| Traumatic brain injury (TBI) is a worldwide public health problem. It not only has conciderable morbility and mortality, but is a major course of epilepsy, and functional impairments severely affect the quality of life of a patient. In China, TBI is responsible for a significant proportion of all traumatic death in individuals yunger than40years old. And in survivors, TBI can lead to severe neurological and behavioral disabilities, great family strain and high cost to society. Despite large efforts, there is currently no specific treatment available for TBI other than supportive care, but aggressive prehospital resuscitation, rapid triage, and intensive care have reduced mortality rates.One way in which TBI can be classified is by either primary or secondary brain injury. In addition to local neuronal destruction resulting from the mechanical primary insult, TBI also induces a progressive cascade of delayed secondary events triggered by the trauma that contribute to neuronal death, including ischemia, Wallerian degeneration secondary to diffuse axonal injury, excitotoxicity, dysregulation of calcium homeostasis, mitochondrial dysfunction, and free radical-mediated damage. Secondary TBI develop over a period of hours or days after the initial impact to the head. The patterns of neuronal loss following these events in TBI are both focal and diffuse. Focal damage is typically seen around hemorrhagic lesions, such as contusions within the gray matter or at gray-white junctions. Such focal neuronal death may occur by both rapid necrotic and slower apoptotic mechanisms. Among diffuse injury sites, the hippocampus is known to be especially vulnerable in humans, with neuronal loss occurring in>80%of fatal TBI, even in the absence of elevated intracranial pressure. These hippocampal changes correlate with the profound memory impairment seen in both human and animal models of TBI.The current therapeutic approach in research to the treatment of TBI is based on a relatively new understanding of the pathophysiological, molecular, and cellular mechanisms causing secondary brain damage, and includes early detection and evacuation of mass lesions, prevention of secondary insults, and attempts to pharmacologically attenuate damaging biochemical and cellular cascades. It is generally believed that the stimulation of regeneration in the injured adult CNS will likely require one or more of the following processes:cellular replacement, neurotrophic factor delivery, promotion of axonal guidance and removal of growth inhibition, manipulation of intracellular signaling, bridging and artificial substrates, and/or modulation of the immune response.Since the demonstration nearly20years ago that the mammalian brain has the capacity for self-renewal of neurons and astrocytes, there has been a gradual paradigm shift in neurobiology research such that cell replacement strategies have become a major focus of research and commercial activity. And increasing evidence suggests that stem cell-based transplantation therapies may be one of the most promising therapeutic strategies for treating functional impairments following TBI. Cell replacement therapies have been based on the concept that neurological function lost to injury or disease can be improved by introducing new cells that can replace lost neurons or glial cells, or via trophic support to the surviving cells to increase survival, plasticity, and functional recovery. To date various types of stem cells, including embryonic stem cells, neural stem cells and mesenchymal stem cells are currently being investigated for transplantation.Transplantation of embryonic stem cells into the injured rat brain can provide functional recovery, however tumor formation raises serious safety concerns about their transplantation use in humans. At the same time, their use in clinical applications is hindered by moral and ethical concerns, as well as the scarcity of fetal tissue. The latest researches show that induced pluripotent stem cells (iPS) have the pluripotency of embryonic stem cells, but it also exist the danger of forming tumors in vivo. Neural stem or progenitor cells (NSC) are also a good candidate to be transplanted into the injured brain, but the production of the NSC is a thorny issue for the use for thansplantation. Human mesenchymal stem cells (MSC) are considered good candidate for transplantation into the injuried brain of the patients. Bone marrow mesenchymal stem cells are currently the most widely used seeding cells for both experimental and clinical studies. However harvesting bone marrow is a highly invasive procedure, and the number, differentiation potential, and maximum life span of mesenchymal stem cells from bone marrow decline with increasing age. Thus, the search for possible alternative MSC is ongoing. Cells derived from the placenta, membranes, amniotic fluid or fetal tissues are higher in number, expansion potential and differentiation abilities compared with mesenchymal stem cells from adult tissues. And some of them have been utilized in the treatment of neurodegenerative and neural traumatic diseases.Although mesenchymal derived from derived from perinatal tissues have been used in the therapies for a variety of neurological diseases in the experimental animal models, it is mot clear whether the cells from different part of the perinatal tissues have the parallel effect on the treatment of neurological diseases. Here we focus our study on the comparison of the biological characteristics of mesenchymal stem cells from human amniotic membrane (AM-MSC) and umbilical cord Wharton’s jelly (WJ-MSC) with respect to their morphology, expansion kinetics, immunophenotype, multipotency. Since NSC are the idealest seed cells to transplant for the cell replacemental treatment for TBI, the capacity of AM-MSC and WJ-MSC to differentiate into neural stem cells were determined in vitro, the easier MSC can differentiat into neural stem cells, the better MSC could be considered as candidate for cell-based seed cells for treatment of TBI.Neurologic benefit resulting from human MSC treatment of nervous system injuries may be due, at least in part, to the increase of growth factors in the ischemic tissue, and neurotrophic factors play crucial roles in the differentiation and survival of neural cells. Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. At the same time, the five important neurotrophic factors which play crucial role in the development and central nervous system restoration, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin3(NT-3), glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), were detected during neural stem cell induction in vitro. These data may in some degree reflect the neurotrophic benefit of MSC on the treatment of TBI between the AM-MSC and WJ-MSC as well as between the MSC and NSC derived from MSC, which may attribute to the selection of seed cells to the cell-based therapy for TBI.The survival capacity of MSCs in host tissues in conditions of ischemia or ischemic reperfusion is another important property to be considered. The use of an MSC graft approach is limited by the fact that most of the transplanted MSC are readily lost, potentially triggered by the ischemic or ischemia-reperfusion environment in vivo. In our study, we investigated the anti-apoptosis ability of these MSC towards oxidative stress induced by hydrogen peroxide (H2O2) or serum deprivation.The study includes three chapters:Chapter Ⅰ Isolation and characterization of MSC from amniotic membrane and umbilical Wharton’s jellyObjective:To establish a simplified culture system to isolate AM-MSC and WJ-MSC, observe their morphology, and investigate the expression of neural stem cell markers and mesenchymal stem cell markers, their karyotype, proliferative capacities and their multi-lineage differentiation capacities, scan their surfacial fine structure.Methods:Amniotic membrane was sequentially digested in2.4U/mL dispase,1.0mg/mL Collagenase A and0.01mg/mL DNase; and Wharton’s jelly was digested in collagenase type Ⅱ followed by further digestion with0.125%trypsin/EDTA. The morphology was observed by an optic microscope and their surfacial fine structures were scaned by an atomic force microscope, the immunophenotype were tested by Immunocytochemistry or flow cytometry. Before we determined their proliferative capacities, their charyotypes were analysed. The differentiation potential to osteogenic, adipogenic and chondrogenic cells were detected.Results:AM-MSC and WJ-MSC were successfully isolated and cultured by the protocols mentioned in the methods section. AM-MSC and WJ-MSC both express the markers of MSC (CD13, CD29, CD44, CD73, CD90and CD105), but not express hematopoietic and endothelial phenotypes (CD19, CD31, CD34and CD45), and they both express HLA-ABC, not HLA-DR, and pocess osteogenic, adipogenic and chondrogenic differentiation potential. The results are in accordance with the criteria of MSC by The International Society for Cellular Therapy. Two kinds of MSC both strongly expressed vimentin (100.00±0.00%vs.100.00±0.00%), while few AM-MSC and WJ-MSC expressed nestin (28.73±2.97%vs.19.22±2.96%,t=2.265, P=0.053), sox2(20.58±2.43%vs.21.11±2.51%, t=0.154, P=0.881) and Musashi1(10.17±3.10%vs.12.62±2.58%,t=0.609, P=0.559). These data were confirmed by quantitative real-time PCR and Western blot analysis. AM-MSC possess stronger adherent capacity to the surface compared with WJ-MSC in culture scanning by atomic force microscope, While WJ-MSC have higher praliferative potential (F=817.948, P<0.001). Normal chariotypes we observed throughout the whole culture in vitro.Conclusion:we have successfully isolate and culture AM-MSC and WJ-MSC, and characterized the cells based on a set of criteria proposed by the International Society for Cellular Therapy. Moreover the two kinds of cells expressed low levels of neural stemness markers. AM-MSC showed stronger adherent capacity to the cultured surface and stronger anti-apoptosis ability compared with WJ-MSC in culture, While WJ-MSC have higher praliferative potential.Chapter Ⅱ Determination of the capacity of AM-MSC and WJ-MSC for neural stem cells differentiationObjective:to establish the protocol to induce AM-MSC and WJ-MSC into neural stem cells. By the above protocol, we firstly want to obtain a new source of neural stem cells, and secondly to compare the neuronal differentiation ability of AM-MSC and WJ-MSC. And the expressing change of NT was investigated during the neural stem induction.Methods:The transdifferentiation protocols:the AM-MSC and WJ-MSC were induced in neural stem cell differentiation medium, composed of KnockOutTM DMEM/F-12Basal Medium supplemented with20ng/mL human epidermal growth factor (EGF),20ng/mL bFGF, StemPro(?) NSC SFM Supplement and GlutaMAXTM-Ⅰ Supplement (1:100) containing1%penicillin/streptomycin at37℃with5%CO2,and plated in Ultra-Low Attachment25cm2culture flasks. The medium was changed every3days. Ten days after induction, neurospheres were collected to determine levels of neurotrophic factors by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) analysis.Results:AM-MSC and WJ-MSC could be induced into neurosphere-like aggregates, a growth form of neural stem cells, successfully (AM-NSC and WJ-NSC respectively). More importantly, these neural stem cells displayed most of the chanracteristics of neural stem cells. Both AM-MSC and WJ-MSC were examined for expression of the three neural stemness markers by immunocytochemistry after neural stem cell differentiation. After differentiation, the fluorescent signal for the stemness markers of NSC derived from MSC significantly upregulated than that of undifferentiated MSC, and a similar results were obtained from the two kinds of MSC. The details are as follows, AM-NSC showed a higher fluorescent signals for nestin, sox2and musashi1than that of undifferentiated AM-MSC (nestin,92.05±2.75%vs. 28.73±2.97%,P<0.001；sox2,70.17±3.16%vs.20.58±2.43%,P<0.001； Musashil,66.94±3.62%vs.10.17±3.10%,P<0.001)；WJ-NSC showed a higher fluorescent signals for nestin,sox2and musashi1than that of undifferentiated WJ-MSC(nestin,83.57±2.60%vs.19.22±2.96%,P<0.001；sox2,71.17±3.63%vs.21.11±2.51%,P<0.001；Musashi,64.15±3.81%vs.12.62±2.58%,P<0.001). Intrestingly,AM-NSC showed a higher fluorescent signal for the nestin but parallel signal for sox2and musashi than that of WJ-NSC(nestin,92.1±2.82%vs.83.6±22.52%,P=0.0497；sox2,70.2±3.26%vs.71.2±3.54%,P=0.816；Musashil,67.0±3.67%vs.64.0±3.89%,P=0.559).These immunocytochemical data were confirmed by quantitative real-time PCR and Western blot analysis.Secreted levels of BDNF,GDNF,NT-3,CNTF and NGF in both populations of MSC to NSC were detected.For AM-MSC,the secreted level of BDNF was BDNF,109.51±15.26pg/mL,GDNF32.85±14.21pg/mL,NT-327.43±11.91pg/mL, CNTF39.62±13.56pg/mL and NGF21.46±9.83pg/mL.After differentiation,for AM-NSC,the secreted level of BDNF was BDNF,477.39±39.95pg/mL,GDNF101.01±11.67pg/mL,NT-3206.33±26.36pg/mL,CNTF160.48±22.69pg/mL and NGF185.23±23.59pg/m.For WJ-MSC,the secreted level of BDNF was BDNF,241.69±25.90pg/mL,GDNF16.21±10.01pg/mL,NT-3172.35±25.20pg/mL, CNTF34.37±11.42pg/mL and NGF105.59±18.24pg/mL.After differentiation, for AM-NSC,the secreted level of BDNF was BDNF,333.66±31.59pg/mL,GDNF93.64±19.17pg/mL,NT-3122.46±20.02pg/mL,CNTF231.28±22.07pg/mL and NGF32.76±10.17pg/gmL.There were significant differences between AM-MSC and WJ-MSC in BDNF(P=0.006),NT-3(P<0.001)and NGF(P=0.002)；between AM-NSC and WJ-NSC in BDNF(P=0.003),NT-3(P=0.015),CNTF(P=0.014) and NGF (P=0.009).These ELISA data were confirmed by quantitative real-time PCR.Conclusion:we successfully established protocols to induce both AM-MSC and WJ-MSC to differentiate into AM-NSC and WJ-NSC in vitro,with AM-NSC expressing higher level of nestin.Before induction,undifferentiated WJ-MSC secret higher levels of BDNF,NT-3and NGF than those of AM-MSC.However after differentiation, the secretions of BDNF, NT-3, CNTF and NGF of differentiated AM-MSC were significantly higher than those of differentiated WJ-MSC. These findings suggested that MSC from different part of placent perssess different reaction to this neural stem cells introduction protocol, and considering the secreting of NTs, AM-NSC may more suitable for transplantation for the treatment of TBI than undifferentiated AM-MSC.Chapter Ⅲ Cytokine antibody array detection and anti-apoptosis abilities of AM-MSC and WJ-MSC in vitroObjective:To investigate the cytokine secreting capacity, a Cytokine antibody array was used to detect the expression of507cytokines in the cells from two sources of MSC. To detecte their anti-apoptosis abilities in vitro, apoptosis was triggered by H2O2and serum deprivation, and the rate of apoptosis was counted.Methods:Total protein was extracted from the AM-MSC and WJ-MSC using a tissue protein extraction reagent (Kangchen, China). Human cytokine antibody arrays (Human L-507Array, RayBiotech Inc.) were used to detect the expression of507cytokines in the cells from two sources of MSC. The signal from the membrane was detected with a chemiluminescene imaging system. The signal intensity was quantified by densitometry. The value altered by twofold or more was statistically significant. A positive control was used to normalize the results from different membranes. Apoptosis triggered by2mmol/L H2O2and serum deprivation. Apoptosis in MSC were identified by nuclear positive staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) and Annexin V and propidine iodide (PI) staining after H2O2or serum deprivation induction.Results:Results of the cytokine antibody array detection:23cytokines in the AM-MSC were detected by cytokine antibody array which were two fold up regulated than that of WJ-MSC; while49cytokines in the WJ-MSC were detected by cytokine antibody array which were two fold up regulated than that of AM-MSC. And expression levels of4cytokines were changed significantly in AM-MSC compared to WJ-MSC; while there was no significant difference in the cytokines that increased by twofold in WJ-MSC compared with AM-MSC. And the4cytokines up regulated in AM-MSC contain interleukins or their receptor that were considered as inflammatory factors. The details are as follows, IL-6(t=3.546, P=0.038), IL-13R alpha2(t=3.336, P=0.045), IL-12p70(t=3.743, P=0.033), IL-22R (t=3.187, P=0.0498).Results of the anti-apoptosis ability detection:AM-MSC showed significantly stronger anti-apoptosis ability not only to the induction by H2O2but by serum deprivation, which were detected by TUNEL and Annexin V/PI staining compared with WJ-MSC in vitro (P<0.05).Conclusion:Cytokines expressed in AM-MSC increased compared with those in WJ-MSC, and these cytokines were growth factors and interleukins, which are related with regulation of cell proliferation and inflammatory response. AM-MSC showed stronger anti-apoptosis ability compared with WJ-MSC in culture. |
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