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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 dev....

Protocol

1. Polymer Synthesis

  1. In a fume hood, weigh out 3,523 mg of butanediol diacrylate (C) and transfer to a glass scintillation vial containing a stir bar.
  2. 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.
  3. Immediately place the vial containing both solutions onto a stir plate. Set stir speed at 600 rpm.
  4. 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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
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  

 

References

  1. Deveza, L., Choi, J., Yang, F. Therapeutic angiogenesis for treating cardiovascular diseases. Theranostics. 2, 801-814 (2012).
  2. Yang, F., et al. Genetic engineering of human stem cel....

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Stem CellsAngiogenesisTherapeutic AngiogenesisBiodegradable Polymeric NanoparticlesVascular GrowthIschemic DiseasesGene TherapyAdipose derived Stem CellsVEGFHindlimb Ischemia Model

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