A Study Of Culture And Differentiation Into Neuro-like Cells For BMSCs On HAM Extracellular Matrix In Vitro

So many diseases such as Glaucoma, age-related macular degeneration, inherited retinitis pigmentosa, optic nerve damage and etc, can cause irreversible loss of vision. The main reason that these diseases lead to the blindness is lack of effective treatment which can make dead retinal ganglion cells regenerative. In recent years, with the advanced researches of embryonic stem cells(ESCs), neuralstem cells(NSCs) and bone mesenchymal stem cells(BMSCs) , the traditional concept that the neurocytes can not regenerate has been broken through. As we know, BMSCs have the characteristics of abundant sources, convient materials, not ethical and legal aspects of the impact. Therefore it is of great advantage, feasibility and necessity to transplant for the study on differentiation of BMSCs into neurons, and on serving them as seed cells for the treatment of neurological diseases. Recently, the study of Jian Ge and associates shows that in vitro retinal cells of rat neonates can induce the cultured BMSCs into retinal neuro-like cells, which offers the hope for the further treatments of many diseases such as retinal and optic nerval damnifications. During the domestic and foreign researches, many scholars attempt the differentiation of BMSCs into retinal-like structures in entoptic environment, culture them in vitro, and try to establish the retinalregeneration of tissue engineering method. While the reports that BMSCs are served as seed cells, induced into neuron-like cells in vitro and the tissure engineering of nerve has been constructed are not so much. In this study, we employed the techniques to isolate, cultivate, proliferate and purify BMSCs of rats, and to identify BMSCs by using the method of immunohistochemistry. Then BMSCs cultured successfully were passaged and proliferated in vitro, so we got a certain number of cells as seed cells. The study is about the feasibility of using human amniotic membrane(HAM) matrix as a vector loading BMSCs, the feasibility of differentiation into neuro-like cells from BMSCs and of constructing three-dimensional culture system in vitro. It may lay the foundation for the further research that is about the application of BMSCs and HAM matrix in nerve tissue engineering, may lay the foundation for the research about intraocular transplantation in the treatments of the diseases such as rhegmatogenous retinal detachment and macular hole. The study uses HAM matrix as a vector loading neuro-like cells induced from BMSCs. Therefore, we have done the following research:Part 1 Isolation and identification of rat BMSCsWe used the combination of Percoll gradient centrifugation and adherence to plastic flask culture to isolate rats BMSCs and establish the system of proliferation and purification, which offered seed cells for the experiment. BMSCs cultured were attached 24 hours after inoculation, after 72 hours, they proliferated, had homogeneous pattern like spindles. On the 7th to 10th days, cells got fusion state, and then passaged. Passaged to the third generation, cells were still spindle cells growth and vigorous. After several generations, BMSCs of rats were more uniform and orderly like spindles. BMSCs got furtherpurification. The experimental result showed that it was a brief and efficient way to isolate BMSCs for the combination of Percoll density gradient centrifugation and adherence to plastic flask culture. It was possible to greatly increase BMSCs.Meanwhile, we used immunohistochemical staining to identify cells cultured by their surface markers. The result indicated: CD71, CD44 was positive, and CD34, CD45 negative. These cells were confirmed BMSCs, which laid the foundation for further differentiation into neuro-like cells and the construction of three-dimensional culture system in vitro.Part 2 Study of BMSCs inoculated on HAM matrix and induced into neuro-like cells to construct three-dimensional culture systemFirstly, we used the same method above to isolate, cultivate and purify rats BMSCs for seed cells as backup. Secondly, Pre-prepared HAM, after treatment by a certain concentration neutral protease, had no epithelial cells, was cut into 2cm×2cm size, laid onto the six-orifice in which cover slip was put into. Then the HAM was covered by little slip around it, and was dipped into 10% fetal bovine serum/Dulbecco's modified Eagle's medium/F12 medium at 4℃as three-dimensional culture vector. We incubated BMSCs of passage 3 and 4 onto HAM matrix in the six-orifice by the density of 1.5×105 cells, and at 37°C for culture. After two days, we washed the cells with PBS, removed them not adherent to the dish and replaced the medium every 2-3 days. We observed the growth of BMSCs loaded on HAM matrix with the inverted microscope. After sedimentation, the prepared neonatal rat retinal cells as inducer would be transferred onto the upper layer of Transwell doubleplate, the second tier was coverslip on which HAM matrix was loading BMSCs(the slide of BMSCs 48 hours after inoculation on the HAM matrix). Two cells were separated by a layer containing microporous membrane 1μmol/L aperture, in a separate co-cultured state. We observed cell morphology on the third and 5th days after being induced by scanning electron microscopy. On the 7th day after induction, immunohistochemical methods were used to detect NF and nestin expressions of the induced cells. The result showed: on inverted microscope observation, BMSCs adhered onto the HAM matrix and growed quickly after inoculated, also had good cell viability and refractive power. By scanning electron microscope observation, on the third day after induction, cell morphology was beginning to change. We could see a small protuberance. On the 5th day, we could see further changes in cell morphology, showing spherical, conical, spindle or astrocytes, neurite growth emerged in the form of neuron-like cells and ends of protuberances connected like network. Identification by immunohistochemical staining, the cells induced showed nestin and NF positive. They were confirmed having character of neurons.In this study the more innovative idea was that we used 1-3 days of rat neonate retinal cells as an inducer, placed it into the upper layer of Transwell double plate. In this way the cell factor and extracellular matrix which promoted the original retinal stem cells to differentiate and develop in the retinal tissue, would spread aroud BMSCs in the lower tier from the membrane pore in the upper layer. That way simulated primitive the micro-environment where retina developed. Meanwhile, we used tissue engineering to plant BMSCs on HAM matrix, so that three-dimensional structure of amniotic membrane matrix could supply a broader space for the growth of neuro-like cells and their axons; so that epidermal growth factor, basic fibroblast growthfactor and other growth factors in matrix could provide rich nutrientsubstance for cell proliferation and differentiation. Amniotic membrane(AM) might have some unknown factor for a strong protective effect to provide more nutritional support for the growth of neuro-like cells, which meaned closer vivo. These results show that it is feasible to load BMSCs on HAM matrix, to induce BMSCs into neuro-like cells, and to construct three-dimensional culture system in vitro. All these above lay the foundation of further research that is about the application of BMSCs and HAM matrix in nerve tissue engineering, lay the foundation of the research about intraocular transplantation in the treatments of the diseases such as rhegmatogenous retinal detachment and macular hole. The research uses HAM matrix as a vector loading neuro-like cells induced from BMSCs.The ultimate goal of this research is to achieve intraocular transplantation. For retina diseases especially retinal detachment caused by macular hole, although the methods at present can close hole, make retinal detachment reduced, it is only the anatomical structure reduction. Retinal ganglion cells loss and death caused by those holes andretinal detachment is irrecoverable through current operation modes. Intraocular transplantation of amniotic membrane matrix which loads neuro-like cells induced from BMSCs provides a new method and idea for the treatment of this kind of diseases. At present the tissue engineering construction of cornea which is loaded by amniotic membrane in vitro has been developed and been used in clinical work step by step. We look forward to the gradual development and usage of retinal ganglion cells in tissue engineering, which will take glad tidings to more patients suffering from retina and optic nerve diseases.