Expression Changes Of Tau Protein After MSCs' Neuro-differentiation

Background and objectiveBesides hemopoietic stem cells (HSCs), there is another kind of stem cells, mesenchymal stem cells (MSCs), in the bone marrow. MSCs are progenitor cells derived from the mesoderm. MSCs not only can support hemopoiesis by participating in the formation of hemopoietic micro-environment, but also can differentiate into cells which are usually derived from mesoderm and ectoderm with the suitable conditions, such as osteoblasts, adipocytes, neural cells, hepatocyte and endothelial cell etc. For the characteristics of highly self-renew and extensive differentiated abilities, MSCs are ideal seed cells of cellular transplant and gene treatments.The focus problems in study of MSCs are as follows: the most specific separated and purified method, regenerating system's functions by transplant, the activity of telomerase, target cells of gene treatment, and the compared study with multipotent adult progenitor cells (MAPCs). But study of neural cells derived from MSCs has still been resting on testing specific markers and assessing behavior changes in disease models of animal.Tau protein is the highest content among microtubule associated protein (MAPs) in neural cells. Tau protein can promote formation of microtubule, hold its stabilization, decrease tubulins' decomposition, and induce microtubules to bind together. Tau protein also plays the important poles in the differentiation,maturation, endocrine activity, and receptor activation of neurons. Tau can regulate microtubules' assembly by its spatio-temporal phosphorylated state. The abnormal phosphorylated Tau protein can make neural cells dysfunctions, lose its physiological functions and lead to diseases of nervous system.Accurate induction is the basis of using MSCs in clinical therapies. And it needs study signal regulations and micro-environment influences on MSCs' development in deep. To date, studies indicate that MSCs cannot differentiate in vitro without the suitable culture conditions or inductors. Some growth factors can promote MSCs' proliferation and differentiation. Cytokines have different effects on MSCs' regulation. Epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) are all the important cellular growth factors in central nervous system and play the crucial poles in neural cells' development.At present, Tau protein and its phosphorylated state is lesser attented by the MSCs' study at home and abroad. With NSE (the specific marker of neurons) made as reference, we observe expression changes of Tau protein during the different phases of MSCs' neuro-differentiation process in vitro, and analyze Ser202's phosphorylated state. In this study, Tau protein and its phosphorylated state integrates with the regulation mechanisms of MSCs, the development of neural cells and keeping their physiological functions. The results of this study can provide experimental data for the deeper study of MSCs and transplanting MSCs for nervous diseases' therapies. MethodsIsolate cavy bone-marrow mononuclear cells (MNCs) by centrifugation over Ficoll and establish MSCs' culture in vitro for their adherent characteristic. Study the proliferation in different passages by BrdU Index and CFU-F test. Condition media with EGF, bFGF and EGF+bFGF were applied for inducing MSCs into neural cells. Make grovrth curves of the neuro-differentiated MSCs according to MTT test. Expression changes of NSE, Tau and pSer202 were identified by immunocytochemistry. ELISA assay verify Tau protein's content. All data were presented in the format of "means±SD" (x ±s). Deal data with SPSS 10.0 software.Results1, MSCs culture in vitro can be established from the samples of cavy bone marrow MNCs about 14 days. MSCs were purified in P4-MSCs. The colony-forming abilities of P4-MSCs were about 1.14×104 folds higher than MNCs in the bone marrow. BrdU Index kept in the high-level (>50%) and showed the proliferation abilities were changeless with 16 passages.2.With MSCs' 14-day culture in conditioned media, cells in EGF+bFGF, bFGF and EGF groups had apparent morphologic changes like neural cells compared with uninduced group (control group), especially in EGF+bFGF group. MSCs in control group still held shuttle-like morphon and vortex-like growth feature.3. Immunocytochemistry indicated that positive rates of NSE and Tau protein were all the highest in EGF+bFGF group, that of bFGF, EGF and control groups decreased in turn. The differences between groups were significant (p<0.001) and passages had not influences on those. There hadn't had expression of pSer202 between uniduced and induced groups in different passages.4. There was an upward tendency in Tau protein's content during the 14-day induction by cytokines. Changes of the contents by ELISA were identical with positive rates of Tau protein: contents were the highest in EGF+bFGF group, which of bFGF, EGF and control groups decreased in sequence. The differences between groups are significant (p<0.001) and passages had not influences on that. Conclusion1. MSCs derived from cavy bone marrow can be established steady and successive passage-cultured in vitro, MSCs still has strong proliferation ability at the 16th passage; MSCs can differentiate into neural cells by cytokines.2. During the development of neural cells derived from bone marrow MSCs, Tau protein can promote neural cells' normal development, form processes and maintain their functions.3. EGF and bFGF could promote MSCs' neuro-differentiation in vitro, they have synergistic effects on the differentiation process of MSCs.