The Experimental Studies On De-differentiation Of Astrocytes Into Neural Stem Cells After Mechanical Injury In Vitro

Neural stem cells(NSCs) are regarded as a perspective candidate for neural transplantation in clinical therapies for central nerve system(CNS) injury and neurological disorders. Unfortunately, NSC sources supply, tumor formation and persistence of pronounced immune response following NSCs transplantation make this technique face great challenge for its therapeutic applications. Astrocyte (AST) within injured CNS region resulting from mechanical, chemical and pathological injury may undergo a process of de-differentiation, these de-differentiated astrocytes express nestin, the marker of neuronal progenitor cells, or sometimes possess neural precursor/NSCs characteristics. How the astrocytes make de-differentiation following injury is largely unclear. It is the present presumption that astrocyte de-differentiation may correlate to intimate microenvironment around them The The data in vitro shows that astrocytes from injured spinal cord may acquire NSCs in culture conditions, the approved viewpoint is of AST de-differentiation profiles. The property of ASTs de-differentiation offers a potentially useful method for treatment of CNS injury, degeneration disease and brain tumor. The objective of present study is investigate the changes of Nestin express in cultured ASTs following injury in scratched–wounded border zone or region that is distal to border in vitro, Meanwhile, nestin positive AST were cultured with stem cells medium to explores their ability of generating neurospheres, the self-renewal and multi-potency of these neurospheres. Additionally, conditioned medium from mechanical injured astrocytes culture were used to investigate wether some diffusible factor derived from injured ASTs could induce de-differentiation of ASTs in culture. To further investigate the mechanisms underlying and find out the diffusible factors, the conditioned medium were selectively separated by protein assay. Eventually these district molecular weight ranges of ingredients were determined and analysed.This present study for de-differentiation of AST might pave a way for further investigation molecular mechanism underlying from protein and gene levels, and lay a foundation for clinical application these viable molecules to treat injury in CNS.The study consisted of 5 parts: PartⅠwe developed a method of preparation of AST from spinal cord with high purity and viability. By using the method, we prepared AST culture and further carried out determination of AST viability and purity. Additionally, the variations in cell cycle was detected and analysed. These results indicate that this methods is very effective in eliminating non-AST contamination, in which we can harvest highly pure (over 99%), viable AST with strong proliferation. Meanwhile, the method has many intrinsic merits including less time- and resource-consumption, simple and convenient manipulation procedures.PartⅡ. The first step is to establish AST mechanical injury model. Further, the expression of Nestin and GFAP in injured AST from 1-7ds were observed and examined by immunocytochemical staining and western-blot assay. At same time, AST proliferation was detected after scratched injury for different time points by BrdU staining. Meanwhile, Nestin-immunoreactive AST were cultured with stem cell medium to explore their ability of generating neurosphere. Eventually, Differentiation of these neurospheres was induced to produce various neural cells. These above results indicate that variations of nestin expression in AST constantly occurred after injury from 1 to 7d in vitro. At beginning of injury, Nestin reactive AST were limted to edge of mechanical injury. With prolongated culture, the number of nestin positive cells gradually increased and extend further distally to lesion zone. The level of nestin expression in AST was also found to increase from 1d to 7d. Their relative optical density values elevated from 0.404 to 0.707.Adversely, the value of GFAP expression decreased from 0.86 to 0.721.As for control group, Nestin expression in AST can not be detected. BrdU staining results revealed that the number of BrdU positive cells continually increased from 1 day to 7days in vitro. The proportion of BrdU positive cells in injury zone ranged from 13.4% to 55.3%, At 7 day injury, the data deceased slightly and still reached 43.2%, and much higher than 7.9% in control group. After these injured AST being cultured with stem cell medium, we found these cells can form neurosphere; Moreover, these neurosphere can differentiate into different type neural cells. The results demonstrated that mechanical injury can induce de-differentiation response of cultured AST.PartⅢ. To further explore which mechanisms cause AST de-differentiation after mechanical injury AST, by some diffusible factors, by network communication (gap junctions) among AST or by either involvement? AST cultured on coverslips were co-cultured with injured AST in dish. Then the changes of these AST characteristics were observed. Furthermore, GFAP and Nestin expression in AST were examined after immunostaining in different time points, Subsequently, these cells of distinct staining type were assessed, meanwhile, BrdU staining were carried out to assess proliferation of AST co-cultured with injurd AST. At same time, changes of cell cycle were determined if these AST co-cultured injured AST have strong division capacity. Finally, Nestin-immunoreactive AST were cultured with stem cell medium to explore their ability of generating neurosphere. Additionly, these differentiation of neurospheres were induced to detect production of various neural cells. We found that a few nestin positive cell occurred in co-culture for 1d in vitro by immunustaining. These nestin positive cells exhibited faint cloudy staining. But the majority of cells show strong GFAP staining. At this time point, the number of GFAP+/Nestin+ is still low and its proportion reach 24.4%. Fortunately, some cells have transformed into GFAP-/Nestin+, the corresponding proporation is 3.6%. After being co-cultured for 3 days, The increasing proporation of GFAP+/Nestin+ cells reached 72.9%, and these cells show strong Nestin reactivity. But the proporation of GFAP+/Nestin- cells decreased to 18.2%, and the proportion of GFAP-/Nestin+ cells gradually elevated to 6.8%., In 5 days, The corresponding proportion reached 78.6%, 10.5% and 6.4%, respectively. As for control groups, the proportion of above cell types in three time points almost remained and changed slightly. Majority of cells are GFAP+/Nestin- reavtive. Meanwhile, the result of BrdU staining show that BrdU+cells percentage in 3 corresponding time reached 13.9%, 19.6% and 25.68%, respectively. But the control group, the percentage remain very low, only reached 3.0%, 5.0% and 7.5%, respectively. Additionally, were we found the normal AST cell cycle significantly change when being co-cultured with injured AST for 1, 3 and 5d. The percentage of G2 phrase increased from normal 7.1% to 23.2% by determination of cell cycle. The result indicated that Injured AST can facilitate transition of G0/G1 to S phase and G2 phase of normal AST. Sequently, these AST were cultured with stem cell medium, we found these cells can form neurosphere; moreover these neurosphere can differentiate into different type neural cells, but differentiation and fate choice of neuropheres are inconsistent, and these neurospheres exhibited distinct heterogenicity. The results above suggest that de-differentiation response of AST to injury occurred by some production of diffusible factors, and neural stem cells from de-differentiation AST has heterogenicity.PartⅣTo further confirm possibility of AST de-differentiation after injury, and investigate AST de-differentiation mechanisms underlying. In the present experiment, we apply two medium, supplement G5 serum-free medium and normal medium, to culture injured AST, Then normal AST were cultured using their production condition medium. Sequently, RG and neural precursor generation were observed and identified by using RC2, nestin and A2B5 immunocytochemical staining as well as Pax6 semi-quantitive RT-PCR. The results are as follows: the two condition mediums can induce AST de-differentiation into RG and A2B5+ cells. In free medium groups, The number of RG gradually increase with time lapse , In 3th and 5th days in vitro, RG-like cells with long ,thin , unbranched processes are present in eye fields, they exhibit RC-2 positive immunoreativity, Pax6 expression in these cells can be detected, and Pax6 relative optical density value reached 0.42 and 0.539, respectively. Meanwhile, the high percentage of RC2 and Nestin double-labeling cell were found in two time points. As for conditioned medium with serum groups, the results are similar to serum-free groups. From 5th to 10th day in vitro, the number of RG increase remarkably either, and level of Pax6 expression reached 0.586 and 1.09 respectively. But the percentage of RC2 positive cells is lower than serum-free medium group. Moreover, by observation of cell A2B5 immunostaining, we found GFAP+/A2B5-cells gradually transformed into GFAP-/A2B5+ cells with prolongated culture. The above results demonstrated that The conditioned culture medium from injured AST led to normal AST de-differentiation into RG and glial precursor, and Serum have no influence to the de-differentiation response.PartⅤ.Conditioned culture medium injured AST were carried out to isolate crudely by centrifugal filtration based on molecular weight using Centricon microconcentrators, and different weight fractions were collected and undertaken to analyse viability. From Bands of SDS-PAGE and viability of determination of mixture, we found that AST after injury in vitro can produce some diffusible molecules which induced de-differentiation of AST. The combination fractions are composed of diversity of molecules, and some ranged different molecular weight, and have synergistic effects on induction AST de-differentiation. After screening, we harvested mixture of molecular weight 30-70kd which can stimulate AST de-differentiation. Unfortunately, the mixture induced AST produce neurosphere with low proliferation capacity, which can differentiate into relative enriched oligodendrocytes. The present results demonstrated that de-differentiation response of these AST to the 30-70kd fraction is only a subpopulation of AST.In summary, the present study revealed that the in vitro AST can de-differentiate into neural stem cell after mechanical injury, and different diffusible factors from these injured AST can evoke normal AST de-differentiation response and even further generate neurosphere, the astrocytic de-differentiation may undergo transitional rejuvenation process from AST to RG and glial precursor cells.Moreover, de-differentiation response of these AST were stimulated by the 30-70kd fractions from mixture of supernatant produced in vitro injured AST, and these cells is only a subpopulation of AST.