Proliferation and committed differentiation into dopamine neurons of neural stem cells induced by the active ingredients of radix astragali
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Abstract:
Neural stem cells (NSCs) are important cellular sources of transplantation therapies for Parkinson's disease. This study aimed to determine the effects of extracts of radix astragali on the proliferation and differentiation into dopamine (DA) neurons in NSCs. NSCs were dealt with astragaloside IV (ASI), astragalus polysaccharide (APS), and astraisoflavan (ASF), the main active ingredients of radix astragali. First, the results from cell-count kit-8 (CCK-8) assay showed that ASI, ASF, and APS had positive effects on the proliferation of NSCs. Next, we also confirmed the effects of ASI, APS, and ASF on BrdU and nestin by immunocytochemistry. Moreover, results from quantitative RT-PCR showed ASI, APS, and ASF could promote the expressions of tyrosine hydroxylase and dopamine transporter mRNA, which are specifically expressed in DA neurons. Simultaneously, sonic hedgehog (Shh), orphan nuclear hormone 1 (Nurr1), and pituitary homeobox 3 (Ptx3) are considered to motivate the formation of DA neurons. Our result showed ASI, APS, and ASF can also promote the expressions of Shh, Nurr1, and Ptx3 mRNAs. In conclusion, our study verifies that the active ingredients of radix astragali can promote the proliferation of NSCs and induce NSC differentiation toward DA neurons in vitro. These phenomena may occur through upregulation of Shh, Nurr1, and Ptx3 in the process of drug treatment.Keywords:
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Objective
To explore the possibility of isolating and culturing in vitro enteric neural stem cells from gut in children with Hirschsprung's disease (HD) and provide experimental rationales for treating HD with neural stem cell transplantation.
Methods
Human gut samples were obtained from one child aged 11 months after pull-through surgery due to HD. After enzyme digestion, single cell suspensions were isolated from postnatal human myenteric plexus. The cells were seeded into serum-free medium and generated neurosphere-like bodies in vitro. Cell proliferation was observed by the curve of optic density (OD) from CCK8 test at the timepoints of 24, 48, 72, 96, 120 and 144h and growth curve diagram plotted. And neurospheres and differentiated cells with immunofluorescent staining of nestin were identified by neuron-specific beta III tubulin (TUJ-1) and glial fibrillary acidic protein (GFAP).
Results
Single cells from colonic muscle formed neurospheres after culturing for 10 days and these neurospheres could be subcultured and maintained for several weeks in vitro. The OD values of CCK8 test increased progressively. Existing neurospheres, identified as nestin positive cells, could be passaged and differentiated into glial cells (GFAP positive) and neurons (TUJ-1 positive).
Conclusions
Enteric neural stem cells may be obtained from human postnatal gut in patients with HD. And it is a potential source of autologous neural stem cells for stem cell transplantation therapy.
Key words:
Hirschsprung's disease; Enteric neural stem cells; Stem cell transplantation
Neurosphere
Nestin
Enteric Nervous System
Stem cell marker
Amniotic epithelial cells
Amniotic stem cells
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Objective: To isolation ,culture and identify the neural stem cells from the hippocampus of the newborn rats. Methods: The neural stem cells were isolated from the hippocampus of the newborn rats and cultured by using serum-free medium containing bFGF and EGF without the substrate. After the neural stem cells gave rise to neuropheres,they were mechanically dissociated to single cells,and after the single cells gave rise to neuropheres again which were identified by immunocytochemistry. Result: Neural stem cells isolated from the hippocampus of the newborn rats could be maintained and propagated in the present of EGF and bFGF .The cells in the neuropheres were identified as neural stem cells by immunocytochemistry,because they expressed nestin which is a specific marker of neural stem cells. Conclusion: The culture method of neural stem cells from the hippocampus of the newborn rats was successfully founded.
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Neurosphere
Neuroepithelial cell
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Stem cells have been the subject of increasing scientific interest because of their utility in numerous biomedical applications. Stem cells are capable of renewing themselves; that is, they can be continuously cultured in an undifferentiated state, giving rise to more specialized cells of the human body. Therefore, stem cells are an important new tools for developing unique, in vitro model systems to test drugs and chemicals and a potential to predict or anticipate toxicity in humans. In the present study, in vitro cultured F3 immortalized human neural stem cell line and in vivo adult Sprague Dawley rats was used to evaluate the cytotoxicity of anticancer drug paclitaxel. In vitro apoptotic activity of paclitaxel was evaluated in F3 cell line by a MTT assay and DAPI test. The cell death was induced with the treatment of 20 nM paclitaxel and chromatin degradation was detected by DAPI staining, which was analyzed by fluorescent microscope. In vivo studies, we also observed nestin immunoreactivity on subventricular zone, which is stem cell rich region in the adult brain of the SD rat. Immunofluorescent staining result shows that pixel intensities of nestin were decreased in a dose dependent manner. These results suggest that paclitaxel is able to induce cytotoxic activity both in F3 neural stem cell line and neural stem cell in SD rat brain.
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DAPI
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Objective To evaluate the effects of bFGF,LIF,BDNF,and the differences from their groupings on neuronal differentiation of neural stem cells(NSCs)from CNS of adult rats in vitro.Methods The serum-free medium containing bFGF,B27 was used to culture NSCs isolated from CNS of adult rats.Immunocytochemistry for Nestin was used to identify monoclonal cells.The passaged monoclonal NSCs were divided into five groups:bFGF,LIF,BDNF,bFGF+LIF and bFGF+BDNF group.Immunocytochemistry for NSE was performed a week later to count the positive cells.Results The cultured monoclonalcells expressed nestin.The percentage of NSE positive cells differentiated from NSCs in bFGF+LIF and EGF+BDNF groups were much higher than those in other groups with the highest in bFGF+BDNF group.Conclusion The ability of BDNF on promoting differentiation of NSCs from CNS of adult rats into neurons is higher than LIF in the media containing bFGF.
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Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc(+)/SV40Tag(+)/Tet-on(+)) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.
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Neurosphere
Neuroepithelial cell
Brain tumor
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Objective:To investigate the proliferation of neural stem cells in the rat model of Parkinsons disease .Methods:Rat model of Parkinsons disease was made by injecting the 6-hydroxydopaminin in the striatum.The rats were killed at different time points,and their brain were taken out.Before they were killed ,the rats were injected Brdu into their abdomen.The dynamic expression of Nestin and Brdu were determined by immunocytochemistry,Brdu labeling method was used to mark the cells of proliferation .Nestin expression was used to identify neuroprogenitor cells.And they both used to mark the diving neural stem cells.Rsults:Campared with the controls,the number of Nestin-positive cells ,Brdu-positive cells,and Brdu-Nestin-positive cells increased strikingly at 3,5,7days,in the hippocampus, then declined at 14 days and almost reached the normal 28 days after.(Conclusion:)The rat model of Parkinsons disease by direct injection of 6-OHDA in the striatum stimulates the proliferation of inherent neural stem cells.
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Bromodeoxyuridine
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Nestin
Neurosphere
Neural cell
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Background and Objectives：Tympanic membrane perforation is an important clinical problem found in various populations of the world. In large number of cases, acute traumatic perforations heal spontaneously, and in the healing process, stem cels appear to play an important role. However, no studies have ben reported regarding somatic stem cells in the tympanic membrane. Herein, we tried to show that guinea pigs tympanic membrane contains cells that display the characteristic features of stem cels. Materials and Method：The tympanic membrane was obtained from the guinea pig. The cels were cultured in a medium with epidermal growth factor (EGF) and fibroblast growth factor (FGF). Proliferating cells were checked with stem cell markers, bromodeoxyu-ridine (BrdU) and nestin. Diferentiated cells from stem cells are checked with βI tubulin and S-100. Results：We observed that some of the cultured cells from the tympanic membrane were stained with both stem cell markers, BrdU and nestin. And we obser-ved that these cells diferentiated into neuron and gilal cells, which expresed βIII tubulin and S-100, respectively. Conclusion：These results suggest that the tympanic membrane of guinea pigs may have neural stem cels. Further study is needed for finding the origin of stem cells. (Korean J Otorhinolaryngol-Head Neck Surg 2008 ;51 :
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Amniotic stem cells
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The cholinergic-inducing effect of BMP4 on isolated and cultivated rat′s cerebral neural stem cells(NSC) was examined. NSC isolated from two months old rat′s brain region like hippocampus and striatum was cultivated in a DMEM/F12 medium containing EGF and bFGF, and was identified with morphological character and nestin immunocytochemistry test.After 24 hours, cultivating the NSC with the BMP4-added medium for 7-8 days, then the microscopical change were observated, ChAT and nestin double-labelling immunocytochemistry test was done. Results showed that about 34% NSC of neuron-like character was observed by microscope in the paper.That ChAT-positive cells coexist with nestin-positive cells was found by immunocytochemistry test.There were 28% ChAT-positive cells and 38% nestin-positive cells in the study.Cholinergic neurons differentiated from NSC could be induced by adding BMP4 to the medium.
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Neurosphere
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[Objective] To isolate and identify neural stem cells derived from hippocampus of neonatal rats. [Methods] The cells obtained from the hippocampus of neonatal rats were incubated in serum-free DMEM/F12 media added with 20ng/ml b-FGF, B27. Some of them were suspension cultured in a 24-well-plate. On day 7 after incubation, some of the cells were fixed and subjected to immunocytochemistry to detect the nestin antigen for the cell clone. The other cells were incubated in DMEM/F12 media added with 10% bovine serum. On day 7 after incubation the cells were fixed and stained by using immunocytochemistry to detect the specific antigen of neuron (Tuj1), astrocyte(GFAP), and oligodendrocyte(Galc). [Result] The cells derived from the hippocampus of neonatal rats expressed nestin antigen and have the potential to form clones and differentiate into neurons, astrocytes and oligodendrocytes. [Conclusion] Multipotent cell line which expressed nestin antigen has been isolated and identified. These cells showed great ability to self-renewal, supporting their character of neural stem cells.
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Neuroepithelial cell
Neurosphere
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