The Research And Application For Schwann-like Cell Differentiation Of Adipose Derived Stem Cells Induced By Neural Leachate

Peripheral nerve injury is common in clinic, because the mature neuron can not proliferate, its recovery and regeneration are not enough after injuring. With the development of tissue engineering, tissue engineering nerve has replaced autologous nerve for repairing peripheral nerve injury. Tissue engineering nerve mainly include three aspects which are the seed cells, scaffold materials, and neurotrophic factor. The ideal seed cells are the premise and foundation of the construction of artificial nerve. Schwann cell plays an important role in nerve regeneration, so it is served as an ideal seed cells in the repair of nerve damage. But it is difficult to culture in vitro, and proliferate slowly, and its purification process is complicated because of fibroblast contamination. So the SCs can not meet the requirements of the tissue engineering neural. Adipose derived stem cells are pluripotent stem cells, which can differentiate into SCs under certain circumstances. ADSCs have many advantages, for example, the wide range of sources, draws materials simply, fewer complications, faster proliferation and stable expansion, so it can be an ideal source of seed cells in nerve tissue engineering cell. However, the current methods used for inducing differentiation of ADSCs into SCs still have some disadvantages. In the resent research, in order to achieve a good method for ADSCs’ differentiation to SCs, we induced ADSCs by neurotrophic factors secreted by peripheral nerve from the co-culture principle, to provide a new idea for the seed cells to resolve the problem of repairing peripheral nerve injury. The experiment is contained five parts.1. Isolation and Identification of rat ADSCs in vitroAdipose tissue was isolated from inguinal fat pads in 3-week-old SD rats. We obtained the single cell suspension by dissociating the tissue using collagenase type I. cultured the cells in DMEM medium containing 10% FBS. Detecting the cells surface marker with flow cytometry and identifing the cells by osteogenic differentiation and adipogenic differentiation. The results showed that after treated by induction medium, ADSCs has formed typical black nodules and alizarin red staining is red; the lipid droplets gradually appeared in adipogenic differentiation and the oil red O staining showed red. Flow cytometric analysis showed that the ADSCs were positive for CD44 and CD90, and negative for CD31 and CD45. These results demonstrate that the cells are ADSCs and it has the characteristics of multipotential differentiation.2. The characteristics of ADSCs differentiation in vitroThe purpose of this section is to determine the differentiation potential of ADSCs’ in vitro, to provide the experimental basis on choosing appropriate passages and the suitable inoculation density. The ADSCs was subcultivated serially until 12 passages, and then the 3、6、9 and 12 generation of ADSCs was selected to differentiate into Adipocytes and Osteoblasts. The differentiation results were detected by alizarin red and oil red O staining. At the same time the 3 generation of ADSCs were inoculated with two different density(5×105/cm2 and 1×103/cm2), then detect the expression of BMP-2 with 3、6、9 and 12 and the different density of ADSCs by Western-blot, and determinate the activity of ALP in 1-12 generation of ADSCs. Results showed that, the 3、6、9 and 12 generation of ADSCs can be induced to osteoblasts and adipocytes, however, with the passage number increasing, the adipogenic ability of ADSCs gradually decreased, and there was no significant difference between the osteogenic ability. The 12 generation and inoculated with 1×103/cm2 density of ADSCs expressed positive of BMP-2; with the passage number increasing, the results showed that ADSCs at the passage 12 underwent a much higher increase in ALP activity compared with ADSCs from the passage 1-10, the ALP activity of ADSCs cultured at a density of 1×103/cm2 is significantly higher than cultured with the density of 5×105/cm2. ADSCs which passaged serially in vitro or inoculated with low density can lead to aging and differentiate into osteoblast spontaneously. So, we’d better selecte the ADSCs in 3-5 generation and the density of 5×105/cm2 for study.3. The preparation of nerve leachate and its influence on neuron-like differentiation of PC12 cells.The rat sciatic nerve was collected under sterile condition, and prepareing nerve leachate by cutting into small pieces, soaking in DMEM medium and filterring. In order to verify the nerve leachate has the potential to promote nerve-like differenation of precursor cells. In this research, PC12 cells were selected as a model of precursor cells, and replaced the PC12 cells medium by nerve leachate. The circumstances of axon differentiation of PC12 cells were observed under the microscop. The length of axons was measured in PC12 cells after replacing with nerve leachate on day 3、6、9、12 and the expression of neuronal markers β3-tubulin and MAP-2 were detected at the same time. The results showed that PC12 cells axons grew significantly when cultured with nerve leaching solution, and the average length of the axons at 3,6,9 dayrespectively reached 28.07 ± 1.76 μm, 58.14 ± 2.60 μm, 170.43 ± 10.08 μm; Axonal markers β3-tubulin and MAP-2 showed posivive expression. Therefore, nerve leachate has the potential to induce neural-like differentiate of precursor cells. 4. The differentiation of ADSCs induced to SCs with nerve leachateADSCs at 3 generation were used for the experiment, culture medium was removed and replaced with nerve leachate. Cells were then incubated under this condition for five days, then detect the expression of SCs specific markers S-100 protein and GFAP with immunofluorescence and Western-blot to confirm the effect of induction. The results showed that the morphology of ADSCs changed significantly after induced by nerve leachate, the cells become to bi- or tri-polar, showed fusiform and similar to mature SCs. Immunofluorescence and Western-blot test results showed that SCs specific markers S-100 and GFAP were positive expression, indicating that the neural leaching solution can induce ADSCs to differentiate into SCs.5. The repairing of rat sciatic nerve injury by using ADSCs induced by nerve leachateWe combined ADSCs and induced ADSCs with collagen gel respectively and injected them into the scaffold materials to prepare tissue engineering nerve graft, then we implanted them into rats with 1 cm sciatic nerve injury. They were marked for group A and group B. The group C: no cells combined with scaffolds and group D:autologous nerve graft were as control. The nerve regeneration of rats were evaluated by electron microscopy, electrophysiology and image analysis after 2 months. The results showed that the repair effect of the sciatic nerve injury in the D group and the B group was the best with no significantly difference, and the effect was significantly higher than that in the A group, the effect of group C was the worst. The results show that the effect of ADSCs on the repair of nerve injury is better than that on the induced ADSCs.