Generation of Integration‐Free Induced Neurons Using Graphene Oxide‐Polyethylenimine

Abstract

Direct conversion of somatic cells into induced neurons (iNs) without inducing pluripotency has great therapeutic potential for treating central nervous system diseases. Reprogramming of somatic cells to iNs requires the introduction of several factors that drive cell-fate conversion, and viruses are commonly used to deliver these factors into somatic cells. However, novel gene-delivery systems that do not integrate transgenes into the genome are required to generate iNs for safe human clinical applications. In this study, it is investigated whether graphene oxide-polyethylenimine (GO-PEI) complexes are an efficient and safe system for messenger RNA delivery for direct reprogramming of iNs. The GO-PEI complexes show low cytotoxicity, high delivery efficiency, and directly converted fibroblasts into iNs without integrating factors into the genome. Moreover, in vivo transduction of reprogramming factors into the brain with GO-PEI complexes facilitates the production of iNs that alleviated Parkinson's disease symptoms in a mouse model. Thus, the GO-PEI delivery system may be used to safely obtain iNs and could be used to develop direct cell reprogramming-based therapies for neurodegenerative diseases.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA

“…However, the low transfection and translation efficiencies of mRNA still remain a challenge. Currently, IVT mRNAs are complexed with structural elements, such as nanoparticles, polymers, or cationic lipids, to further improve intracellular stability and translational efficiency 23 , 43 , 44 .…”

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…Since higher animals, including humans, do not have reverse transcriptase, there are no risks of integration of the transduced mRNAs into their genome. Many studies using mRNA transduction have been reported, e.g., hepatocytes, 59),115) cardiomyocytes, 116) neuronal cells, 40) endothelial progenitor cells, 117) and neural stem/ precursor cells. 34),118) Nevertheless, several difficulties of this method, such as stability and transfection efficiency, remain.…”

Graphene-Based Nanomaterials for Theranostic Applications

“…Two very recent works demonstrated the application of GO-PEI complexes as mRNA and gene delivery vectors for direct reprogramming of human somatic cells into cells of neural and mesodermal lineage. 64,65 Baek et al demonstrated the reprogramming of¯broblasts into integration-free induced neurons (iNs) through the transfer of mRNA of neural lineage speci¯c reprogramming factors using GO-PEI complexes, that alleviated Parkinson's disease symptoms in a mouse model ( Fig. 3 (iii)).…”

“…3 (iii)). 64 In another study, Chiang et al demonstrated the direct reprogramming of human peripheral blood mononuclear cells into cells of the mesodermal lineage using GO-Fe 3 O 4 -PEI complexes carrying three episomal plasmids encoding pluripotent related factors via a process of combined magnetic stirring and photothermal stimulation to enhance the transfection e±ciencies of the complexes in suspension culture. 65 The transfected PBMCs were reprogrammed into iPSCs, which rapidly transdi®erentiated into mesodermal lineages without full reprogramming.…”

“…Graphene-based nanomaterials have also been explored as substrates for radiolabel imaging because of the surface properties of graphene that makes functionalization and loading of molecules and imaging agents much easier. Hong et al developed a novel PET contrast agent by labeling nGO-PEG with 64 Cu via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) linkage. 136 They further conjugated this nanocomposite with a CD105 speci¯c antibody TRC105 and demonstrated targeted PET imaging of tumor vasculatures in 4T1 murine breast tumors (Fig.…”