Regulatory effects of ADT-OH on the proliferation of neural precursor cells / 中国组织工程研究

BACKGROUND: Researchers believe that hydrogen sulfide (H2S), as an important cell protective molecule, may become a new treatment method to restore the physiological function of diseased cells or organ systems through the artificial regulation of endogenous H2S biosynthesis or in vitro administration of H2S donor. ADT-OH is a slow-release donor of H2S that can improve the survival rate of hippocampal nerve cells with glutamate-induced injury, but studies on the proliferation of cerebral cortical neural precursor cells are rare. OBJECTIVE: To investigate the effect of ADT-OH on the proliferation of neural precursor cells in embryonic cerebral cortex. METHODS: Neural precursor cells from cerebral cortical ventricular zone and subventricular zone of embryonic mice at embryonic 14.5 days were isolated. Neural precursor cells from one fetal mouse were inoculated into one well (24-well plate), and cultured with the medium containing 100 μmol/L ADT-OH. The size and number of neural spheres per well were measured at 3 days after culture. The proliferation rate of cultured neural precursor cells was detected by BrdU labeling. The proliferation of the cells was further verified by immunofluorescence staining with the specific antibody Ki67. The expression of cyclin D1 was finally detected by western blot assay. RESULTS AND CONCLUSION: Our experimental results showed that ADT-OH could promote the formation of neural spheres, and further detection by BrdU and Ki67 antibody showed that ADT-OH could promote the proliferation rate of neural precursor cells. Meanwhile, the expression of cyclin D1, a proliferation-related gene, was up-regulated in neural precursor cells after ADT-OH treatment. Overall, ADT-OH may promote the proliferation of neural precursor cells by regulating the expression of cyclin D1.

Generation of Retinal Pigmented Epithelium-Like Cells from Pigmented Spheres Differentiated from Bone Marrow Stromal Cell-Derived Neurospheres

BACKGROUND: Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. METHODS: BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65 kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. RESULTS: The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7–14 days were positive for RPE65 (92.76–100%), CRALBP (95.21–100%) and OTX2 (94.88–100%), and after 30 days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. CONCLUSION: Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performancemanner over a short period of time. These cells due to expressing theRPELCgenes andmarkers can be used in cell replacement therapy for degenerative diseases including age-relatedmacular degeneration as well as retinitis pigmentosa.

Effect of oxygen glucose deprivation-reperfusion in astrocytes overexpressing endothelin-1 on the proliferation of neural stem/progenitor cells / 器官移植

Objective To investigate the effect of oxygen glucose deprivation-reperfusion (OGD-R) in astrocytes overexpressing endothelin (ET)-1 on the proliferation of neural stem/progenitor cells (NSPCs). Methods OGD-R models of negative control astrocytes (C6-Mock) and astrocytes over-expressing ET-1 (C6-ET-1) were constructed. Transwell co-culture system of astrocytes and NSPCs was established. Morphologic observation and identification of the astrocytes and primary NSPCs were performed. The cells were divided into four groups: C6-Mock+NSPCs, OGD-R+C6-Mock+NSPCs, C6-ET-1+NSPCs and OGD-R+C6-ET-1+NSPCs groups and co-cultured for 0, 24, 48 and 72 h respectively. The diameter of neurosphere was measured in each group. Results In the C6-Mock and C6-ET-1 cells, type Ⅰ astrocytes in fibrous morphology were observed. Glial fibrillary acidic protein (GFAP) was expressed in the cytoplasm of these two types of cells. Primary NSPCs were positive for nestin staining. After co-culture for 48 and 72 h, the neurosphere diameter in the OGD-R+C6-Mock+NSPCs group was significantly greater than that in the C6-Mock+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was considerably greater than that in the C6-ET-1+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was significantly greater compared with that in the OGD-R+C6-Mock+NSPCs group (all P<0.05). Conclusions OGD-R astrocytes can promote the proliferation of NSPCs. ET-1 over-expression further accelerates the proliferation of NSPCs.

Induce and differentiation of pig Müller cells into photoreceptors in vitro / 中华实验眼科杂志

Background Recent studies indicated that rat and mouse Müller cells can be induced and differentiated into photoreceptor-like cells in vitro, but it is not known whether this also happens to adult pig Müller cells nowadays.Objective This study was to test whether adult pig Müller cells can be differentiated to the retinal photoreceptors (the primary transmission neurons of the retina) in vitro.Methods Müller cells were isolated from the neural retina of adult pig eyes and cultured and passaged.The 3rd and 4th generation of cells were themonolayerly cultured,and the cells forced to form spheres in suspension in altra-low adherent dishes for 2-3 days first and then reseeded in normal adherent plates,and both of them were cultured in a specifically formulated medium to induce the differentiation of retinal photoreceptor.The cells was verified by immunocytochemistry.Cell morphology and immunofluorescence staining were utilized to measure the efficacy of the differentiation.Results The 2nd,3rd and 4th generation of Müller cells expressed glutamate synthetase (GS) , a specific maker of Müller cells.Inaddition, the 3rd generation of cells also expressed glial fibrillary acidic protein (GFAP) and another specific maker of Müller cells.Three visual fields under fluorescence-microscope were randomly chosen to calculate the average positive ratio of rhodopsin,a specific marker of mature photoreceptors.The photoreceptor differentiation ratios of the 2nd generation of cells for monolayer culture only and with additional sphere suspension culture were (27.99±6.53 (％ and (16.54±3.40) ％ , respectively.With passages, the number of rhodopsin positive cells gradually decreased, and the intensity of rhodopsin expression gradually weakened.The directed rhodopsin positive ratios of the 2nd,3rd and 4th generation of cells from sphere formation were (56.23±7.32)％ , (36.26 ±8.55)％ and (12.68 ±3.18)％ , respectively.Although the rhodopsin expression was weakened over passages,the differentiated cells were more slender and elongated.There was no statistic ally significant difference between different groups (F =2.618, P =0.099).Conclusions Adult pig Müller cells can be differentiated into retinal photoreceptors in vitro.The morphology of the differentiated cells appears moreslender and elongates if the sphere-induced differentiation method is used and/or the directed differentiation time is further extended.

Elimination of the geomagnetic field stimulates the proliferation of mouse neural progenitor and stem cells

Living organisms are exposed to the geomagnetic field (GMF) throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field (HMF), leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mechanisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF (<200 nT), produced by a magnetic field shielding chamber, promotes the proliferation of neural progenitor/stem cells (NPCs/NSCs) from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres (NSs) were significantly larger in size, and twice more NPCs/NSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multipotency of the NSs were maintained, as HMF-exposed NSs were positive for NSC markers (Nestin and Sox2), and could differentiate into neurons and astrocyte/glial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells in the subventricular zone. These findings indicate that continuous HMF-exposure increases the proliferation of NPCs/NSCs, in vitro and in vivo. HMF-disturbed NPCs/NSCs production probably affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the bio-HMF response.

Amniotic fluid exerts a neurotrophic influence on fetal neurodevelopment via the ERK/GSK-3 pathway

BACKGROUND: The fetus is surrounded by the amniotic fluid (AF) contained by the amniotic sac of the pregnant female. The AF is directly conveyed to the fetus during pregnancy. Although AF has recently been reported as an untapped resource containing various substances, it remains unclear whether the AF could influence fetal neurodevelopment. RESULTS: We used AF that was extracted from embryos at 16 days in pregnant SD rat and exposed the AF to the neural cells derived from the embryos of same rat. We found that the treatment of AF to cortical neurons increased the phosphorylation in ERK1/2 that is necessary for fetal neurodevelopment, which was inhibited by the treatment of MEK inhibitors. Moreover, we found the subsequent inhibition of glycogen synthase kinase-3 (GSK-3), which is an important determinant of cell fate in neural cells. Indeed, AF increased the neural clustering of cortical neurons, which revealed that the clustered cells were proliferating neural progenitor cells. Accordingly, we confirmed the ability of AF to increase the neural progenitor cells through neurosphere formation. Furthermore, we showed that the ERK/GSK-3 pathway was involved in AF-mediated neurosphere enlargement. CONCLUSIONS: Although the placenta mainly supplies oxygenated blood, nutrient substances for fetal development, these findings further suggest that circulating-AF into the fetus could affect fetal neurodevelopment via MAP kinases-derived GSK-3 pathway during pregnancy. Moreover, we suggest that AF could be utilized as a valuable resource in the field of regenerative medicine.

The effects of repetitive transcranial magnetic stimulation on proliferation and differentiation of neural stem cells / 대한해부학회지

Repetitive transcranial magnetic stimulation (rTMS) is a new method for treating many neurological conditions; however, the exact therapeutic mechanisms behind rTMS-induced plasticity are still unknown. Neural stem and progenitor cells (NS/PCs) are active players in brain regeneration and plasticity but their behavior in the context of rTMS therapy needs further elucidation. We aimed to evaluate the effects of rTMS on proliferation and differentiation of NS/PCs in the subventricular zone (SVZ) of adult mouse brain. Adult male mice (n=30) were divided into rTMS (1-Hz and 30-Hz) and sham groups and treated for 7 or 14 consecutive days. Harvested NS/PCs from the SVZ were cultured in the neurosphere assay for 8 days and the number and size of the resulting neurospheres as well as their in vitro differentiation capacity were evaluated. After one week of rTMS treatment at 1-Hz and 30-Hz compared with sham stimulation, the mean neurosphere forming frequency per brain was not different while this measure significantly increased after two weeks (P<0.05). The mean neurosphere diameter in 1-Hz treatment paradigm was significantly larger compared with sham stimulation at both 1 and 2 weeks. In contrast, 30-Hz treatment paradigm resulted in significantly larger neurospheres only after 2 weeks. Importantly, rTMS treatment at both frequencies increased neuronal differentiation of the harvested NS/PCs. Furthermore, one week in vitro rTMS treatment of NS/PCs with both 1-Hz and 30-Hz increased NS/PCs proliferation and neuronal differentiation. It is concluded that both 1-Hz and 30-Hz rTMS treatment increase NS/PCs proliferation and neuronal differentiation.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines / 대한해부학회지

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Effect of Schisandra lignans on the apoptosis of SHG-44 glioma neurospheres / 中国药学杂志

OBJECTIVE: To explore the mechanism of Schisandra lignans for inducing the apoptosis of human glioma SHG-44 neurospheres. METHODS: The effects of Schisandra lignans on the SHG-44 neurospheres proliferation were detected by MTT. The apoptotic rate was analyzed by Annexin-V/PI double staining. The secretion of the bax, bcl-2 and caspase 3 protein was detected by Elisa assay. The expression of bcl-2 protein was examined by Western blot. RESULTS: Schisandra lignans could inhibit the proliferation of SHG-44 neurospheres with the concentration of 50,100,200 mg · L-1 in a dose-dependent manner. Schisandra lignans could induce the apoptosis of SHG-44 neurospheres in a dose-dependent manner. The secretion of bcl-2 was decreased, but ratio of bax/bcl-2 was increased, caspase 3 was increased, the expression of bcl-2 protein was down-regulated. CONCLUSION: Schisandra lignans could induce the apoptosis of human glioma SHG-44 neurospheres. Its mechanism is correlated with down-regulation of the expression of bcl-2 and up-regulation of the ratio of bax/bcl-2, and then activated caspase 3.

Isolamento e cultivo de neurônios e neuroesferas de córtex cerebral aviar / Isolation and culture of neurons and neurospheres from chicken brain cortex

Métodos de cultivo celular são convenientes na realização de análises funcionais de alterações/interações protéicas das células neuronais, auxiliando a decifrar o interactoma de proteínas chaves na neurogênese de doenças do Sistema Nervoso Central. Por esse motivo, culturas de neurônios e neuroesferas isolados do córtex cerebral aviar representam um modelo acessível para o estudo de diversas doenças neurológicas, tal como a epilepsia. A espécie aviar apresenta peculiaridades em seu proteoma neuronal, visto a presença de uma expressão diferenciada de proteínas chaves no metabolismo energético cerebral, algumas destas (VDAC1 e VDAC2) desempenham papel importante na compreensão do mecanismo da epilepsia refratária. A metodologia estabelecida no presente estudo obteve cultivo de neuroeferas, onde as células cresceram tipicamente em aglomerados atingindo, dentro de 7 dias, o diâmetro ideal de 100-200 µm. A diferenciação celular das neuroesferas foi obtida após a aderência destas às placas tratadas com poli-D-lisina, evidenciada pela migração de fibras do interior da neuroesfera. Ao contrário das neuroesferas, os neurônios em cultivo extenderam seus neuritos após 11 dias de isolamento. Tal modelo in vitro pode ser utilizado com sucesso na identificação das variáveis neuroproteômicas, propiciando uma avaliação global das alterações dinâmicas e suas interações protéicas. Tal modelo pode ter aplicações em estudos dos efeitos de indutores da morte celular e bloqueadores de canais de membrana mitocondriais em proteínas chaves do metabolismo energético cerebral.

Cell culture methods are used for studies of protein interactions in neural cells, helping to detect the interactome of proteins linked to generation of central nervous system diseases. For this reason, neural cells and neurospheres isolated from cortical chicken brain are a current model for studies of neurological diseases, such as epilepsy. Chicken brain has key characteristics on its proteome, with a differential expression of proteins linked to energy metabolism, some of them (VDAC 1 and VDAC 2) play an important role in understanding mechanism of refractory epilepsy. Using the methods described, we found neurospheres, in which cells grow in structures with the ideal diameter of 100-200µm within seven days after isolation. Neurospheres differentiation was obtained after adhesion of these cells to surfaces coated with poly-D-Lysine, detected by migration of fibers inside them. Unlike neurospheres, neurons extended neurites after 11 days of isolation. Here we describe a method to isolate and culture neurons and neurospheres from chicken cerebral cortex. Such "in vitro" model can be utilized on studies of neuronal protein differential expression and interaction. Cultures of isolated neurons represent an accessible model on studies of apoptosis and channel blockers of key proteins linked to brain metabolism.

Brain Tumor Stem Cells as Therapeutic Targets in Models of Glioma

At this time, brain tumor stem cells remain a controversial hypothesis while malignant brain tumors continue to present a dire prognosis of severe morbidity and mortality. Yet, brain tumor stem cells may represent an essential cellular target for glioma therapy as they are postulated to be the tumorigenic cells responsible for recurrence. Targeting oncogenic pathways that are essential to the survival and growth of brain tumor stem cells represents a promising area for developing therapeutics. However, due to the multiple oncogenic pathways involved in glioma, it is necessary to determine which pathways are the essential targets for therapy. Furthermore, research still needs to comprehend the morphogenic processes of cell populations involved in tumor formation. Here, we review research and discuss perspectives on models of glioma in order to delineate the current issues in defining brain tumor stem cells as therapeutic targets in models of glioma.

Effects of FGF-2 and EGF removal on the differentiationof mouse neural precursor cells

Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.

As terapias celulares para doenças neurológicas têm avançado e células precursoras neurais (NPC) surgem como candidatas ideais para o transplante de células neurais em muitas doenças. No entanto, trabalhos adicionais devem ser feitos para determinar o ambiente de cultivo ideal para a expansão in vitro das NPC, sem alterar sua plasticidade, e os mecanismos de sinalização celular do fator de crescimento epidérmico (EGF) e fator de crescimento de fibroblasto 2 (FGF-2) no crescimento, sobrevivência e diferenciação da neuroesfera. Nesse trabalho avaliamosNPCcultivadas na presença e na ausência de FGF-2 e EGF e mostramos que esses fatores de crescimento são responsáveis pela proliferação das NPC. Também foi demonstrado que a produção endógena de fatores de crescimento alterna de FGF-2 a fator de crescimento de insulina 1 (IGF-1) e fator de crescimento derivado de plaquetas b (PDGFb) após remoção de EGF e FGF-2. NPC de camundongo cultivadas em suspensão mostraram padrões de localização neuronal distintos (centro versus borda) tanto no grupo controle como no grupo sem EGF e FGF-2. Juntos, esses resultados mostram que a remoção de EGF e FGF-2 exerce importante ação na diferenciação de NPC e possivelmente contribui para melhor compreensão dos mecanismos envolvidos na diferenciação. Nossos achados sugerem que, privando as NPC de fatores de crescimento antes do transplante, talvez aumente as chances de que as células efetivamente se integrem ao hospedeiro.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signáis that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signáis involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of reléase from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Establishment of a Method for Cryopreservation of Neural Stem Cells / 대한해부학회지

Neural stem cells are multipotent stem cells that can differentiate into neurons and glial cells. Neural stem cells are found in not only developing nervous system but some restricted regions in adult brain. Here, we presented an effective method that allows a long-term preservation of neural stem cells without losing multipotency. First, we isolated neural stem cells from the developing forebrain of nestin-EGFP transgenic mice carrying green fluorescence protein (GFP) driven by nestin promoter and enhancer. Primary neurospheres isolated from these mice highly expressed GFP. The expression of GFP in neurospheres was sustained for several passages. In order to investigate the effect of freezing on the stem cell properties, we cryopreserved the primary neurospheres for 2 wks in liquid nitrogen. GFP expression pattern as well as differentiation potential of the secondary neurosphere formed after cryopreservation were not that different from those of the primary neurosphere formed before cryopreservation. When the same cryopreservation method was applied to neural stem cells isolated from human fetal brain (gestation 13 ~15 wks), the expression of nestin, a stem cell marker, and differentiation patterns were not changed after cryopreservation. We also performed isolation of neural stem cells from long-term cryopreserved human fetal brain tissues. The neurospheres were successfully formed and showed similar differention properties with neurospheres isolated from fresh brain tissue. In addition, we demonstrated multipotentiality of neural stem cells was not changed with the duration of cryopreservation of brain tissue, suggesting the self renewality and multipotentiality of neural stem cells were not affected by long-term cryopreservation, The present results provide an useful information for the development of stem cell expansion which is essential factor in clinical application of stem cells.

ULTRASTRUCTURE OF NEURAL STEM CELL NEUROSPHERE CULTURED in vitro / 解剖学报

Objective To study the ultrastructure of neural stem cell neurosphere cultured in vitro. Methods Neural stem cell neurospheres from the rat brain were observed under transmission electron microscope or with stain of lanthanum nitrate, ruthenium red and tannic acid. Results The conjunction between the cells within neurosphere was loose and no tight junction was observed. Neural stem cells were proliferating lively. Some neural stem cells differentiated into cells with process and structure of axon, even showed the structures similar to myelin.Conclusion The ultrastructure of neural stem cell neurosphere cultured in vitro was revealed.