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* These authors contributed equally
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.
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.
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 dev....
1. Polymer Synthesis
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.......
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.......
The authors would like to acknowledge American Heart Association National Scientist Development Grant (10SDG2600001), Stanford Bio-X Interdisciplinary Initiative Program, and Stanford Medical Scholars Research Program for funding.
....Name | Company | Catalog Number | Comments |
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 |
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