Performance of the Polydopamine-Graphene Oxide Composite Substrate in the Osteogenic Differentiation of Mouse Embryonic Stem Cells

Graphene oxide (GO) is a biocompatible material considered a favorable stem cell culture substrate. In this study, GO was modified with polydopamine (PDA) to facilitate depositing GO onto a tissue culture polystyrene (PT) surface, and the osteogenic performance of the PDA/GO composite in pluripotent embryonic stem cells (ESCs) was investigated. The surface chemistry of the PDA/GO-coated PT surface was analyzed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A high cell viability of ESCs cultured on the PDA/GO composite-coated surface was initially ensured. Then, the osteogenic differentiation of the ESCs in response to the PDA/GO substrate was assessed by alkaline phosphatase (ALP) activity, intracellular calcium levels, matrix mineralization assay, and evaluation of the mRNA and protein levels of osteogenic factors. The culture of ESCs on the PDA/GO substrate presented higher osteogenic potency than that on the uncoated control surface. ESCs cultured on the PDA/GO substrate expressed significantly higher levels of integrin α5 and β1, as well as bone morphogenetic protein receptor (BMPR) types I and II, compared with the control groups. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was observed in ESCs culture on the PDA/GO substrate. Moreover, BMP signal transduction by SMAD1/5/8 phosphorylation was increased more in cells on PDA/GO than in the control. The nuclear translocation of SMAD1/5/8 in cells was also processed in response to the PDA/GO substrate. Blocking activation of the integrin α5/β1, MAPK, or SMAD signaling pathways downregulated the PDA/GO-induced osteogenic differentiation of ESCs. These results suggest that the PDA/GO composite stimulates the osteogenic differentiation of ESCs via the integrin α5/β1, MAPK, and BMPR/SMAD signaling pathways.

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Faculty Opinions recommendation of Integration of external signaling pathways with the core transcriptional network in embryonic stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1115772.571853 ◽

2008 ◽

Author(s):

Deyou Zheng

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Signaling Pathways ◽

Embryonic Stem ◽

Transcriptional Network ◽

The Core

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Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

Disease Models & Mechanisms ◽

10.1242/dmm.009696 ◽

2012 ◽

Vol 5 (6) ◽

pp. 956-966 ◽

Cited By ~ 9

Author(s):

P. Serup ◽

C. Gustavsen ◽

T. Klein ◽

L. A. Potter ◽

R. Lin ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Signaling Pathways ◽

Embryonic Stem

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Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

Cellular Physiology and Biochemistry ◽

10.1159/000495330 ◽

2018 ◽

Vol 51 (2) ◽

pp. 746-762 ◽

Cited By ~ 3

Author(s):

Jin-Sun Lee ◽

Hong Jae Lee ◽

Jae Won Lee ◽

Sang Cheon Lee ◽

Jung Sun Heo

Keyword(s):

Nitric Oxide ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Nitric Oxide Synthase ◽

Cell Viability ◽

Osteogenic Differentiation ◽

Inducible Nitric Oxide Synthase ◽

Embryonic Stem ◽

Oxide Synthase ◽

Inducible Nitric Oxide

Background/Aims: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). Methods: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. Results: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. Conclusion: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

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