ABSTRACT
Transcription factors (TFs) are multidomain proteins that play a critical role in orchestrating stem cell differentiation, but several limitations hinder the full potential of TF-based gene regulation. Here we report a unique strategy to emulate TFs and differentiate stem cells in a nonviral approach using an artificial, nanoparticle-based transcription factor called NanoScript. The NanoScript platform consists of a gold nanoparticle functionalized with small molecules that mimic the various domains of TFs. As a result, NanoScript mimics the function and structure of TF proteins. Specifically, NanoScript was designed to regulate muscle cell differentiation by targeting myogenic regulatory factors (MRFs), which play an important role in inducing myogenesis. This NanoScript-MRF is stable in physiological environments, localizes within the nucleus, induces differentiation of adipose-derived mesenchymal stem cells into mature muscle cells in 7 days, and is naturally excreted from induced muscle cells. As such, NanoScript represents a safe and powerful tool for applications requiring gene manipulation.
Subject(s)
Mesenchymal Stem Cells/cytology , Metal Nanoparticles , Muscle Cells/cytology , Muscle Development , Myogenic Regulatory Factors/administration & dosage , Cell Differentiation , Cell Line , Gold , Humans , Mesenchymal Stem Cells/metabolism , Muscle Cells/metabolism , Myogenic Regulatory Factors/geneticsABSTRACT
BACKGROUND: In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. METHODS AND RESULTS: Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF-All6A(+)) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12 ± 2% vs 24 ± 3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4-fold. GMT-treated animals also demonstrated greater density of cardiomyocyte-specific marker beta myosin heavy chain 7(+) cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12 ± 3% vs -7 ± 3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4-fold greater in GMT/VEGF vs GMT/null animals (17 ± 2% vs 4 ± 1%, P<0.05). CONCLUSIONS: VEGF administration to infarcted myocardium enhances the efficacy of GMT-mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.
