In Vitro Differentiation Of Bone Marrow Stromal Cells Into Neural Cells By SHH

Spinal cord injury(SCI) is commonly encountered in clinic, and with the development of industrialization the number of SCI patients is significantly increasing nowadays. However, the therapeutic effect is not ideal. All the researchers are searching for effective treatment. Stem cell transplantation therapy has heightened our interest recently with the development of tissue engineering. This would be a new therapy for SCI. A great many seed cells of high quality is choosed, isolated and cultured. And it is the key point in tissue engineering. BMSCs is a kind of stem cells in marrow which has capacity of self-renew and multipotency of differentiation. They are conveniently available and easy to be separated from marrow as well as proliferated in vitro, which are helpful to avoid immune rejection and ethic problem. It is the reason that BMSCs is an ideal kind of seed cells for tissue engineering.In the first place, the density gradient centrifugation method was adopted to isolate and culture the BMSCs , as well as to evaluate the biological characteristics. In the second place, RA (all-trans-retinoic acid), FGF-8(Fibroblast Growth Factor-8) and SHH(sonic hedgehog) were added in different concentration and combination in order to induce the BMSCs to neural cells. The influence of different concentration and combination on cell quantity and activity were investigated. In conclusion, the best way of directional induction and differentiation was confirmed and it is proved the method would be helpful to get high quantity and stabilized seed cells.To isolate and cultivate the BMSCs by density gradient centrifugation method were the first part of this experiment. The feature of growth and other biological characteristics were observed and recorded. The cell (p5) type was identified by morphological feature, surface-specific antigens and other biological characteristics.Results and conclusions:1. The density gradient centrifugation method was adopted as an easy and unexpensive way, as well as 100% live rate for primary cells. The cells growed stably and proliferated fast with high adherence rate. Meanwhile, the morphological feature of seed cells was remained. The structure of BMSCs was observed by electro microscope. We found that BMSCs cultured in vitro showed strong synthesizing and proliferating abilities and other features, which stem cells had. It is confirmed that we can acquire BMSCs steadily by this method.2. The morphological features, surface-specific antigens and osteogenic ability of the cells(p5) were identificated. We found that external morphology became fibroblast-like, spindle and satellite formation; Surface-specific antigens of bone marrow stromal cells were positive; the cells had stable osteogenic ability. In conclusion, we acquired bone marrow stromal stem cells with capacity of self-renew and multipotency of differentiation.To induce and differentiate the BMSCs directionaly into neural cells by SHH, RA, FGF-8 in different combinations were the second part of experiment. We investigated the quantity and activity of cells induced by different combination and found out the best way to induce the human BMSCs to neural cells .Results and conclusions:1. After a 7-day-induction, some BMSCs cells exhibited immunoreactivity. SHH could induce human BMSCs to neural cells in certain concentration in vitro. In Group C(SHH 500ng/ml,FGF-8 100 ng/ml), the best differentiation rate was got with low concentration .We can draw a conclusion that SHH had a significant linear effect on differentiation rate.2. After a 7-day-induction, some BMSCs cells exhibited immunoreactivity. NSE, MAP-2,β-tubulin positive cells were increased significantly in groupⅢ( which BMSCs was treated with SHH plus RA)(P<0.05). SHH plus RA may imitate the embryonic development process and induce human BMSCs to neural cells in certain concentration. It is also confirmed that SHH plus RA could promote the differentiation of BMSCs into neural cells and the proliferation of these nerve cells in vitro.In conclusion, we have established a set of separating, purificating and identificating methods of steady acquirement and proliferation of BMSCs. The features of growth and other biological characteristics were observed and recorded. BMSCs could be induced to neural cells by SHH plus RA with high differentiation rate in vitro. The protocol may be useful in studying early steps of neural cell differentiation of BMSCs in vitro and in employing the quantity seed cells for tissue engineering transplantation therapy.