| Background:Drug control was the main treatment for the central nervous system diseases in the past. With the progress of studies on tissue engineering, stem cells replacement has raised new hopes. So far, the neural stem cells (NSCs) transplantation has achieved a great success in restoring the normal functions of the nervous system. The discovery, isolation and culture of NSCs from the hippocampus, cerebral cortex, cerebellum and spinal cord. made the treatment for nervous system diseases possible, but the acquisition of seed cells is limited. With the success in separation, culture and multipotent differentiation of BMSCs in vitro, most papers on the BMSCs differentiating into neurons were reported and the results displayed that BMSCs could be induced to differentiate into neuron-like cells in vivo and vitro. Although BMSCs are good seed cells for stem cells transplantation, they are not easily acquired in clinic so that its application was restricted. Adipose tissue-derived stem cells (ADSCs) as another adult stem cells deriving from the mesoderm were separated from the fat tissue. They can adhere to the flask and form fibroblast-like cell clones. Compared with BMSCs, they have similar surface antigens and culture condition, but ADSCs are more suitable for clinic because of their rich source, easy approach and little damage Zuk et al found that ADSCs induced in certain conditions can be differentiated into neuron-like cells. The early induced cells expressed Nestin and neuron specific factors including enolase (NSE), and neurofilament are expressed in late stage. Cell morphology and expression of surface markers proved that the corresponding differentiated cells were neurons. Adult stem cells'transdifferentiation into neuron-like cells was first reported by Woodbury. The typical chemical induction protocol uses chemical reagents to induce BMSCs to differentiate into neuron-like cells. This way is proved by Deng and Safford. However, this method is controversial, the formation of neuron-like morphology may be a cytoskeleton change caused by cytotoxic. Another way is to use cytokines. It avoids DMSO-mediated cytotoxicity, but yields low rate of differentiation. A literature in 2004 reported that rat BMSCs could form neurosphere-like structures in DMEM/F12 medium containing B27, EGF and bFGF, and the neurospheres expressed neural stem cell marker Nestin, and could differentiate into neurons and glial cells, so they were called stem-like cells.Current studies showed that the differentiation of ADSCs was regulated by many factors, including extracellular environment signals and cell gene expression. These niches include extracellular matrix (ECM), cells that interact with each other, signal transduction between cells and so on. They play an important role in providing nutrition for cells, and regulating the proliferation, differentiation of cells and myelination of neurons. The relatively accepted view is that cytokine regulation is very important. Different cytokines can direct cell differentiation fates. Cytokines may be derived from blood serum or be secreted by adult stem cells themselves. They may also arise from the extracellular matrix. It is unclear about the interaction between cytokines. The main research method was to observe the regulation effect of cytokines on the differentiation of ADSCs. A statement that gene regulation plays a role in the differentiation of stem cells was proposed in recent years, which remains unsolved.This study contains two parts. PartⅠis to study how to separate and culture ADSCs. We get fat tissue from the rat epididymis, and then purify the isolated cells using differential adhesion methods. We observe the cell morphology, do adipogenic differentiation and flow cytometry to identify ADSCs. PartⅡis to study how to induce ADSCs to differentiate into neurons. In our study, we use several cytokines as the suitable inducible factors and explore the optimal concentration to induce ADSCs to differentiate into neurons. Cell morphology and neuron-specific markers identification are used to identify the differentiated cells.PartⅠCulture and identification of rat ADSCsObjectiveThis experiment aimed at isolating ADSCs from rat adipose tissue, before in vitro culture and identification for the induction.Methods1,The isolation, purification, and passage of ADSCs:The adipose tissues were harvested by aseptic mechanical operation from the epididymal fat pads in male SD rats weighting 120-150 g. The tissues were shaken and digested with collagenase 1 at 37℃for 45 min after removing the blood vessels. Digestion was terminated and the digestive juice was centrifuged at 1000 rmp for 10 minutes, the upper layer adipose tissue and the middle layer cell culture medium were discarded, then the lower layer ADSCs were resuspended by 3 ml DMEM/F12 containing 10% FBS. The cell suspension was cultured in incubator with 5% CO2 after filtered though a 6 cm sieve and seeded into a 6 cm Petri dish. In 4 h, the cell suspension was transferred into a new Petri dish in order to remove some adhered cells. This procedure was repeated twice. The medium was changed twice a week. Cells then were passaged according to 1:3 when they showed the 80%-90% of fusion growth. The cells in passage 4 were used for later experiment.2,The adipogenic induction of ADSCs and the identification of fat cells:ADSCs of the 4th generation were induced in adipogenic induction medium containing 1μmol/L Dexamethasone,10 mg/L insulin and DMEM/F12 and cultured in incubator with 5% CO2. The medium was changed twice a week. The induced fat cells were identified by oil O staining.3,Flow cytometric identification of the cell surface antigens of ADSCs:We used flow cytometry to detect the surface antigens of ADSCs of passage 4 through direct immunofluorescence labeling. The selected markers were CD 106, CD11b, CD45, CD49d, CD90 and CD29.ResultsA small number of ADSCs separated from the epididymal fat pads adhered rapidly.4 hours after removal of such cells, the remaining hererogeneous cells were of different morphology, in the shape of short spindle, triangular or flat-shaped. ADSCs could survive for over 20 passages with stable cell proliferation and unified, long spindle-shaped. After adipogenic induction, fat droplets of verified size could be observed under phase-contrast microscope, with positive oil red O staining. The immunofluorescence staining by flow cytometry showed positive rate of CD 106 was 28.48%, CD450.45%, CD11b 0.41%, CD49d 0.21%, CD2998.96% and CD90 82.53%.ConclusionsADSCs can be obtained from adipose tissue of the rats using enzyme digestion. In vitro cells proliferate stably and rapidly. ADSCs are identified to have stem cell ability through adipogenic induction and flow cytometric identification, which will require further research.Part 2 The differentiation induction of ADSCs into neuron-like cells Object:To explore the possibility of isolated and purified in vitro ADSCs differentiating into neuron-like cells.Methods:1 The induction of ADSCs differentiating into NSC:The ADSCs of generation 4 wrer resuspended in DMEM/F12 containing 2% B27,10 ng/ml bFGF and 20 ng/ml EGF, then seeded into 24 well plate according to the density of 1×105/ml. The half of medium was changed every day and new factors were added every 3 days. The next step was performed until the clone spheres were formed.2 The passage and culture of NSCs:When ADSCs induced neurosphere differentiated into 20-25 NSC, they were digestion with digestion with 2 ml TrypLETM and mechanical percussion. The digestive juice was centrifuged at 500rmp for 5 minutes and resuspended with DMEN/F12 containing 2% B27,10 ng/ml bFGF,20 ng/ml EGF and cultured in incubator with 5% CO2.3 The induction of NSC-like cells differentiating into neurons:Floating neurospheres were seeded into 24 well plate covered with cover-slip which was pre-coated with polylysine. We replaced the culture medium with DMEM/F12 containing 10 ng/ml GDNF,10 ng/ml BDNF,1μmol/L RA after the neurospheres adhered.1 day later, the medium was replaced again with neurobasal medium containing 10 ng/ml GDNF,10 ng/ml BDNF,1μmol/L RA. It was changed every 3 days.4 The identification of surface antigen of induced NSC-like cells and NLCs: The positive expression of Nestin on NSC-like cells and expression of Nestin, MAP2, NeuN and B-tubμlinⅢon NCLs were identified by Immunofluorescence.ResultsAfter ADSCs were seeded with high density, some of them were floating, formed into small spheres in 3 days. They grew up gradually and the spheres had strong refraction, especially around the spheres and velvet-like processes under the microscope. The single cells could form neurospheres again after centrifugal and passage in 3 days. They can survive for over 5 passages and the Nestin expression is positive. The neurosphere adhered to the cover-slip after differentiated into NCLs. A small number of cells moved out from the centre to periphery, with cell body highly refractive,2 to 3 short processes were founded around some cells under inverted microscope. The morphology of induced cells was similar to neurons and they arranged in network with each other by low magnification. The expression of Nestin, MAP2, NeuN and B-tubulin were all positive.ConclusionsThe isolated and purified ADSCs could be induced to differentiate into neurons like cells by two-step protocol using cytokine induction method. Nestin was highly expressed on cells differentiated from ADSCs in step 1. It proved that the induced cells do have the characteristics of neural stem cells. The expression of Nestin was still expressed after the stem cells were induced to differentiate into neurons in step 2, but with reduced expression. On the contrary, the expression of MAP2, NeuN and B-tubulinⅢpositive. It proved that the induced cells do have the characteristics of neurons. So ADSCs have plasticity and can be differentiate into neurons. |
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