A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells

2015 ◽  
Vol 47 ◽  
pp. 47-53 ◽  
Author(s):  
Claudia Ceci ◽  
Maria Luisa Barbaccia ◽  
Giuseppa Pistritto
Keyword(s):  
Neuronal Differentiation ◽  
Nickel Concentration ◽  
Differentiation Markers ◽  
Nt2 Cells

scholarly journals Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 609
Author(s):  
Nunzia Mollo ◽  
Matteo Esposito ◽  
Miriam Aurilia ◽  
Roberta Scognamiglio ◽  
Rossella Accarino ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Mitochondrial Dysfunction ◽  
Neuronal Differentiation ◽  
Expression Profiles ◽  
Meta Analysis ◽  
Atp Synthesis ◽  
Differentiation Markers ◽  
Differentiation Potential ◽  
Atp Production ◽  
Mitochondrial Alterations

Background: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction. Methods: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer. Results: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. Conclusions: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells

1990 ◽  
Vol 10 (1) ◽  
pp. 361-370
Author(s):  
C Bjelfman ◽  
G Meyerson ◽  
C A Cartwright ◽  
K Mellström ◽  
U Hammerling ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Kinase Activity ◽  
Phorbol Esters ◽  
Neuroblastoma Cells ◽  
Functional Differentiation ◽  
Differentiation Markers ◽  
Human Neuroblastoma ◽  
Fourfold Increase ◽  
Phosphopeptide Mapping

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

scholarly journals Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Thunwa Binlateh ◽  
Supita Tanasawet ◽  
Onnicha Rattanaporn ◽  
Wanida Sukketsiri ◽  
Pilaiwanwadee Hutamekalin
Keyword(s):  
Cell Differentiation ◽  
Neuronal Differentiation ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cells ◽  
Metformin Treatment ◽  
Neurite Length ◽  
Differentiation Markers ◽  
Morphological Criteria ◽  
Phosphorylated Akt ◽  
Ros Scavenger

Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, β-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.

scholarly journals Otic Neurogenesis in Xenopus laevis: Proliferation, Differentiation, and the Role of Eya1

2021 ◽  
Vol 15 ◽  
Author(s):  
Suad Hamdan Almasoudi ◽  
Gerhard Schlosser
Keyword(s):  
Xenopus Laevis ◽  
Neuronal Differentiation ◽  
Underlying Mechanism ◽  
Otic Vesicle ◽  
Transcriptional Coactivator ◽  
Differentiation Markers ◽  
Apicobasal Polarity ◽  
Ganglion Neurons ◽  
Peripheral Cells

Using immunostaining and confocal microscopy, we here provide the first detailed description of otic neurogenesis in Xenopus laevis. We show that the otic vesicle comprises a pseudostratified epithelium with apicobasal polarity (apical enrichment of Par3, aPKC, phosphorylated Myosin light chain, N-cadherin) and interkinetic nuclear migration (apical localization of mitotic, pH3-positive cells). A Sox3-immunopositive neurosensory area in the ventromedial otic vesicle gives rise to neuroblasts, which delaminate through breaches in the basal lamina between stages 26/27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (as judged by EdU incorporation), while central cells differentiate into Islet1/2-immunopositive neurons from stage 29 on and send out neurites at stage 31. The central part of the neurosensory area retains Sox3 but stops proliferating from stage 33, forming the first sensory areas (utricular/saccular maculae). The phosphatase and transcriptional coactivator Eya1 has previously been shown to play a central role for otic neurogenesis but the underlying mechanism is poorly understood. Using an antibody specifically raised against Xenopus Eya1, we characterize the subcellular localization of Eya1 proteins, their levels of expression as well as their distribution in relation to progenitor and neuronal differentiation markers during otic neurogenesis. We show that Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) vestibulocochlear ganglion neurons and in the developing sensory areas. Morpholino-based knockdown of Eya1 leads to reduction of proliferating, Sox3- and Islet1/2-immunopositive cells, redistribution of cell polarity proteins and loss of N-cadherin suggesting that Eya1 is required for maintenance of epithelial cells with apicobasal polarity, progenitor proliferation and neuronal differentiation during otic neurogenesis.

scholarly journals Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures

2021 ◽  
Vol 333 ◽  
pp. 07008
Author(s):  
Otome Kitano ◽  
Kohji Nakazawa
Keyword(s):  
Cell Proliferation ◽  
Neuronal Differentiation ◽  
Monolayer Culture ◽  
Cultured Cells ◽  
Spheroid Formation ◽  
Promising Technique ◽  
Retinoic Acid Treatment ◽  
Spheroid Culture ◽  
Proliferation Ability ◽  
Nt2 Cells

Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.

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