Abstract
BackgroundDuring differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage. In this study, using microarray-based technology, gene expression profiling was examined during the process of chondrogenic differentiation of human mesenchymal stem cells (hMSCs). To induce chondrogenic differentiation of hMSCs, the cationic polymer polyethyleneimine (PEI) was coupled with the synthetic glucocorticoid dexamethasone (DEX). DEX/PEI could be polyplexed with anionic plasmid DNAs (pDNAs) harboring the chondrogenesis-inducing factors SOX5, SOX6, and SOX9. These are named differentiation-inducing nanoparticles (DI-NPs).MethodsA DI-NP system for inducing chondrogenic differentiation was designed and characterized by dynamic light scattering and scanning electron microscopy (SEM). Chondrogenic induction of hMSCs was evaluated using various tools such as reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, confocal fluorescent microscopy, and immunohistochemistry analysis. The gene expression profiling of DI-NP-treated hMSCs was performed by microarray analysis.ResultsThe hMSCs were more efficiently transfected with pDNAs using DI-NPs than using PEI. Moreover, microarray analysis demonstrated the gene expression profiling of hMSCs transfected with DI-NPs. Chondrogenic factors including SOX9, collagen type II (COLII), Aggrecan, and cartilage oligometric matrix protein (COMP) were upregulated while osteogenic factors including collagen type I (COLI) was downregulated. Chondrogenesis-induced hMSCs were better differentiated as assessed by RT-PCR, Western blotting analyses, and immunohistochemistry.ConclusionDI-NPs are good gene delivery carriers and induce chondrogenic differentiation of hMSCs. Additionally, comprehensive examination of the gene expression was attempted to identify specific genes related to differentiation by microarray analysis.Graphical abstract
Highlights
During differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage
Preparation and characterization of trio-coated PEI and differentiation-inducing nanoparticles (DI-NPs) Here, branched PEI (bPEI) was conjugated with TRITC to generate PEI, which was subsequently conjugated with DEX to generate DEX-conjugated bPEI (DEX/PEI)
Thereafter, DEX/PEI were complexed with SOX5, SOX6, and SOX9 plasmid DNAs (pDNAs) to generate DI-NPs
Summary
During differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage. DEX/PEI could be polyplexed with anionic plasmid DNAs (pDNAs) harboring the chondrogenesis-inducing factors SOX5, SOX6, and SOX9. Cells have been transfected with genes encoding specific transcription factors in an attempt to enhance differentiation towards the desired lineage and prevent differentiation towards other lineages [11, 12]. In this regard, we designed and synthesized dexamethasone (DEX)-conjugated polyethyleneimine (PEI), called DEX/PEI, as a gene carrier. By conjugating DEX to PEI, the efficiency of gene delivery could be improved, facilitating induction of differentiation with low cytotoxicity and low effects of inflammation when applied to an in-vivo study [15]
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