Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

Cell cycle exit and neuronal differentiation 1-engineered embryonic neural stem cells enhance neuronal differentiation and neurobehavioral recovery after experimental traumatic brain injury

Our previous study showed that cell cycle exit and neuronal differentiation 1(CEND1)may participate in neural stem cell cycle exit and oriented differentiation.However,whether CEND1-transfected neural stem cells can improve the prognosis of traumatic brain injury remained unclear.In this study,we performed quantitative proteomic analysis and found that after traumatic brain injury,CEND1 expression was downregulated in mouse brain tissue.Three days after traumatic brain injury,we transplanted CEND1-transfected neural stem cells into the area surrounding the injury site.We found that at 5 weeks after traumatic brain injury,transplantation of CEND1-transfected neural stem cells markedly alleviated brain atrophy and greatly improved neurological function.In vivo and in vitro results indicate that CEND1 overexpression inhibited the proliferation of neural stem cells,but significantly promoted their neuronal differentiation.Additionally,CEND1 overexpression reduced protein levels of Notch1 and cyclin D1,but increased levels of p21 in CEND1-transfected neural stem cells.Treatment with CEND1-transfected neural stem cells was superior to similar treatment without CEND1 transfection.These findings suggest that transplantation of CEND1-transfected neural stem cells is a promising cell therapy for traumatic brain injury.This study was approved by the Animal Ethics Committee of the School of Biomedical Engineering of Shanghai Jiao Tong University,China(approval No.2016034)on November 25,2016.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells

Background The production of neural stem cells （NSCs） derived from embryonic stem （ES） cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.Methods Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells＇ morphology and formation of teratoma in severe combined immunodeficiency disease （SCID） mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 （Oct-4） and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription- polymerase chain reaction （RT-PCR） method to monitor the process of cell differentiation.Results The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2±3.5 neurospheres per plate formed in astrocyte-induced group and only 0. 8±0.3 per plate in the control group （ elonogenic assay, P 〈 0.01 ）, and the ratio of nestin positive cells was （50. 2±2. 8） % in astrocyte-induced group and only （ 1.4±0. 5） % in the control group （flow cytometry, P 〈0. 01 ）. With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was （42. 7±2. 6） % in astroeyte-indueed group and only （ 11.2 ±1.8 ） % in the control group （P〈0.01）.Conclusions Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

Assessment of Bisphenol A(BPA) neurotoxicity in vitro with mouse embryonic stem cells

The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell（ESC） properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A（BPA）, a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.