ABSTRACT
First messengers and viral transfection are the two most common ways to stimulate cells for signal output, although their applications are limited. We investigated mechanisms of inducing neural stem cell differentiation using recombinant ephrin-B2/Fc and found that it acted as a ligand and inhibited endogenous ephrin-B2, which maintenance of the neural progenitor cell state, by direct interference. Our results showed the movement of ephrin-B2/Fc within the cell and indicated that it recycled to the plasma membrane surface, revealing a possible pattern of ephrin trafficking. Our results also serve as proof of concept for the reconstruction of the intracellular domain of ephrin using an artificial receptor to direct input signals in future studies.
Subject(s)
Ephrin-B2/physiology , Signal Transduction/physiology , Stem Cells/physiology , Animals , Cells, Cultured , Endocytosis/drug effects , Ephrin-B2/immunology , Immunoglobulin Fc Fragments/pharmacology , Neurons/cytology , Neurons/metabolism , RatsABSTRACT
Objective To investigate the effect of extracellular signal-regulated kinases(ERK) signal transduction in the process of NSCs differentiating into neurons in the fimbria-transected hippocampi's extracts. Methods Twelve Sprague-Dawley rats'right fimbrias were transected. The extracts were gained from the fimbria-transected hippocampi at the 14th day normal rat, and the extracts supernatant fluid was collected after centrifugal process, then the protein concentration in the extracts was determined. In the serum-free medium,NSCs from the fetal hippocampus were planted on 24 well culture plate, then were divided into three group and eight wells for each group as follows: the transected group contained the extracts of the fimbria-transected hippocampi;the normal group contained the extracts of the normal hippocampi;the pure control group have no extracts. After cultured for 14 days,the cells were detected by using MAP-2 and p-ERK immunofluorescence. Result The number, area, perimeter of MAP-2 positive neurons were all declined in transected group, the normal group and the control group orderly. Statistic results showed significant difference between every two groups. The number of MAP-2/p-ERK double-positive neurons were decreased in transected group, the normal group and the control group orderly, but the percentage of double-labeled neurons in total MAP-2 positive neurons were increased in turn. In these two aspect, there were also significant difference between every two group. And most of the MAP-2/p-ERK double-positive neurons were immature. Conclusion The extracts of the fimbria-transected hippocampi had obvious effects on promoting NSCs differentiating into neurons and speeding up the maturation of neurons than those of the normal hippocampi. The morphological results showed that ERK signal transduction might be related to the differentiation of NSCs into neurons.
ABSTRACT
Objective To evaluate the effects of IL-1? and combination of IL-1?,IL-11,LIF and GDNF on inducing human neural stem cells(hNSCs)to differentiate into dopaminergic(DA)neurons in vitro. Methods A great deal of neurospheres was obtained by the technology of serum-free culturing and single-cell cloning,and was splitted into 3 groups,which were cultured in different media.In control group,the differentiation medium used only contained 10%FBS.In IL-1? group,the medium contained IL-1? and 10%FBS.In united factors group,the medium contained IL-1?,10%FBS supplemental with IL-11,LIF and GDNF.After 3 weeks,the tyrosine hydroxylase(TH)positive cells were detected by using immunofluorescence,and image processing about the number,the induced differentiational rate,the cell bodies' areas and the cells' perimeters of TH positive neurons was carried out.TH/MAP-2 double-immunofluorescence labeling was used to calculate the percentage of DA neurons in total neurons. Results In the control group,there were few TH positive neurons with poorly developed morphology.The presence of IL-1? induced more DA neurons,but these cells were still immature.In the united factors group,the number of maturer TH positive DA neurons was the most. Conclusion IL-1? has an obvious effect on inducing hNSCs derived from human fetal mescenphalon to DA neurons.The utilization of IL-1?,IL-11,LIF and GDNF in combination has a cooperative effect on inducing differentiation of hNSCs to mature DA neurons.