The overall goal of this procedure is to demonstrate the use of polymeric nanoparticles for the overexpression of therapeutic genes in stem cells using a marine hind limb ischemia model. This is accomplished by first synthesizing biodegradable polymers through a two-step mic edition reaction, followed by fabricating polymeric nanoparticles encoding therapeutic genes. The second step is to transfect human adipose-derived stem cells in vitro to overexpressed vascular endothelial growth factor.
Next programmed stem cells are injected intramuscularly into an ischemic hind limb for NC two production of therapeutic factors. The final step is to monitor cell survival and blood reperfusion in the ischemic limb over time using bioluminescence imaging and laser doppler imaging. Ultimately, quantitative gene expression and histology can be performed to show localized expression of therapeutic factors and efficacy of tissue regeneration.
The advantage of this technique over conventional methods for angiogenesis is the use of stem cells as vehicles for drug delivery. This strategy allows us to employ the natural hoing capacity of stem cells to migrate towards ischemia, and also allows dynamic responses to microenvironmental cues. The nanoparticles used for plasma DNA delivery are highly efficient and biodegradable affording for lower cytotoxicity and a larger number of intracellularly delivered DNA copies.
This technique can be applied to many clinically relevant cell-based therapeutics because it utilizes a biodegradable non-viral vector to insert genes into autologous cells Working in a fume hood, weigh the butane dial DLI, and transfer the material to a glass inhalation vial containing a stir bar. Next, add preheated five amino one pentol to the same vial. Immediately place the vial onto a stir plate and set the speed to 600 RPM.
Transfer the vial to an oven set at 90 degrees Celsius and increase the stirring speed to 1000 RPMs after four hours, lower the speed to 300 RPM and stir for an additional 12 to 16 hours. When ready, add 10 milliliters of anhydrous tetra hydro uran to five grams of C 32 in a glass vial containing a stir bar. After wrapping in foil vortex on high until fully dissolved in a separate glass vial containing a stir bar, add 10 millimolar of tetra ethylene glycol diamine.
Add 40 milliliters of THF to this vial place both vials on a stir plate for five minutes before combining them together. Cover the resultant in foil and leave it to stare room temperature for 24 hours. The final product is termed C 32 1 22.
Next transfer 30 milliliters of anhydrous dathyl ether to each of five 50 milliliter falcon tubes. After adding C 32 1 22 to anhydrous dyl ether, a white cloudy solution forms vortex the tube and then centrifuge it. The extracted polymer will collect at the base of the tube, discard the upper solution and repeat these steps two more times.
Once extracted, place the open tubes in the desiccate and vacuum overnight, ensure that the tubes are protected from light. The next day weigh the tubes containing C 32 1 22 to determine the final mass. Finally dissolve the extracted polymer in anhydrous DMSO at a concentration of 100 milligrams per milliliter.
For nanoparticle preparation. First dilute plasma DNA to a final concentration of 120 micrograms per milliliter in sodium acetate in a second tube dilute C 32 1 22 to a final concentration of 3.6 milligrams per milliliter. In sodium acetate, combine the contents of each tube into a single 15 milliliter Falcon tube and vortex immediately on high for 10 seconds.
Let the tube sit at room temperature for 10 minutes for nanoparticle formation to occur while the nanoparticles are forming. Replace the medium in a previously seated tissue culture flask with 7.8 milliliters. Fully supplemented DMEM.
Transfer the nanoparticle solution to the tissue culture flask and spread it evenly before placing in the incubator. After two hours, replace the nanoparticle containing media with DMEM. After four hours, the cells are ready for in vivo injection.
When the transfected cells are ready, count spen the cells at a density of 10 times 10 of the six cells per milliliter in PBS to ensure that each mouse receives an equal amount. Separate the cell suspensions into einor tubes at a volume of 110 microliters. Next, anesthetize a mouse that has previously undergone hind limb ischemia.
After confirming anesthesia by toe pinch, clean the injection site with a 27 gauge syringe. Re suspend the cells and draw up 100 microliters of the suspension. Inject half of the cell suspension into the adductor muscle region, and then inject the other half into the calf muscle region.
After injecting, leave the syringe in place for at least 15 to 30 seconds to prevent leakage. When the injections are complete, keep the animal warm until fully recovered. For bioluminescence imaging, confirm anesthesia with a toe pinch and then clean the injection site as shown earlier.
Fill an insulin syringe with 100 microliters of Lucifer and inject it intraperitoneal. Before placing the animal in the ivus Luciferase imaging chamber, image the mice with an exposure time of one minute. When the image is acquired, mark the region of interest.
In this case, the ischemic limb at the cell injection site continue to measure the luciferase signal every three to five minutes until the signal reaches a peak and begins to decline. This is the value used for comparison across mice and over time points when complete, remove the mice from the chamber and keep the animal warm until fully recovered. Tissues are harvested at select time.
Points after transfection after euthanasia, cut the skin surrounding the hind limb circumferentially at the distal abdominal region and proximal to the hind limb. After pulling the skin back, amputate the limb between the pelvic and femur joint. Finally, the medial adductor and the gastroc muscles are isolated.
For further analysis, these images portray the synthesis of C 32 1 22. Here, ACR related terminated C 32 AC was formed by a mic edition reaction between a monomer with DAL end groups and a monomer with a primary am mean end group. In this example and modified PBAE polymers can be formed by adding a mean terminated monomers for enhanced transfection efficiency.
A successful transfection is apparent by the overexpression of green fluorescent protein as seen here. This bioluminescence imaging data shows a mouse at day zero and day 14. After injecting GFP luciferase positive adipose-derived stem cells into the hind limb, representative Doppler images demonstrate the induction of ischemia in one side of the mirroring hind limb at day zero and successful blood reperfusion.
14 days after the injection of VEGF over expressing adipose-derived stem cells. R-T-P-C-R confirmed the successful upregulation of vgf, the encoded therapeutic protein in the treated group four days after the cell injection, whereas no expression was detected in the PBS control. Other methods that apply a combined stem cell and gene delivery approach can be performed in order to identify novel therapeutic targets for promoting tissue regeneration.
After watching this video, you should have a good understanding of how to synthesize biodegradable polymers for non-viral gene therapy and use these polymers to program stem cells for the treatment of ischemia and vivo Using non-viral engineered stem cells for over expressing therapeutic factors in situ may be broadly useful for treating a variety range of degenerative diseases.
