Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Summary

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Abstract

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

Introduction

The overall goal of this technique is to promote therapeutic angiogenesis using non-virally programmed stem cells overexpressing therapeutic factors at the site of ischemia. Stem cells were modified ex vivo first using biodegradable nanoparticles synthesized in the lab, and then transplanted in a murine model of hindlimb ischemia to validate their potential for enhancing angiogenesis and tissue salvage.

Controlled vascular growth is an important component of successful tissue regeneration, as well as for treating various ischemic diseases such as stroke, limb ischemia, and myocardial infarction. Several strategies have been developed to promote vascular growth, including growth factor delivery and cell-based therapy.¹ Despite the efficacy observed in the animal disease models, these methods still face limitations such as the need for supraphysiological doses for growth factor delivery, or insufficient paracrine release by cells alone. One potential strategy to overcome the above limitations is to combine stem cell therapy and gene therapy, whereby stem cells are genetically programmed ex vivo prior to transplantation to overexpress desirable therapeutic factors. This approach has been demonstrated in various disease models including hindlimb ischemia², heart disease³, bone healing⁴ and neural injury⁵, etc. However, most gene therapy techniques rely on viral vectors, which are associated with safety concerns such as potential immunogenicity and insertional mutagenesis. Biomaterials mediated non-viral gene delivery may overcome these limitations, but often suffer from low transfection efficiency. To speed up the discovery of novel biomaterials for efficient non-viral gene delivery, recent studies have employed combinatorial chemistry and high-throughput screening approach. Biodegradable polymer libraries such as poly(β-amino esters) (PBAE) have been developed and screened, which led to the discovery of leading polymers with markedly enhanced transfection efficiency compared to the conventional polymeric vector counterparts.^6-7

Herein, we describe the synthesis of PBAE and verification of their ability to transfect adipose-derived stem cells (ADSCs) in vitro, followed by subsequent transplantation of genetically-modified ADSCs overexpressing vascular endothelial growth factor (VEGF) in a murine model of hindlimb ischemia. The outcomes were evaluated by tracking cell fate using bioluminescence imaging, assessing tissue reperfusion using laser Doppler perfusion imaging (LDPI), and determining angiogenesis and tissue salvage by histology.

Protocol

1. Polymer Synthesis In a fume hood, weigh out 3,523 mg of butanediol diacrylate (C) and transfer to a glass scintillation vial containing a stir bar. Pre-heat 5-amino-1-pentanol (32) to 90 °C to solubilize the salt, then in a fume hood, weigh out 1,533 mg 32 and add to the scintillation vial containing C. This method will result in a molar ratio of C:32 = 1:1.2. Immediately place the vial containing both solutions onto a stir plate. Set stir speed at 600 rpm. Transfer the s…

Representative Results

Upon mixing together, the positively-charged polymer (C32-122) and negatively-charged DNA plasmid self-assembles into nanoparticles. Nanoparticle formation may be confirmed through electrophoresis analysis i.e. the complexation between C32-122 and plasmid DNA will prevent mobilization of the DNA during electrophoresis. The polymer serves as a transfection reagent to facilitate enhanced uptake of DNA into the target cells and the subsequent expression of encoding proteins (Figure 2). Cells can be…

Discussion

Here we report a method to program adult stem cells to overexpress therapeutic factors using non-viral, biodegradable nanoparticles. This platform is particularly useful for treating diseases where stem cells can naturally home, such as ischemia and cancer.^9-10 Furthermore, the non-viral gene delivery platform allows for transient overexpression of therapeutic factors, which is suitable for most tissue regeneration and wound healing processes. The transfection process depends upon efficient DNA entry into cell…

Materials

Name of the Reagent	Company	Catalogue Number	Comments (optional)
DMEM	Invitrogen	11965
Fetal Bovine Serum	Invitrogen	10082
Penicillin/Streptomycin	Invitrogen	15070
Basic Fibroblast Growth Factor	Peprotech	100-18B
1,4-Butanediol Diacrylate (90%)	Sigma Aldrich	411744	Acronym: C
5-amino-1-pentanol (97%)	Alfa Aesar	2508-29-4	Acronym: 32
Tetraethyleneglycoldiamine >99%)	Molecular Biosciences	17774	Acronym: 122
Sodium Acetate	G-Biosciences	R010
Phosphate Buffered Saline	Invitrogen	14190-144
Tetrahyofuran Anhydrous (>99.9%)	Sigma Aldrich	401757
Diethyl Ether Anhydrous (>99%)	Fisher Scientific	E138-4
DMSO Anhydrous (>99.9%)	Sigma Aldrich	276855
Gelatin	Sigma Aldrich	G9391
Trypsin-EDTA	Invitrogen	25200
D-luciferin	GoldBio
Optimal Cutting Temperature (O.C.T)	Tissue-Tek	4583
Rat anti-Mouse CD31	BD Pharmingen	550274
Alexa Fluor 594 anti-rat IgG	Invitrogen	A11007