Study On Cholinergic Neuron Differentiation Induced By Growth Factors From Bone Marrow Stromal Stem Cells And Its Signal Transduction Mechanisms

Study on bone marrow stromal stem cells (BMSCs) plasticity has been progressed rapidly. BMSCs may differentiate to a few types of cells including hemopoietic, osteoblast, chondroblast, adipocyte, fibrocyte, epithelial cell, bone marrow stromal cells, which brings new hope in neural degenerative disease therapy.Differentiation from the BMSCs to neuron, however, was evidenced until 2000 by Brazelton and Mezey using BMSCs allotransplantation in vivo. Afterwards, many studies demonstrated that several differentiation promoting factors of BMSCs, such as beta—mercaptoethanol ( BME ) , dimethylsulfoxide ( DMSO ) , butylated hydroxyanisole (BHA), 5-azacytidine, retinoic acid (RA), etc., could induce BMSCs differentiating to neuron-like cells and gliacytes. Some nerve growth factors e.g., epidermal growth factor (EGF), brain derived neurotrophic factor (BDNF), and retinoic acid or cell suspension from foetus mice midbrain and corpus striatum were also successfully verified in inducing rat and human BMSCs differentiation to immature neuron and gliacytes in vitro. Other studies reported that committed differentiation of the BMSCs from rat bone marrow could induce production of 5-hydroxytryptamine-sensitive neurons and dopaminergic neuron-like cells in vitro, however, the cholinergic neuron differentiation from BMSCs is still unclear so far.Neuron differentiating mechanisms from the BMSCs are rather complicated due to BMSCs mRNA expressions not only in mesodermic cells but also in germocytes and endodermal or ectodermal cells. In addition, the differentiation of BMSCs to neuron was regulated by the changes in neuronal specific genetic expressive quantity rather than the specific genetic expressive on / off. Genes like Sox, Pax, Notch, delta, frizzled, erbB, RB and RB2/P130 all played some important roles in regulating the differentiation. Recent research displayed that mitogen-activated protein kinase (MAPK) signal pathway might stimulate the BMSCs differentiation.There are several open issues in studies on BMSCs differentiation to neuron :First, present method of inducing BMSCs differentiation to neuron have some intrinsic defects. For instance, toxic chemical inducers are not able for clinical use, some growth factors can induce differentiation of BMSCs to neurons but fail to commit differentiation to specific neuron. Some experimental protocols have been used to induce BMSCs differentiating to dopaminergic neuron and 5-hydroxytryptamine-sensitive neuron successfully, but not to cholinergic neuron.In addition, mechanisms of the BMSCs differentiation into neurons are still obscure, especially in terms of signal transduction pathway. Previous studies suggested that a specific signal transduction process in different cells might turn out to be contrary effects. Moreover, roles of PI3K-Akt-mTOR and MAPK pathways in the process of BMSCs differentiation to neurons require further investigation.Moreover, cell differentiation is a complicated process involved in its structure restitution and organelle recombination, and apoptosis and autophagy are important in cell renewing. Study on apoptosis and autophagy related to the BMSCs differentiation into neurons, however, is obviously insufficient.Objective:To screen and optimize the methods of BMSCs differentiation into cholinergic neurons induced by growth factors in vitro. To elucidate the signal transduction mechanism of BMSCs differentiation into cholinergic neurons induced by growth factors, a series of experiments were conducted by using specific antagonist of LY294002 to inhibit PI3K activity investigating PI3K-Akt-mTOR pathway protein Akt activity, MAPK pathway protein extracellular signal-regulated kinase (ERK1/2) activity, and by detecting downstream gene p53 protein expression and autophagy level.Methods:(1) BMSCs culture and proliferation were established by using nerve growth factors (NGF) with different concentration gradients to induce BMSCs differentiation. Morphology of cell differentiation was observed and photographed by inverted phase contrast microscope to screen and determine the optimal concentration of NGF. Three experimental groups were treated by NGF alone, NGF with basic fibroblast growth factors (bFGF) and NGF with bFGF combined epidermal growth factors (EGF), and immunofluorescence identification of mitogen-activated protein 2 (MAP-2) expression of induced BMSCs differentiation to select optimal combination of the treatments.(2) A specific antagonist, LY294002 was used to inhibit PI3K activity. Effects of PI3K on BMSCs differentiation rate induced with growth factor were detected and photographed under the inverted phase contrast microscope. Expressions of MAP-2 and choline acetyl transferase (ChAT) were identified by immunofluorescence, and then the proportion of ChAT-positive cells was calculated with computer. Moreover, the BMSCs differentiated survival rate was determined by method MTT. Statistical comparison of differences among the effects of growth factors, LY294002 and both on BMSCs differentiated rate and survival rate with other three treatments, i.e., NGF with bFGF combined EGF, LY294002 alone and three growth factors with LY294002, were carried out.(3) Changes in Akt and ERK1/2 activated degree, p53 protein quantitative expression and autophagy level determined by detecting expression of Cathepsin-B and LC3-II/LC3-I, in terms of inductive effects of three growth factors alone, LY294002 alone and both on BMSCs differentiation into cholinergic neurons, were tested with Western blot analysis. Furthermore, the proportion of ChAT-positive cells and survival rate were then inspected in order to explore the signal transduction pathway mechanisms regulating the BMSCs differentiation into cholinergic neurons induced by growth factors.Results:(1) NGF with concentration of 100 ng/mL could induce BMSCs differentiation more effectively. Combination of NGF with bFGF and EGF showed a remarkable effect in inducing BMSCs differentiating into cholinergic neurons. Proportion of ChAT-positive cells at 3 h and 5 h after induction were obviously higher than at 1 h but there was no statistical difference after 3 h (P>0.05, vs 5 h). Inhibition alone of PI3K also induced BMSCs differentiating into cholinergic neurons and the proportion of ChAT-positive cells did not increase after 3 h induction. Compared to group NGF with bFGF and EGF, the proportion of ChAT-positive cells in group PI3K inhibition alone significantly decreased in all times (P<0.05). Inhibition of PI3K activity reinforced growth factors inducing effects and the proportion of ChAT-positive cells after 3 h and 5 h induction in combining treated group of growth factors with LY294002 was remarkably higher than that in group of growth factors treatment alone (P<0.05).(2) Cell survival rate did not decline obviously in process of time (P>0.05) after BMSCs differentiating into cholinergic neurons induced by growth factors. Inhibition of PI3K activity reduced cell survival rate apparently not only in group of PI3K inhibition alone (using LY294002) but also in group of growth factors with LY294002. Cell survival rate after 3 h and 5 h induction in combining treated group of growth factors with LY294002 was remarkably lower than that in group of growth factors treatment alone (P<0.05).(3) Expression of Akt phosphorylated protein increased notably and lasted for 5 h after BMSCs differentiating into cholinergic neurons induced by growth factors and displayed some extent level of PI3K inhibition alone, then decreased after the fifth hour but no difference detected in comparison with control group. After interrupting PI3K activity, the level of growth factors activating Akt phosphorylated protein did not markedly change within the first hour, but was higher than untreated group after the third hour, and remained to the fifth hour.(4) Expression of ERK1/2 phosphorylated protein in control group exhibited higher level and was more increasing in process of time. Growth factor induction for 1 h more increased ERK1/2 activity than control group but the activity showed a gradual decrease after growth factors induction from 3 h to 5 h. Inhibition alone of PI3K activity obviously weakened ERK1/2 activation whose activity was also remarkably lower than other groups. ERK1/2 activation induced by growth factors was more depressed than the uninhibited group after PI3K activity was alone inhibited for 3 h and 5 h. (5) Expression of p53 protein in three treated groups were obviously higher than those in control group. Expression of p53 protein showed great increase after inhibiting PI3K activity alone. Induced by growth factors, p53 protein expression with inhibiting PI3K activity was lower than that with inhibiting PI3K activity alone. Growth factors obviously reduced cell autophagy level after BMSCs differentiation, and manifested a synergistic action diminishing cell autophagy level remarkably with inhibition of PI3K.Conclusion:(1) Combination of NGF with bFGF and EGF is an effective way in inducing BMSCs differentiation to cholinergic neurons.(2) Akt activation is involved in BMSCs differentiation to cholinergic neurons induced by growth factors. BMSCs differentiation may require Akt phosphorylated protein to reach to a given level as a"inducing threshold".(3) Survival of the differentiated cells depend on activated level of ERK1/2. High level of Akt activation results in inhibition of ERK1/2 activation, and decreasing survival rate of neurons differentiated from BMSCs. Balance in activation of Akt and ERK1/2 is nessesary for the neuron differentiation from BMSCs.(4) Increased expression of p53 protein is not a determinant of cell death, and autophagy is not the trigger factor in BMSCs differentiation induced by growth factors.