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RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells.

ABSTRACT: Stem cell differentiation toward a specific lineage is controlled by its microenvironment. Polymer scaffolds have long been investigated to provide microenvironment cues; however, synthetic polymers lack the specific signaling motifs necessary to direct cellular responses on their own. In this study, we fabricated random and aligned poly(?-caprolactone) nanofiber substrates, surface-functionalized with RGD viastrain-promoted azide-alkyne cycloaddition, that were used to investigate the role of a covalently tethered bioactive peptide (RGD) and nanofiber orientation on neural differentiation of mouse embryonic stem cells. Gene and protein expression showed neural differentiation progression over 14 days, with similar expression on RGD random and aligned nanofibers for neurons and glia over time. The high levels of glial fibrillary acidic protein expression at early time points were indicative of neural progenitors, and occurred earlier than on controls or in previous reports. These results highlight the influence of RGD binding versus topography in differentiation.

SUBMITTER: Philip DL

PROVIDER: S-EPMC6650284 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells.

Philip Diana L DL Silantyeva Elena A EA Becker Matthew L ML Willits Rebecca K RK

Biomacromolecules 20190218 3

Stem cell differentiation toward a specific lineage is controlled by its microenvironment. Polymer scaffolds have long been investigated to provide microenvironment cues; however, synthetic polymers lack the specific signaling motifs necessary to direct cellular responses on their own. In this study, we fabricated random and aligned poly(ε-caprolactone) nanofiber substrates, surface-functionalized with RGD viastrain-promoted azide-alkyne cycloaddition, that were used to investigate the role of a ...[more]

PMID: 30726667

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Project description:IntroductionNpas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis.MethodsTwo different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation.ResultsWe found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation affected their ability to differentiate appropriately, resulting in delayed neural differentiation.ConclusionsHere we provide the first evidence that Npas4 is expressed during embryonic development and that it may have a developmental role that is unrelated to its function in the adult brain.

Dataset Information

RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells.

Publications

RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells.

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