Effect Of Math5 In Inducing Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Retinal Ganglion-like Cells

Effect of Math5 in inducing the differentiation of bone marrow mesenchymal stem cells into retinal ganglion-like cellsThere are many refractory ophthalmologic diseases, such as glaucoma, ischemic optic neuropathy, optic nerve atrophy etc, all of which can cause irreversible damage to optic nerve and retinal ganglion cells, and subsequently serious visual dysfunction and even blindness. Because of the non-renewable nature of nerve cells, human beings are at the end of their resources for these diseases. However, the recent discovery of stem cell differentiation potential has provided new ideas for the treatment of neurodegenerative diseases, and it is expected to regain the optic function by supplying exogenous neural cells.Bone marrow mesenchymal stem cells (BMSCs) are mesenchymal stem cells with multi-directional differentiation potential in the bone marrow mesenchyma. They are also considered as the seed cells in the tissue engineering, because of the advantages such as, easy to be obtained, highly efficient proliferation, stable genetic background, and mild in vivo implantation reaction etc. A large quantity of studies have demonstrated that BMSCs can be differentiated into osteoblast, adipose cells, hepatocytes and myocardial cells etc, and can even be transdifferentiated into neural cells with relevant functions. BMSCs induced by NGF and BDNF expressed B-tubulin III and NSE. RT-PCR testing of CoCL2 induced BMSCs found that the expression of MAP2, Tuj1, NF200 and NSE was increased and after 3 days of induction the cells responded to several neurotransmitters, demonstrating that BMSCs differentiated into neuron-like cells. In a rat mechanic retinal injury model experiment in which concentrated BMSCs were injected into vitreous cavity, BMSCs were able to integrate and express rhodopsin and vimentin in retina, demonstrating that BMSCs could differentiate into retinal cells and help injured retina to repair, and that BMSCs are able to be differentiated into retinal neuron-like cells. Though the neurodifferentiation of BMSCs is satisfactory, but due to its low differentiation efficiency, only a small amount of neuron-like cells can be obtained by induction, which is not enough for tissue engineering. Thus, the critical problem is to find a way to increase BMSCs differentiation efficiency. Although there are many approaches to increasing differentiation efficiency, the most practical and feasible one is to provide exogenous key neurodifferentiation gene to induce BMSCs differentiation. Nowadays, there are many genes which can induce the neuron differentiation, including those that can induce stem cells into retinal ganglion-like cells. Therefore, how to choose the appropriate gene is important.Math5 is a pro-neuron gene of bHLH family. It was first discovered by Browm in 1998 who also found that Math5 could be first detected at E11 in a small patch of cells in the central dorsal optic cup, from E11 to E12, the dorsal expression domain expands circumferentially, by E12.5, Math5-expressing cells are distributed throughout the developing retina.The first neurons to differentiate was retinal ganglion cells, which appeared at the late E12.5 or early E13 stage. Math5 expression begins a full day prior to this time. The period of Math5expression thus preceded and overlaed retinal neurogenesis, demostrating that Math5 maybe the key gene for retinal ganglion cells formation. In Math5 knock-out mice, retinal ganglion cells and optic tissue are not developed, further indicating that Math5 is a key pro-neuron gene and regulates stem cell differentiation into retinal ganglion cells. Math5 exerts its function not only by activating genes required for retinal ganglion cells differentiation, such as Brn3b, GAP43; but also by repressing other proneural bHLH genes, such as Mashl, Math3, NeuroD and Ngn2, to prevent the specification of other cell fates. It has been clear and definite that Math5 is the key gene for retinal ganglion cells differentiation. Therefore, based on this characteristics, transfect the BMSCs by Math5, and if the potential of retinal ganglion cells differentiation is improved, it will be a new method for solving the low differentiation effenciency.In this experiment, use optimized adherent culture method and the first fluid exchange method to obtain a large amount of relatively pure BMSCs, and perform passage when cells' fusion came to 90%, followed by further purification by controlling trypsinization time and multiple digestion. Observing the third passage cells by light microscope found that the shape of these cells was uniform. As the number increased, these cells presented colony. The test of flow cytometry assay showed that CD44 was possitive(50.0%) and CD34 was negetive(0.7%). After the induction, the Oil Red-O stain showed that some cells were positive. All the result demonstrated that the cultured cells were homogeneous BMSCs. BMSCs neurodifferentiation inducted by growth factor and retinal conditioned differentiation medium was observed respectively. Comparatively, BMSCs inducted by retinal conditioned differentiation medium demonstrated higher differentiation rate with better morphology. Immunocytochemistry and RT-PCR scanning showed that induced cells could express specific nerve cell markers of nestin, NSE and Thy 1.1, demonstrating the differentiation ability of BMSGs into retinal ganglion-like cells.In the subsequent study, Math5 was obtained by chemical synthesis according to gene bank and successfully constructed to the shuttle plasmid pShuttle-IRES-hrGFP-2, and named with pSGFP/math5. Use Pme I enzyme to linearize pSGFP/math5, and then electrotranformate it to the competent bacteria BJ5183 which had contained pAdEasyl backbone p lasmid. Then the p roductwas selected by kanamycin and identified by digestion with Pac I. The pAd/math5 was amplified in XL10-Gold ultracompetent cells and identified by digestion with Pac I. Then package pAd/math5 in AD293 cells and obtain the infectious adenovirus. Infect BMSCs by different titer. The result showed that using the 5x 106 PFU/mL virus leaded to high transfection efficiency and lower toxic effect. RT-PCR showed that Math5 begun to express one days after transfection, and the expression increased to the peak in three days.Use recombined adnovirus vector pAd/math5 to infect BMSGs. Four days after the transfection, the shape of BMSCs begun to change. The cytoplasm begun to contract. Seven days later, the contraction was more obvious, and synapses came out, and extended as branch, showing the classic type of neuron cells. RT-PCR detected the expression of NSE, nestin and Thy 1.1, demostrating that the differentiated cells were retinal ganglion-like cells. Under the induction of retinal conditioned differentiation medium, contrasted with the contral group, the percentage of Thy 1.1 positive cells in the transfected group was significantly increased, the result had significance. Besides, RT-PCR testing showed the expression of nerve cell markers like nestin, NSE, and Thy 1.1 was much higher than that of control group, the result hadsignificance. The results suggested that Math5 promoted BMSGs differentiation into retinal ganglion-like cells, and that the enhancement was more remarkable under certain induction conditions.Brn-3b is the important gene for RGCs axon growth and survival, which has also been proved to be the downstream gene of Math5 during the formation of RGCs. During the experiment, RT-PCR scanning showed that without induction, the BMSCs transfected by Math5 didn't express Brn-3b. But with the retinal conditioned differentiation medium, the BMSCs transfected by Math5 expressed Brn-3b. This result showed that with the synergistic effect of retinal conditioned differentiation medium, Math5 exerted its effect by activating the downstream gene Brn-3b. GAP-43 is also the downstream gene of Math5, which is also the important growth associated protein. It has been demonstrated to have a close relationship with neural development, axon regeneration, synapse reconstruction. Without induction, RT-PCR showed that BMSCs transfected by Math5 expressed small amount of GAP-43, indicating that GAP-43 participated the process. With the retinal conditioned differentiation medium, the Math5+BMSCs expressed larger amount of GAP-43 than the control group, which also indicating that Math5 activating the downstream gene GAP-43. Therefore, it showed that Maybe Math5 exerted its function by activating the downstream gene GAP-43 and Brn-3b.To sum up, BMSGs have the potential of differentiating into retinal ganglion like cells and exogenous introduction of Math5 and its expression enhances directed differentiation of BMSGs which is more obvious under induction conditions. Math5 exertes its function by activating the downstream gene GAP-43 and Brn-3b. These results make it possible to obtain a large quantity of cells needed in tissue engineering. However, it is necessary to study the synergistic induction condition and induction mechanism of Math5 further, in order to illustrate better the mechanism of BMSGs-directed differentiation, providing theoretical basis for application.