Study On Immunogenicity Of Mouse Neural Stemcell

IntroductionFor the past few years, with the development of cell biology and molecular biology, NSC transplantation has become a hot topic of neuroscience research. NSC is capable of self-renewal and differentiation into neurons and glias, which participate in repairing damaged neural tissue;thus, it is a promising donor cell source for injury, infarction, and degeneration disease in central nervous system. Recently, some exciting results in NSC transplantation have been achieved. However, as for the clinical application of NSC transplantation, the problem of immune rejection cannot be easily overcome. After more than a decade of human neural transplantion studies, our knowledge of the immunological properties of NSC remain inadequate. This study tries to investigate the immunogenicity of NSC as the first step toward solving the immune rejection problem after stem cell transplantation. PartⅠCultivation and identification of neural stem cellsBackgroundA large number of studies over the past decade have shown that neural stem cells (NSCs) with multi-lineage differentiation potential can be found in the brains of adult mammals. This discovery was identified as one of the most important advancements in the field of neuroscience in 1990s. Although it was observed that, under the natural conditions, very few NSC in the body contributes little to the self-recovery of damaged nerve tissues, many scientists believe that transplantation of high concentration of NSC can be realized by growing a large number of NSCs outside the body, which has a promising prospect in treating various diseases such as the brain and spinal cord injuries, clogging, and degeneration of nerve system.Large numbers of NSCs with high purity need to be extracted from the in vitro cell culture for the purpose of a successful transplantation of NSC with high concentration. Despite extensive efforts having been made in summarizing and improving the method of NSC suspension culture since its establishment in 1992, it is still far from being satisfactory in many aspects, including the quantity, viability and adherent differentiation. On the basis of the experience and lessons of the previous researchers, a set of practical methods for culturing and identifying NSC have been explored with the help of learning the experience of our laboratory over the last decade. The results and conclusions are presented as follows: Materials and methods1 MaterialsBALB/c inbred strain mice were provided by Laboratory Animal Center, Zhejiang University. DMEM/F12 medium, B27supplement and trypsinase were purchased from Gibco. EGF, bFGF and MMC were purchased from Sigma. Disposable plastic culture flasks were purchased from Orange. Agarose gels were purchased from Sangon Biotech(shanghai) Co.Ltd.2 MethodsCultivation and identification of neural stem cells:Newborn BALB/c mice were sacrificed under deep anesthesia with pentobarbital. The hippocampal tissues were dissected from the mouse brains and washed in sterile phosphate-buffered saline (PBS, pH7.4) with 0.6% glucose. Tissue pieces were incubated in 0.05% trypsin with 0.012% EDTA for 15 min at 37℃and then triturated in the same solution using a polished Pasteur pipette. Cells were collected by centrifugation and seeded in substrate-free tissue culture flasks at a density of 50,000/mm. Passaging was carried out every week and consisted of a gentle mechanical dissociation, after which the mixture of small spheres and single cells were re-seeded into fresh medium. After passage and monoclonal expansion Immunocytochemistry for CD133 was performed to verify the neural stem cells.Result1 Cultivation of NSCsOn the first day of culturing NSC, it took on a single cell suspension. Then the cells gradually concentrated into a small neurosphere on the second day. It was around on the third day that the neurosphere gradually expanded due to NSC cloning and formed a large one. However, the NSCs in the center had to be re-digested and sub-cultured, as it was difficult for them to obtain the nutrient.2 Identification of NSCsUnder the condition of serum-free culture, NSC which was cultured as per clone density proliferated and gradually formed a cloned neurosphere, indicating that the NSC under this experimental group was capable of reproducing themselves. Cultured in the medium of DMEM/F12 containing 10% of fetal bovine serum, NSC cloned sphere gradually grew adherently, and differentiated into neuroglial cells and a small amount of nerve cells. This showed that NSC under this experimental group had a potential of multi-lineage differentiation. After being digested in a short span of time and blown away mechanically, the NSC cloned sphere formed into small cloned NSC spheres and single cell suspension. Then, the cell smears were collected and all found to be positive in CD 133 immunofluorescence staining, suggesting that all the cells under this experimental group were NSCs.ConclusionSufficient quantity of NSCs with high purity can be obtained in a long-term basis by adopting the cell isolation and culture method of combining mechanical isolation with the improved enzymatic digestion, with the serum-free culture medium adding mitogen, the culture bottle covered with agarose gel, as well as the appropriate cell density and the right timing of subculture. PartⅡDifference between immunogenicity of mouse NSC and it's HepatocytesBackgroundWith the development of cell biology and molecular biology, NSC transplantation has become a hot topic of neuroscience research. NSC is capable of self-renewal and differentiation into neurons and glias, which participate in repairing damaged neural tissue; thus, it is a promising donor cell source for injury, infarction, and degeneration disease in central nervous system. Recently, some exciting results in NSC transplantation have been achieved. However, as for the clinical application of NSC transplantation, the problem of immune rejection cannot be easily overcome. After more than a decade of human neural transplantion studies, our knowledge of the immunological properties of NSC remain inadequate. This part of study tries to investigate the difference between immunogenicity of mouse NSC and it's Hepatocytes. This may be helpful to explore the immunogenicity of mouse NSC,then be helpful to solve the immune rejection problems after NSC transplantation.Materials and methods1 MaterialsC57BL/6 and BALB/c inbred strain mice were provided by Laboratory Animal Center, Zhejiang University. DMEM/F12 medium, B27supplement and trypsinase were purchased from Gibco. EGF, bFGF and MMC were purchased from Sigma. Disposable plastic culture flasks were purchased from Orange. [3H]-TdR was purchased from Amersham Pharmacia Biotec. Agarose gels were purchased from Sangon Biotech(shanghai) Co.Ltd. 2 Methods2.1 Cultivation and identification of neural stem cells:Newborn BALB/c mice were sacrificed under deep anesthesia with pentobarbital. The hippocampal tissues were dissected from the mouse brains and washed in sterile phosphate-buffered saline (PBS, pH7.4) with 0.6% glucose. Tissue pieces were incubated in 0.05% trypsin with 0.012% EDTA for 15 min at 37℃and then triturated in the same solution using a polished Pasteur pipette. Cells were collected by centrifugation and seeded in substrate-free tissue culture flasks at a density of 50,000/mm. Passaging was carried out every week and consisted of a gentle mechanical dissociation, after which the mixture of small spheres and single cells were re-seeded into fresh medium. After passage and monoclonal expansion, Immunocytochemistry for CD 133 was performed to verify the neural stem cells.2.2 Immunization of C57BL/6 inbred strain miceFive C57BL/6 mice were immunized by NSCs through intraperitoneal inoculation at a cell density of 1×106/mouse, once per week.3 weeks late, mice were sacrificed 4 days after the last immunization. C57BL/6 mice hepatocytes were used as a control between NSCs in this study2.3 One-way mixed lymphocytes culture testSpleens of immunized C57BL/6 mice were harvested under sterile conditions. Lymphocyte suspensions were then extracted after removal of monocyte and diluted to a final density of 1×106/ml with RPMI 1640 containing 10% FCS. 100μl lymphocyte suspension was added to the wells of the 96-well plates, serveing as responding cell. Then the prepared C57BL/6 NSCs suspension was diluted to a final density of 2×105/ml with culture medium, supplemented with MITO at a concentration of 40-50ug/105, weaved for 30 minutes away from light, washed 3 times with PBS, re-suspended with RPMI1640, and added at a density of 100μl/well into 96-well plates which has already been mounded with host lymphocyte, serveing as stimulating cell. The proportion between stimulating cell and responding cell is 1:5. Then the mixed culture was centrifuged for 5 minutes at 200G to make NSCs and lymphocytes fully contacted, and cells were further cultured for 120h in 5% CO2 incubator at 37℃. [3H]-TdR (0.8μci/ well) was added into the 96-well plates 16h before the end of culture.2.4 Liquid scintillation counterCultured cell were collected with filter paper and counted by using liquid scintillation counter and expressed as counts per minute (cpm).2.5 Hepatocytes control groupHepatocytes were prepared in Seglen Perfusion from the same BALB/c embryonic mouse, and used to immunize five C57BL/6 mice through intraperitoneal inoculation, and then carried out "one-way MLC test" as method mentioned above.2.6 StatisticsData are expressed as X±S, and t test is used to compare the results between two groups.Results1 Morphology and idification of NSC in vitroNSCs were seeded according the clonal culture density (5×103 cells in 30μl culture medium) in 96-well culture plates. After 15 days, the NSCs grow as a neurosphere floating in the culture medium. When added fetal calf erum, the neurosphere begin to adhere and differentiate into glial cell like cells. Both the single cells and neurosphere are CD133-positive.2 Compared with the hepatocytegroup, the NSC group absorbed fewer [3H]-TdR, so the scores counted by liquid scintillation counter were smaller. Analysed with t test, it showed that the cpm values of NSC were significantly lower than that of hepatocyte (16592.8±2865.3 vs.27815.0±2416.3, P<0.001). So it revaled that the proliferative leval of T lymphocytes in NSC group was greatly lower than that of hepatocyte group. Then it can be concluded that the immunogenicity of mouse NSCs is weaker than that of hepatocyte from the same body.ConclusionAbove all, we believe that mouse NSCs show weaker immunogenicity, and their immunogenicity are significantly weaker than hepatocytes, whose immunogenicity is already well known as a weak one. Therefore we can deduce that the immunogenicity of NSCs may be weaker than most of the normal cells. However, further studies are needed for the precise evaluation on immunogenicity of NSCs. PartⅢExpression of MHC-Ⅰand MHC-Ⅱin NSC by flow cytometryBackgroundWith the development of cell biology and molecular biology, NSC transplantation has become a hot topic of neuroscience research. NSC is capable of self-renewal and differentiation into neurons and glias, which participate in repairing damaged neural tissue; thus, it is a promising donor cell source for injury, infarction, and degeneration disease in central nervous system. Recently, some exciting results in NSC transplantation have been achieved. However, as for the clinical application of NSC transplantation, the problem of immune rejection cannot be easily overcome. After more than a decade of human neural transplantion studies, our knowledge of the immunological properties of NSC remain inadequate. This part of study tries to investigate the expression of MHC-Ⅰand MHC-Ⅱin NSC by flow cytometry,and to investigate the change in the expression of MHC-Ⅰand MHC-Ⅱwhen IL-2 were added into the medium. This may be helpful to explore the immunogenicity of mouse NSC and the change of immunogenicity when the environment was changed,then be helpful to solve the immune rejection problems after NSC transplantation.Materials and methods1 MaterialsC57BL/6 and BALB/c inbred strain mice were provided by Laboratory Animal Center, Zhejiang University. DMEM/F12 medium, B27supplement and trypsinase were purchased from Gibco. EGF, bFGF and MMC were purchased from Sigma. Disposable plastic culture flasks were purchased from Orange. Agarose gels were purchased from Sangon Biotech(shanghai) Co.Ltd, monoclonal antibody of mouse MHC-Ⅰand MHC-Ⅱwere purchased from eBioscience.2 Method2.1 Cultivation and identification of neural stem cells:Newborn BALB/c mice were sacrificed under deep anesthesia with pentobarbital. The hippocampal tissues were dissected from the mouse brains and washed in sterile phosphate-buffered saline (PBS, pH7.4) with 0.6% glucose. Tissue pieces were incubated in 0.05% trypsin with 0.012% EDTA for 15 min at 37℃and then triturated in the same solution using a polished Pasteur pipette. Cells were collected by centrifugation and seeded in substrate-free tissue culture flasks at a density of 50,000/mm. Passaging was carried out every week and consisted of a gentle mechanical dissociation, after which the mixture of small spheres and single cells were re-seeded into fresh medium. After passage and monoclonal expansion Immunocytochemistry for CD 133 was performed to verify the neural stem cells.2.2 Expression of MHC-Ⅰand MHC-Ⅱin NSC by flow cytometryA bottle of well-grown BALB/c's NSC was taken to collect the cells with centrifugation. After dissociating for about 30 minutes, the cells were gently and intermittently dispersed into single cell suspension. Then the cells were again collected by centrifugation and re-suspended in serum-free medium (SFM). They were removed into flow tubes and then divided into 12 tubes at random, with approximately 5,000 cells each tube. Firstly, two tubes were taken, and a microlitre of'MHC-Ⅰantibody with FITC mark/isotype control with PE mark'and'MHC-Ⅱantibody with PE mark/ isotype control with FITC mark'was added into the tubes respectively, then the machine background was set. After that, the other 10 tubes were centrifuged and added with 1 microlitre of anti-mouse MHC classⅠwith FITC mark and anti-mouse MHC classⅡwith PE mark respectively. After reacting for 15 minutes under the normal temperature, the cells were rinsed and re-suspended in PBS 400 microlitre of solution. Subsequently, flow cytometry was used to measure the proportions of positive cells of MHC-Ⅰand MHC-Ⅱin NSCs by calculating the average value of 10 tubes.2.3 The change in the expression of MHC-Ⅰand MHC-Ⅱunder the existence of inflammatory factor IL-2 by flow cytometry by flow cytometryThree bottles of BALB/c's NSC were taken and incubated into 25 bottles of 5ml culture medium after dissociation. These bottles were then divided into five groups randomly, with five bottles for each group. As per time line, e.g.24 hours,3 days,7 days and 14 days, four groups of bottles were added with IL-2 in a concentration of lOng/ml. The fifth group was set to be the negative control group (without adding IL-2). All these groups of cells were then collected and transferred into flow tubes, with about 5,000 cells each tube. Following that, each group of cells were centrifuged and added with 1 microlitre of anti-mouse MHC classⅠwith FITC mark and anti-mouse MHC classⅡwith PE mark respectively. Marked as MHC-Ⅰand MHC-Ⅱ, the cells were then cultured for 15 minutes under the normal temperature. Finally, the flow cytometry was used to measure the proportions of positive cells of MHC-Ⅰand MHC-Ⅱin NSCs by calculating the average value of five tubes.2.4 StatisticsData are expressed as x±s, and t test is used to compare the results between two groups.Result1 Expression of MHC-Ⅰand MHC-Ⅱin NSC by flow cytometryShowed as the data:Most of the NSCs have no expression of MHC-Ⅰand MHC-Ⅱ, and the average percentage of MHC-Ⅰpositive cell is slightly high than that of MHC-Ⅱ. The average percentage of MHC-Ⅰor MHC-Ⅱpositive cell in mice NSCs is 4.12% and 1.75%, and 6.58% NSCs co-expressed both MHC-Ⅰand MHC-Ⅱ, while 87.54% mice NSCs expressed neither MHC-Ⅰnor MHC-Ⅱ. 2 The change in the expression of MHC-Ⅰand MHC-Ⅱunder the existence of inflammatory factor IL-2 by flow cytometry by flow cytometryShowed as the data:Under the existence of inflammatory factor IL-2, the expression of MHC-Ⅰand MHC-Ⅱin each experimental group were found to increase at different degrees compared with the control group, P<0.01, suggesting the difference was of statistical significance. The test also showed no significant changes in expression of MHC-Ⅰand MHC-Ⅱin NSC at four different time lines from 24 hours to 14 days.ConculdeAbove all, it is found that mouse NSCs show a weak immunogenicity. Under the existence of inflammatory factor IL-2, the expression of MHC-Ⅰand MHC-Ⅱin each experimental group were found to increase at different degrees. But there is no significant changes in expression of mouse NSCs'MHC-Ⅰand MHC-Ⅱat different time lines from 24 hours to 14 days.