Preparation and Characterization of Genetically Engineered Mesenchymal Stem Cell Aggregates for Regenerative Medicine

ABSTRACT − Combining cell- and gene-based therapy is a promising therapeutic strategy in regenerative medicine. The aim of this study was to develop genetically modified mesenchymal stem cell (MSC) aggregates using a poly(ethylene glycol) (PEG) hydrogel micro-well array technique. Stable PEG hydrogel micro-well arrays with diameters of 200 to 500 μm were fabricated and used to generate genetically engineered MSC aggregates. Rat bone marrow-derived MSCs were transfected with a green fluorescent protein (GFP) plasmid as a reporter gene, and aggregated by culturing in the PEG hydrogel micro-well arrays. The resultant cell aggregates had a mean diameter of less than 200 μm, and maintained the mesenchymal phenotype even after genetic modification and cell aggregation. Transplantation of MSC aggregates that are genetically modified to express therapeutic or cell-survival genes may be a potential therapeutic approach for regenerative medicineope

허혈성 심장질환이 유발된 동물모델에서 중간엽 줄기세포와 혈관신생인자를 이용한 세포기반 유전자치료 개발

Dept. of Science for Aging/석사In primary cultured cells including bone marrow-derived MSCs, most transfection techniques achieve very low efficiencies, generally no more than a few percent. Thus, preparing effective transfection methodologies is crucial to the success of MSC-based gene therapy. Facial amphiphiles having hydrophilic and hydrophobic groups located on two opposite faces have known to destabilize plasma membrane and enhance their cellular permeability. To develop efficient transfection methodologies for MSCs, herein different types of bile acids having facial amphiphilicity were conjugated to low molecular weight polyethyleneimine (PEI1.8,1.8kDa) (BA-PEI1.8). The purpose of the present study is to enhance in vitro gene transfection efficiencies in rat MSCs with various bile acid-modified cationic gene carriers. Herein, hypoxia inducible VEGF plasmid modified MSCs was used as an effective cell-based gene therapy strategy for salvaging myocardial ischemia and infarction.First, we synthesized different types of bile acid-conjugated PEIs (DA-PEI, CA-PEI, LA-PEI) to use as gene carriers for primary cultured MSCs. Gene transfection efficiency and cell toxicity in mesenchymal stem cells (MSCs) was examined with different BA-PEI1.8 conjugates. Compared with conventional transfection reagents, DA-PEI1.8 exhibited more than 10-fold higher transfection efficiency in rat MSCs. In particular, DA-PEI1.8 conjugate could increase VEGF gene expression in MSCs up to 50-fold higher than commercialized transfection reagent (Lipofectamine). The transplantation of MSCs genetically modified to overexpress VEGF by BA-PEI1.8 enhanced the capillary formation in the infarction region and eventually attenuated left ventricular remodeling after myocardial infarction in rats. This study demonstrates the applicability of the BA-PEI1.8 conjugates for the efficient transfection of therapeutic genes into MSCs and the feasibility of using the genetically engineered MSCs in regenerative medicine for myocardial infarction.restrictio

MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine

Mesenchymal stem cells (MSCs) have attracted much attention in regenerative medicine owing to their apparent usefulness as multi-potent replacement cells. The potential of MSC therapy can be further improved by transforming MSCs with therapeutic genes that maximize the efficacy of gene therapy and their own therapeutic ability. Since most conventional transfection methodologies have shown marginal success in delivering exogenous genes into primary cultured cells, efficient gene transfer into primary MSCs is a prerequisite for the development of MSC-based gene therapy strategies to achieve repair and regeneration of damaged tissues. Herein, facially amphipathic bile acid-modified polyethyleneimine (BA-PEI) conjugates were synthesized and used to transfer hypoxia-inducible vascular endothelial growth factor gene (pHI-VEGF) in MSCs for the treatment of rat myocardial infarction. Under the optimized transfection conditions, the BA-PEI conjugates significantly increased the VEGF protein expression levels in rat MSCs, compared with traditional transfection methods such as Lipofectamine™ and branched-PEI (25 kDa). Furthermore, the prepared pHI-VEGF-engineered MSCs (VEGF-MSCs) resulted in improved cell viability, particularly during severe hypoxic exposure in vitro. The transplantation of MSCs genetically modified to overexpress VEGF by BA-PEI enhanced the capillary formation in the infarction region and eventually attenuated left ventricular remodeling after myocardial infarction in rats. This study demonstrates the applicability of the BA-PEI conjugates for the efficient transfection of therapeutic genes into MSCs and the feasibility of using the genetically engineered MSCs in regenerative medicine for myocardial infarction.ope

Anti-apoptotic cardioprotective effects of SHP-1 gene silencing against ischemia–reperfusion injury: Use of deoxycholic acid-modified low molecular weight polyethyleneimine as a cardiac siRNA-carrier

The cardiomyocyte apoptosis plays a critical role in the development of myocardial injury after ischemia and reperfusion. Thus, alteration of the major apoptosis-regulatory factors during myocardial ischemia-reperfusion is expected to have favorable cardioprotective effects. Herein, we report ischemic-reperfused myocardial infarction (MI) repair with siRNA against Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), which is known as a key factor involved in regulating the progress of apoptosis in many cell types. A low molecular weight polyethyleneimine modified with deoxycholic acid (PEI1.8-DA)-based delivery strategy was suggested for the cardiac application of SHP-1 siRNA to overcome the poor gene delivery efficiency to myocardium due to the highly charged structures of the compact cardiac muscles. The PEI1.8-DA conjugates formed stable nanocomplexes with SHP-1 siRNA via electrostatic and hydrophobic interactions. The PEI1.8-DA/SHP-1 siRNA polyplexes effectively silenced SHP-1 gene expression in cardiomyocytes, leading to a significant inhibition of cardiomyocyte apoptosis under hypoxia. In comparison to conventional gene carriers, relatively large amounts of siRNA molecules remained after treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes. Cardiac administration of the PEI1.8-DA/SHP-1 siRNA polyplexes resulted in substantial improvement in SHP-1 gene silencing, which can be explained by the enhancement of cardiac delivery efficiency of the PEI1.8-DA conjugates. In addition, in vivo treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes induced a highly significant reduction in myocardial apoptosis and infarct size in rat MI models. These results demonstrate that the PEI1.8-DA/SHP-1 siRNA polyplex formulation is a useful system for efficient gene delivery into the compact myocardium that provides a fundamental advantage in treating ischemic-reperfused MI.ope

Deoxycholic acid-modified polyethylenimine based nanocarriers for RAGE siRNA therapy in acute myocardial infarction

The activation of receptor for advanced glycation end products (RAGE) signaling is mainly associated with myocardial ischemia/reperfusion injury. Thus the blockade of RAGE-ligands axis can be considered as a potential therapeutic strategy to protect myocardial infarction after ischemia/reperfusion injury. Herein, we strengthened the cardioprotective effect with combinatorial treatment of soluble RAGE (sRAGE) and RAGE siRNA (siRAGE) causing more effective suppression of RAGE-mediated signaling transduction. For pharmacological blockade of RAGE, sRAGE, the extracellular ligand binding domain of RAGE, acts as a pharmacological ligand decoy and inhibits the interaction between RAGE and its ligands. For genetic deletion of RAGE, siRAGE suppresses the expression of RAGE by participating in RNA interference mechanism. Therefore, we combined these two RAGE blockade/deletion strategies and investigated the therapeutic effects on rat ischemic and reperfused myocardium. According to our results, based on RAGE expression level analysis and infarct size/fibrosis measurement, co-treatment of sRAGE and siRAGE exhibited synergic cardioprotective effects; thus the newly designed regimen can be considered as a promising candidate for the treatment of myocardial infarction.ope

Hypoxia-inducible vascular endothelial growth factor gene therapy using the oxygen-dependent degradation domain in myocardial ischemia.

PURPOSE: A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease. METHODS: Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively. RESULTS: The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments). CONCLUSION: An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.ope