MRNA as a Tool for Gene Transfection in 3D Cell Culture for Future Regenerative Therapy.

Satoshi Uchida,Kayoko Yanagihara,Kazunori Kataoka,Keiji Itaka,Akitsugu Matsui

doi:10.3390/mi11040426

Satoshi Uchida, Kayoko Yanagihara + Show 3 more

Open Access

https://doi.org/10.3390/mi11040426

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Abstract

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

Highlights

Cell transplantation therapy has demonstrated excellent potential in a variety of medical fields, both in preclinical and clinical research [1,2,3]
We showed that messenger RNA (mRNA) introduction provided protein expression in a larger percentage of cells in mesenchymal stem cell (MSC) spheroids compared to introduction of plasmid DNA (pDNA), leading to enhanced outcomes after in vitro osteogenic induction using mRNA encoding an osteogenic transcription factor
In this study, using MSC spheroids, we revealed that compared with pDNA transfection, mRNA transfection induced protein expression in a larger percentage of 3D cultured cells (Figure 1)

Summary

Introduction

Cell transplantation therapy has demonstrated excellent potential in a variety of medical fields, both in preclinical and clinical research [1,2,3] Transplanted cells exert their therapeutic effect by integrating into host tissues and replacing endogenous cell functions [4], and by secreting paracrine factors [5]. For introduction of mRNA and pDNA, poly[N’-[N-(2-aminoethyl)-2-aminoethyl]aspartamide] [PAsp(DET)] polycation was employed, which has two notable features: pH-responsive protonation behavior allows for efficient endosomal escape of polyplexes and its biodegradable nature contributed to its low cumulative toxicity [27,28,29] Using these platforms, we showed that mRNA introduction provided protein expression in a larger percentage of cells in mesenchymal stem cell (MSC) spheroids compared to introduction of pDNA, leading to enhanced outcomes after in vitro osteogenic induction using mRNA encoding an osteogenic transcription factor

Spheroid Preparation

Preparation of mRNA and pDNA

Transfection of MSCs with GFP mRNA and pDNA

In Vitro Osteogenic Differentiation

Findings

Discussion