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Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

ABSTRACT: Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito), at holding potential of -80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.

SUBMITTER: Subbarao RB

PROVIDER: S-EPMC4463684 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

Subbarao Raghavendra Baregundi RB Ullah Imran I Kim Eun-Jin EJ Jang Si-Jung SJ Lee Won-Jae WJ Jeon Ryoung Hoon RH Kang Dawon D Lee Sung-Lim SL Park Bong-Wook BW Rho Gyu-Jin GJ

International journal of molecular sciences 20150514 5

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipo ...[more]

PMID: 26006231

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Project description:ObjectiveTo investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.MethodsRat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.ResultsMSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.ConclusionmiR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.

Dataset Information

Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

Publications

Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

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