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Neuron-Specific Fluorescence Reporter-Based Live Cell Tracing for Transdifferentiation of Mesenchymal Stem Cells into Neurons by Chemical Compound.

ABSTRACT: Although transdifferentiation of mesenchymal stem cells (MSCs) into neurons increases the possibility of therapeutic use of MSCs for neurodevelopmental disorders, the use of MSCs has the limitation on differentiation efficiency to neuronal lineage and lack of an easy method to monitor the transdifferentiation. In this study, using time-lapse live cell imaging, we assessed the neuronal differentiation of MSCs induced by a small molecule "NHPDQC (N-hydroxy-2-oxo-3-(3-phenylprophyl)-1,2-dihydroquinoxaline-6-carboxamide, C18H17N3O3)." Plasmid vector containing red fluorescence reporter genes under the control of the tubulin α1 (Tα1) promoter (pTα1-DsRed2) traced the neuronal differentiation of MSCs. Two days after NHPDQC treatment, MSCs showed neuron-like phenotype with neurite outgrowth and high expression of neuron-specific markers in more than 95% cells. The fluorescence signals increased in the cytoplasm of pTα1-DsRed2-transfected MSCs after NHPDQC treatment. In vitro monitoring of MSCs along the time courses showed progressive increase of fluorescence till 30 h after treatment, corresponding with the increase in neurite length. We examined an efficient neuronal differentiation of MSCs by NHPDQC alone and monitored the temporal changes of neuronal differentiation by neuron-specific fluorescence reporter along time. This method would help further our understanding of the differentiation of MSCs to produce neurons by simple treatment of small molecule.

SUBMITTER: Hwang DW

PROVIDER: S-EPMC5541830 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-?C4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8-6.1 in the early endosomes to 6.0-4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.

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Neuron-Specific Fluorescence Reporter-Based Live Cell Tracing for Transdifferentiation of Mesenchymal Stem Cells into Neurons by Chemical Compound.

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