| BACKGROUND AND OBJECTIVEIt has been demonstrated that bone mesenchymal stem cells (BMSCs) are capable of differentiating into neurons and astrocytes in vitro and in vivo. The cells are interesting because they are readily accessible and grow rapidly in culture. Thus, BMSCs may provide an unlimited source of cells for the treatment of neurodegenerative diseases.Recently, it is reported that BMSCs could differentiate into neuron and the differentiated cells were identified, but the molecular mechanisms of BMSC differentiation have not been clarified. So, it is in urgent need of elucidating the apoptotic mechanisms.Neural stem cells (NSCs) are capable of both self-renewal and differentiation into neurons and glial cells. The cellular replacement therapies using NSCs would be promising approaches for the treatment of several chronic or acute neurological diseases such as Parkinson's, Huntington's diseases and spinal cord injuries.Phosphatidylcholine-specific phospholipase C (PC-PLC) might play a key role in the signal transduction. The PC-PLC hydrolyses phosphatidylcholine to DAG. DAG is the second message in the cells and activates protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). The signal transduction via PC-PLC is important in cell proliferation, differentiation and apoptosis.In the previous researches, we accidentally found that D609 could induce some of human umbilical vein vascular endothelial cells (hUVECs) and human marrow stromal cells (hMSCs) to differentiate into neuron-like cells. Did D609 induce several kinds of MSCs isolated from different tissues and species to differentiate into neural cells? By which way did D609 regulate MSC differentiation? how did PC-PLC work in the neural differentiation of the MSCs? Were some important signal molecules (ROS,NADPH oxidase,NF-κB,c-fos and Rb) involved in this process? Did D609 induce NSC to differentiate into neural cells? These problems need to be solved.Based on the backgrounds mentioned above, the objective of this study was as follows: To investigate PC-PLC mediated BMSC and NSC differentiation into neuron and the molecular mechanisms of neuronal differentiation. The research on neuronal differentiation will offer theory basics for the treatment of neurodegenerative diseases.At present, the research of PC-PLC on differentiation signal transduction was rarely reported. In this research, our data provide the first evidence that PC-PLC mediate BMSC neuronal differentiation. To clarify signal transduction pathway mediated by PC-PLC in neural differentiation of BMSCs would help us to further understand the molecular mechanisms of cell differentiation.We used BMSCs and NSCs as the experiment models to study differentiation signal transduction pathway of adult stem cells. The researches would provide a theoretical basis for studying the molecular mechanisms of transdifferentiation among adult stem cells. To induce MSCs and NSCs to differentiation into neurons would provide a theoretical basis for the neurological diseases theraphy in clinic.STUDY CONTENTS1. MSC neuronal differentiation induced by D609, including the three kinds of MSCs, rat bone marrow derived MSCs (rBMSCs), human bone marrow derived MSCs (hBMSCs) and human placenta-derived MSCs (hPMSCs).2. Improving the differentiation induction protocol and researching the functional characters of the differentiated cells in rBMSCs.3. Analysis of some important signal molecules in neuronal differentiation and the differentiation pathways mediated by PC-PLC in rBMSCs.4. Analysis of differentiation and apoptosis mediated by PC-PLC in NSCs.METHODS: 1. Rat BMSCs culture and identification:1) Rat BMSCs were isolated and cultured as described [Pittenger et al, 1999].2) Rat BMSCs were phenotypically characterized by flow cytometry.2. Methods for neuronal differentiation identification:1) Observation of cell morphological changes by Phase Contrast Microscope2) Analysis the expressions of neural special marker NSE, NF-L and synapsin by immunofluorescence assay3) The mitochondrial membrane potential (MMP) was measured by the TMRM staining as described previously [Falchi et al, 2005]4) The resting membrane potential (Vm) was measured by the TMRM staining as described previously [Mao and Kisaalita 2004]3. PC-PLC activity assay was performed as described by Wu X [Wu et al., 1997]4. Analysis ROS level by the fluorescence probe, DCHF5. SOD activity assay was performed by the SOD detection kit6. NADPH oxidase activity was examined as described [Li et al, 2002]7. Analysis of expression and distribution of proteins:1) Examination of the changes in Rb, integrinβ4, NF-κB and c-fos protein level and distribution by Immunocytochemistry.2) Analysis the levels of Rb and c-fos protein by Western blot assay8. NSCs were isolated and cultured as described previously [Gritti et al, 1996]9. Cell apoptosis measurement:1) Observation of cell morphological changes by Phase Contrast Microscope2) The cell growth was determined by MTT-assay3) Analysis of DNA nuclear fragmentation by acridine orange staining4) LDH assay to determine whether cells treated with D609 underwent necrosis5) TUNEL assay to determine the apoptotic indexRESULTS:1. D609 induced MSC differentiation into neuron1.1 After passaging three times, rBMSCs isolated from rat bone was a homogeneous cell population. The cells were uniformly positive for CD29, CD44, CD105, HLA-ABC. In contrast, these cells were negative for CD14, CD34, CD45 and HLA-DR.1.2 rBMSCs were exposed to 5, 10, 15μg/ml D609; hBMSCs and hPMSCs were exposed to 3, 5, 8μg/ml D609. When the cells were exposed to D609, they exhibited a typical neuronal appearance at 6 h. At 48 h, the processes formed extensive networks and the differentiated cells exhibited intensive positive NSE, NF-L and synapsin. The effect of D609 on MSC differentiation was in a dose-dependent manner (P<0.01, P<0.05). The viability of the control and experimental groups decreased significantly with the increased D609 concentration and the treatment time.2. Improving the differentiation induction protocol in rBMSCs2.1 Treatment with D609 for 48h, in the presence of FGF-2, the cell differentiation rate was partly increased (P<0.05), and the viability was increased (P<0.05), and the apoptotic index was significantly lower (P<0.05); in the presence of B27, the viability was significantly increased (P<0.01) and the percentage of apoptotic cells decreased markedly (P<0.05), but rBMSC neuronal differentiation were inhibited (P<0.01); in the presence of 10% FBS, D609 was unable to induce neuronal differentiation.2.2 Over the subsequent 8 days, in FGF-2 treatment group, the differentiated cells displayed typical neuronal morphology. However, large numbers of the cells fell off and died in the absence of FGF-2. The viability of the cells was significantly increased by FGF-2 (P<0.01, P<0.05).3. The functional characters of the differentiated cells.With the treatment time, the MMP of the differentiated cells was increased significantly (P<0.05), while the Vm gradually decreased and these characters were consistent with those of the cultured neurons from mouse embryo forebrains in vitro.4. The differentiation-related mechanisms 4.1 The PC-PLC activity assay showed that there were at least two isoforms of PC-PLC in rat BMSCs. They were the Ca2+-dependent PC-PLC and the Ca2+-independent PC-PLC. Following the treatment with 10μg/mL D609, the activities of the both PC-PLC decreased significantly (P<0.01).4.2 When rBMSCs differentiated into neurons, the intracellular ROS level was increased obviously (P<0.05); the activity of NADPH oxidase were elevated to 3 folds (P<0.01); Cu/ZnSOD and MnSOD activities were not remarkably altered (P>0.05).4.3 Immunocytochemistry assay showed that during the neuronal differentiation of rBMSCs, NF-κB nuclear translocation were not observed; the integrin 84 level was increased (P<0.01). Western blot assay showed that during the neuronal differentiation of rBMSCs, the c-fos level was elevated (P<0.05). Immunocytochemistry and western blot assay showed that during the neuronal differentiation of rBMSCs, the Rb level was increased (P<0.05).5. The effects of D609 on NSC differentiation and apoptosis5.1 The NSCs were isolated and cultured from embryonic day 14 rat cortex. Immunocytochemistry assay showed the cells expressed Nestin positive. Moreover, the neurons derived from the stem cells exhibited intensive positive NSE, NF-L and synapsin.5.2 When the cells were exposed to D609 5-15μg/mL for 24-72h, the viability were decreased in the concentration- and time-dependent manner (P<0.01, P<0.05); under phase microscope, when the cells were incubated with D609 10μg/mL for 24 h, cell shrinkage, membrane blebbing and apoptotic bodies were observed; AO staining showed that nuclear fragmentation, chromatin condensation and apoptotic bodies occurred in D609 treatment group; TUNEL assay showed that the D609 treatment group showed an overwhelming majority of apoptotic NSCs (P<0.01); when cells were treated by D609 15μg/mL for 72 h, there was no significant difference in LDH activity present in the mediums between the control and the D609 treatment group (P>0.05).5.3 When NSCs were treated with D609 15μg/mL for 72 h, the intracellular ROS level was decreased obviously (P<0.05)CONCLUSION:1. D609 could induce rBMSC, hBMSC and hPMSC differentiation into neurons.2. FGF-2 enhanced the function of D609 and was necessary for maintaining the neuronal differentiation state.3. The differentiated cells have the functional characters of neurons cultured in vitro. With the treatment time, the MMP of the differentiated cells was increased significantly, while the Vm gradually decreased.4. Suppressing PC-PLC activity induced rBMSC neuronal differentiation. PC-PLC might activate ROS pathway, and NADPH oxidase and c-fos could be included in this pathway; integrinβ4 and Rb protein could be involved in this differentiation process; SOD and NF-κB might be irrelevant to this differentiation process.5. D609 could not induce NSC differentiation, but induce the cell apoptosis. The modest ROS level might be necessary for NSC survival.
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