culture system consisted of embryonic fibroblasts and LIF significantly enhanced the total number of BMMNC, CFC, Sca-1(+) cells, suppressed cell apoptosis (P < 0.05). In control without MEF and LIF, the total number of BMMNC was reduced remarkedly, CFC and Sca-1(+) cells were completely dead, the majority of cells produced apoptosis (P < 0.01). The expression of CD49d, Cd44 and CD61L on Sca-1(+) cells were similar to that befor expression (P < 0.05), but the expression of CD49e and CD11a on Sca-1(+) cells were remarkably increased (P < 0.05). It is concluded that culture system from embryonic fibroblasts and LIF can only significantly expand mouse bone marrow hematopoietic progenitor cells ex vivo, but the expanded hematopoietic progenitor may well sustain the expression of homing-related adhesion molecules. The homing functions of these expanded hematopoietic progenitors kept no change.

Effect of angiotensin II on cord blood CD34(+) cells expansion in vitro / 中国实验血液学杂志

Angiotensin II (Ang II) is one active substance of renin-angiotensin system. In order to explore the effect of Ang II combined with various cytokines on proliferation and differentiation of cord blood CD34(+) cells, in vitro experiments of cell cultures of Ang II with or without cytokines were taken place. The results showed that Ang II stimulated both BFU-E and CFU-GM expansion. The numbers of BFU-E and CFU-GM raised with increase of Ang II concentrations ranged from 0.01 - 0.1 micro mol/L. In semi-solid culture assay, Ang II stimulated CFU-GM production but no effect on BFU-E occurred. The multiple number of CFU-GM increased from 2.3 +/- 0.8 to 7.8 +/- 1.9 times when Ang II was added into SCF + G-CSF + GM-CSF + IL-3 combination. Similarly, the multiple number of BFU-E increased from 3.1 +/- 1.8 to 9.2 +/- 2.3 times when Ang II was combined with SCF + EPO + TPO + IL-3. In conclusion, Ang II stimulated cord blood hematopoietic stem/progenitor cell expansion in vitro the in presence of various cytokines.

The growth characteristics of mesenchymal stem/progenitor cells in human umbilical cord blood / 中国实验血液学杂志

This study was done for investigating the frequency and proliferative feature of mesenchymal stem/progenitor cells (MSPC) in human umbilical cord blood (CB) and for searching a new seed cell for tissue engineering. Mononuclear cells was separated by Ficoll-Hypaque from cord blood and suspended in DMEM culture medium supplemented by 2% fetal bovine serum. The adherent CB cells were cultured and expanded at same medium. The results showed that the frequency of CB-MSPC was 0.5 x 10(-6) [(0.2 - 0.8) x 10(-6)]. The CB-MSPC showed a fibroblast-like morphology and retained their morphological feature at least after 20 sub-passages, and could extensively be expanded by about 1.3 x 10(7) times as much. The conclusion is that low serum DMEM culture could maintain the proliferation and differentiation potential of CB-MSPC. CB-MSPC might be a favorable seed cell for tissue engineering and regeneration.