Experimental Studies On The Neural Stem Cell-like Features Of Leptomeningeal Cells

Traditionally, meninges have been thought to be only a protective apparatus and structurally and functionally separated from brain parenchyma. In 1990s, Mercier et al challenged this prevailing view by using immunohistochemical methods to vividly mark the cellular network formed by leptomeningeal cells, glial cells and neurons. This conception of meningeo-glial network calls for a reconsideration of the role of meninges and appears to reflect the possibility of leptomeningeal cells'involvement in physiological or pathological processes in CNS. Increasing evidences show that meninges are composed by various cells besides fibroblasts, which facilitate their complicated functions. Leptomeningeal cell, a special kind of fibroblast-like cell in CNS, has become a hot topic. For example, it is recognized that leptomeningeal cells can express and secret various cytokines and biological molecules, participate in the neuroimmunomodulation in CNS, be actively involved in neuronal cells differentiation and migration, and play key role in cerebellum maturation and enzyme-barrier constitution.However, the role of leptomeningeal cells in inducing neuronal growth and differentiation is still unclear. Some researchers held the opinion that leptomeningeal cells produced inhibiting molecules to form the barriers of neural regeneration. In contrast, other evidences demonstrated that leptomeningeal cells mainly promote the neurites growth by secreting neurotrophic factors, such as neural growth factors, and served as"feeder-layers"to sustain the proliferation of neural stem cells. Recently, it was showed that Lex/ssea-1, a specific marker for neural stem cell, was also expressed by cells in leptomeninges in adult rats. All of these results indicate that leptomeningeal cells might play special roles in neurogenesis and interact with neural stem cells, unveiling the study of leptomeningeal cells'characteristics and novel potentials.Accordingly, aiming to elucidate the possible identity of leptomeningeal cells as neural stem cells, we designed five sets of experiments in this study, which were made on animal models and cultured cells, respectively. The methods used were leptomeningeal wholemount preparation, cell culture, immunohistochemistry, immunofluorescence, BrdU incorporation and Western blotting analysis. In the first part of study, we detected the expression of nestin protein, a specific marker for neural stem cell, by the leptomeningeal cells in rats of different developmental stages. In the second part of study, after establishment of a relatively pure culture system of leptomeningeal cells, we detected whether these cells could exhibit self-renewal and differentiation potentials. Then in the following experiments, we observed the effects of three kinds of regulation factors, which included interleukin-6, anti-depression drugs and mechanical injury respectively, on the proliferation and differentiation of leptomeninges-derived neural stem cells.The main results are as follows:Part One 1.It was showed that leptomeningeal cells in adult SD rats and adult Balb/c mice could express nestin, a specific marker for neural stem cell. However, the expression level was quite low and the positive cells were only limited in special location of leptomeninges, mainly referred to which overlaid on the ventral brain and cerebral falx. In addition, the morphology and the distribution of positive cells in leptomeninges were different between two species of animals.2.It was detected that leptomeningeal cells expressed nestin in rats at different developmental stages and the expression level diminished gradually as development advancing. Moreover, we found that cells in wholemount preparation were not only double-immunostained by nestin/Thy1.1, but also by GFAP/nestin.These results indicate that nestin, a specific marker for neural stem cells, could be expressed by leptomeningeal cells in rats of different developmental stages. That is to say, leptomeningeal cells possessed stem cell-like proliferation potential, although this feature was diminished as the development advancingPart Two1.After leptomeningeal cells were cultured in different condition, we compared the purity and cellular property in these culture systems. When cultured in DMEM containing 5% FCS and 5% HS, leptomeningeal cells were mixed with 30% of astrocytes and few neurons, But in DMEM/1%FCS/9%HS, the leptomeningeal cells accounted for more than 90% of the total cells and glia and neurons were rarely seen. In addition, sequential passages of the latter system improved the cellular purity to more than 97% and only a few astrocytes were found in this culture system. Therefore, changing media and passage during culture processes were effective to establish a pure culture system for leptomeningeal cells.2.Accordingly, we detected whether cultured leptomeningeal cells could exhibit essential characteristics of neural stem cell. The results showed that in the presence of EGF/bFGF containing serum-free conditioned medium, leptomeningeal cells proliferated to develop into neurosphere-like morphology. This process was in a bFGF-dependent manner. Then serum administration induced these leptomeningeal cells to differentiate into the neuronal and glial lineage cells, marked byβ-tubulinIII, GFAP and RIP respectively. Moreover, it was showed that after repetitious passages, leptomeningeal cells still generated neurospheres and multipotentially differentiated, though such capacity declined gradually.These results indicate that the mesenchyma-derived leptomeningeal cells possess the capacity of neural stem cell to self-renew and differentiate multipotentially, especially producing neurons.Part Three1.It was revealed that Interleukin-6 administration not only led the leptomeninges-derived neurospheres to generate neurons and glial cells, but also elevated the percentage of neuronal lineage cells. Then using different neuronal markers, these newly formed neurons were found to be 5-HT positive immature neurons.2.It was revealed by the immunocytochemical and Western blotting results that IL-6 administration up-regulated the expression of its own receptor(sIL-6Rαand gp130)in these leptomeninges-derived neurospheres. In this regard, the presence of IL-6 receptors enabled cells to sense and react to IL-6 stimulation. 3.In above system, IL-6 also induced the phosphorylation of ERK1/2 and STAT3 molecules in the cells. Then we administrated pharmacological inhibitors individually before IL-6 incubation, as PD98059 and AG490. It was revealed that PD98059 pre-treatment decreased the generation of neurons and glial cells, while AG490 administration only down-regulated the GFAP level. In addition, the phosphorylation of STAT3 molecules also indicated the activation of gp130.These data suggest that IL-6 could promote the differentiation of leptomeninges-derived neural stem cell into neurons, which were mainly serotonergic immature neurons. In addition, IL-6 administration increases the expression of its own receptors and the phosphorylation of signal molecules, ERK1/2 and STAT3, in these cells. Thus, these molecules may cooperatively involve in the differentiation of leptomeninges-derived neurospheres induced by IL-6 stimulation.Part Four1 . After incubated the leptomeninges-derived neurospheres with citalopram, one kind of anti-depression drugs, we detected that citalopram not only supported the clonal morphology and the proliferation of cells, but also induced the phosphorylation of ERK1/2 and STAT3 signal molecules. Moreover, using pharmacological inhibitors of these two molecules further indicated an ERK1/2-dependent effect of citalopram on the cellular proliferation.2.On the other hand, we also detected an effect of citalopram on the differentiation potentials of leptomeninges-derived neural stem cells. It was showed that when the neurospheres had attached to the coverslip and tended to differentiate, citalopram administration could not only significantly promote their differentiation processes, but also improve the neurogenesis. Similarly, it induced the phosphorylation of ERK1/2 and STAT3 in these cells, while the gliogenesis and neurogenesis exhibited these two pathways related manner respectively.These data suggest that citalopram could promote the proliferation and neurogenesis of leptomeninges-derived neural stem cells. But the regulation mechanisms are quite different. In other word, the cellular proliferation involves ERK1/2 pathways only, while the differentiation is dependent on both pathways.Part Five1.We established a leptomeningeal cells'mechanical injury model and examined the capacity of these cells to proliferate or de-differentiate. It was revealed that, though the injury promoted the proliferation of cells, the expression of nestin couldn't be detected simultaneously in these cells. However, in this injury model, cells were found to be able to generate neurospheres in the presence of growth factors containing serum-free medium, and differentiate into neuronal or glial lineage cells when re-incubated with serum.2.To explore the effects of injured leptomeningeal cells on the normal astrocytes, we co-cultured these two cells to detect the latter cells'capacity of proliferation and de-differentiation. It was showed that normal astrocytes could proliferate and express nestin protein in the presence of injured leptomeningeal cells. In addition, astrocytes in this system could also develop into neurospheres and then generate neurons or glial cells when cultured in serum-free or serum containing medium respectively.These results indicate that though the mechanical injury couldn't induce the leptomeningeal cells to de-differentiate into neural progenitors directly, it increases the production of certain diffusible factors by the cells which then induced the de-differentiation and proliferation of astrocytes. Moreover, the environmental factors play key roles in directing cells to exhibit neural stem cell-like potentials.ConclusionIt can be verified by the present study that the mesenchyma-derived leptomeningeal cells possess the essential characteristics of neural stem cells. They can self-renew and proliferate, and then differentiate mutipotentially into neuronal and glial lineage cells under certain circumstances. In addition, different kinds of stimuli, such as cytokines, anti-depression drugs and mechanical injury, can involve in their proliferation or differentiation processes. Also, injury may lead the leptomeningeal cells to secret some factors to induce the activation and proliferation of neural stem cells in situ in CNS. In a word, it is helpful for us to establish a comprehensive and in-depth understanding of leptomeningeal cells'functions, which will well ground their clinical application.