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.

1. Polymer Synthesis
<ol>
	<li>In a fume hood, weigh out 3,523 mg of butanediol diacrylate (C) and transfer to a glass scintillation vial containing a stir bar.</li>
	<li>Pre-heat 5-amino-1-pentanol (32) to 90 &#176;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.</li>
	<li>Immediately place the vial containing both solutions onto a stir plate. Set stir speed at 600 rpm.</li>
	<li>Transfer the scintillation vial to an oven set at 90 &#176;C. Set stirring speed to 1,000 rpm for 4 hr. If the stir bar gets stuck, lower the speed. After 4 hr, lower speed to 300 rpm and maintain at 90 &#176;C for another 12-16 hr.</li>
	<li>Add 5 g of C32 to 10 ml of anhydrous tetrahydrofuran (THF) in a glass scintillation vial containing a stir bar. Wrap in foil and vortex on high until fully dissolved.</li>
	<li>In a separate glass scintillation vial containing a stir bar, add 10 mM of Tetraethyleneglycoldiamine (122). Add 40 ml of THF.</li>
	<li>Place both vials (C32 and 122) on a stir plate for 5 min then combine together. Cover in foil and leave at room temperature stirring at 400 rpm for 24 hr. The final product is termed C32-122.</li>
	<li>Weigh out 5 x 50 ml Falcon tubes for each 5 g batch of C32-122. Note the mass of each tube in a notebook.</li>
	<li>Transfer 30 ml of anhydrous diethyl ether to each 50 ml Falcon tube.</li>
	<li>Transfer 10 ml of C32-122 to each 50 ml Falcon tube.</li>
	<li>Vortex Falcon tubes vigorously then centrifuge at 2,500 rpm for 2 min. Note: The extracted polymer will collect at the base of the tube.</li>
	<li>Discard the upper solution and repeat steps 1.9 and 1.11 two more times.</li>
	<li>Place the open tubes containing the extracted C32-122 in the desiccator and vacuum overnight. Ensure all tubes are protected from light.</li>
	<li>Weigh all tubes containing C32-122 to determine the final mass of the extracted polymer.</li>
	<li>Dissolve the extracted polymer in anhydrous DMSO at a concentration of 100 mg/ml.</li>
	<li>Store the dissolved polymer at -20 &#176;C protected from light and moisture.</li>
</ol>
2. Cell Seeding
<ol>
	<li>Pre-coat the base of a T-150 tissue culture flask with 0.1% (wt/vol) gelatin. The gelatin should remain in the flask for 30-45 min. The gelatin coating helps adhesion of ADSCs.</li>
	<li>Aspirate gelatin from the pre-coated T-150 flask.</li>
	<li>Add 4 x 106 ADSCs (&#8804; passage 3) to the flask in 20 ml of DMEM containing 10% FBS, 1% Penicillin/Streptomycin and 10 ng/ml bFGF.</li>
	<li>Place the T-150 flask in the incubator at 37 &#176;C, 5% CO2 overnight.</li>
	<li>Begin transfection procedure the following day.</li>
</ol>
3. Nanoparticle Preparation and Transfection
<ol>
	<li>The following procedure is for the preparation of nanoparticles containing 16.1 &#956;g plasmid DNA per 1.0 x 106 cells at a polymer to plasmid weight ratio of 30:1.</li>
	<li>Dilute plasmid DNA (1 mg/ml, pVEGF165, Aldevron, ND, USA) to a final concentration of 120 &#956;g/ml in sodium acetate (25 mM) in a 15 ml Falcon tube.</li>
	<li>Dilute C32-122 (100 mg/ml) to a final concentration of 3.6 mg/ml in sodium acetate (25 mM) in a second 15 ml Falcon tube.</li>
	<li>Combine the contents of each Falcon tubes into a single 15 ml falcon tube and vortex immediately on high for 10 sec.</li>
	<li>Let the tube sit at room temperature for 10 min for nanoparticle formation to occur.</li>
	<li>While incubating, replace medium in tissue culture flask with 7.8 ml fully supplemented DMEM per 1.0 x 106 cells transfected.</li>
	<li>Transfer the nanoparticle solution to the tissue culture flask and tilt forward and backwards to spread evenly.</li>
	<li>Place the tissue culture flask in the incubator at 37 &#176;C, 5% CO2 for 4 hr.</li>
	<li>After 2 hr, replace the nanoparticle containing media with DMEM.</li>
	<li>Following a further 2 hr incubation at 37 &#176;C, 5% CO2, the cells are ready for in vivo injection.</li>
</ol>
4. Hindlimb Ischemia Procedure
<ol>
	<li>All experiments were performed in accordance with the Stanford University Animal Care and Use Committee Guidelines and approved APLAC protocols. Generating the murine model of hindlimb ischemia is performed as previously described.8 Please refer to the reference for detailed instructions. A short description is provided below.</li>
	<li>Place the mouse under anesthesia with 1-3% dosage isoflurane by inhalation and oxygen flow rate of 1 L/min. Ensure anesthetic depth by toe pinch, reduction in respiratory rate, and lethargy.</li>
	<li>Remove hair from the abdominal region and both hindlimb areas of the mouse with shaving cream.</li>
	<li>Make incision at center of medial thigh towards midline and then cut through overlying fat pad to expose the femoral artery.</li>
	<li>Ligate the artery at two sites: distal site (close to the knee) and proximal site (just distal to the inguinal ligament).</li>
	<li>To create the ligations, separate the artery from the vein and thread a 5-0 silk suture around the artery. Tie off the artery with two knots to make the ligation.</li>
	<li>Remove the artery between the two ligation points.</li>
	<li>Wash the surgical area with PBS and then suture the incision closed.</li>
	<li>The anesthesia can now be removed. Keep the mouse warm until re-awakening. For post-operative care, 2-4 mg/kg Lidocaine can be injected subcutaneously at the incision site prior to re-awakening. Further pain management may include subcutaneous delivery of 0.05-0.1 mg/kg Buprenorphine at 12 and 24 hr. Monitor the health status of the mice once a day for seven days by observing changes in breathing pattern, overt pain or distress, and skin color.</li>
	<li>Confirm hindlimb ischemia by laser Doppler perfusion imaging.</li>
</ol>
5. Cell Injections
<ol>
	<li>When the transfected cells and the mice are ready, wash transfected cells with PBS, trypsinize, centrifuge, and then count.</li>
	<li>Resuspend cells at a density of 10 x 106 cells per ml in PBS.</li>
	<li>Cell suspensions should be separated into separate Eppendorf tubes at a volume of 110 &#956;l. This will ensure that each mouse receives an equal amount of cells.</li>
	<li>Place each mouse under anesthesia (2.5% isoflurane, 1 L/min oxygen flow rate).</li>
	<li>Clean the injection site with alcohol wipes.</li>
	<li>Utilizing a 27 G tuberculin syringe, draw the cells up and down into the syringe to ensure a homogeneous suspension is achieved. Draw up 100 &#956;l of the cell suspension volume into the syringe.</li>
	<li>Inject half the cell suspension into the adductor muscle region and then inject the other half into the calf muscle region. Upon injecting, leave the syringe in place for at least 15-30 sec to prevent leakage of the cell suspension.</li>
	<li>Proper controls for the animal study include: 1) cells transfected with a non-coding plasmid (e.g. plasmid with no biological activity); 2) cells alone; and 3) PBS.</li>
	<li>When injections are complete, remove the mouse from anesthesia and keep warm until the mouse re-awakes.</li>
</ol>
6. Bioluminescence Imaging
<ol>
	<li>To begin bioluminescence imaging (BLI), anesthetize mice as described above.</li>
	<li>Clean the injection site with alcohol wipes.</li>
	<li>Utilizing insulin syringe, load 100 &#956;l of 15 mg/ml D-luciferin (the substrate for firefly luciferase). Keep substrate away from light.</li>
	<li>To perform intraperitoneal injections, hold mice by the skin of their back to pull their anterior skin taut. Insert needle intraperitoneally into the abdominal region and inject 100 &#956;l of substrate.</li>
	<li>Place the mouse in the IVIS luciferase imaging chamber under anesthesia.</li>
	<li>Image the mice with an exposure time of 1.0 min. When parameters are set, begin to acquire images.</li>
	<li>When the image is acquired, mark the region of interest to measure the luciferase signal. In this case, mark the ischemic limb at the cell-injection site.</li>
	<li>Continue to measure the luciferase signal every 3-5 min until the signal reaches a peak and begins to decline. This is the value utilized for comparison across mice and over several time points.</li>
	<li>When complete, remove the mice from the chamber and anesthesia. Keep mice warm until re-awakening.</li>
</ol>
7. Laser Doppler Perfusion Imaging
<ol>
	<li>Laser Doppler perfusion imaging is performed as previously described.8 The procedure is briefly described here.</li>
	<li>Prior to Doppler imaging the mouse should be cleanly shaven at the areas overlying both hindlimbs and the abdominal region to prevent artifact due to hair.</li>
	<li>When prepared to Doppler image, the mouse should be anesthetized as described above.</li>
	<li>Warm the mouse to 36 &#176;C on a hot plate while monitoring body temperature by rectal thermometer.</li>
	<li>Place the mouse on a non-reflective black surface and keep anesthetized by nose cone. The mouse should be placed on its dorsal surface with its limbs exposed evenly.</li>
	<li>Position the laser Doppler sensor roughly 15 cm above the mouse and directing the laser pointer from the sensor towards the groin region of the mouse.</li>
	<li>When the mouse and the Doppler sensor are in position, images can be taken utilizing the LDPIwin software by pressing the Start button.</li>
	<li>Relative perfusion measurements could be taken by indicating both hindlimbs (ischemic and non-ischemic) as region of interests (ROI). The ratio of mean perfusion of the ischemic to the non-ischemic hindlimb will indicate relative perfusion.</li>
</ol>
8. Tissue Harvest Procedure
<ol>
	<li>When prepared to harvest mouse tissue for histology and/or biochemical processing, the mouse should be euthanized. Here, the mouse is euthanized by exposing to 5% isoflurane for 3-5 min, then euthanizing the mouse by cervical dislocation.</li>
	<li>Cut the overlying skin surrounding the hindlimb circumferentially at the distal abdominal region and proximal to the hindlimb. The skin is cut from the ventral to the dorsal surfaces, such that the skin can be pulled back over the hindlimb to the foot.</li>
	<li>Upon pulling the skin back, the limb could be amputated by utilizing blunt dissection scissors to cut at the pelvic and femur joint.</li>
	<li>Two main tissues are taken for analysis: the medial adductor muscles and the gastrocnemius muscles.</li>
	<li>For histology, embed one or both tissues in optimal cutting temperature (OCT) for cryosectioning.</li>
	<li>For biochemical analysis, collect one or both tissues into an 1.5 ml Eppendorf and submerge into a bath of liquid nitrogen for at least 2 min. The samples can be stored at -80 &#176;C for future processing.</li>
</ol>

<ol>
	<li>Deveza, L., Choi, J., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+for+treating+cardiovascular+diseases.">Therapeutic angiogenesis for treating cardiovascular diseases.</a> Theranostics. 2, 801-814 (2012).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+human+stem+cells+for+enhanced+angiogenesis+using+biodegradable+polymeric+nanoparticles.">Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 3317-3322 (2010).</li><li>Mangi, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+modified+with+Akt+prevent+remodeling+and+restore+performance+of+infarcted+hearts.">Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts.</a> Nat Med. 9, 1195-1201 (2003).</li><li>Lee, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+bone+healing+based+on+ex+vivo+gene+therapy+using+human+muscle-derived+cells+expressing+bone+morphogenetic+protein+2.">Enhancement of bone healing based on ex vivo gene therapy using human muscle-derived cells expressing bone morphogenetic protein 2.</a> Hum. Gene Ther. 13, 1201-1211 (2002).</li><li>Park, K. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+may+be+uniquely+suited+for+combined+gene+therapy+and+cell+replacement:+Evidence+from+engraftment+of+Neurotrophin-3-expressing+stem+cells+in+hypoxic-ischemic+brain+injury.">Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury.</a> Exp. Neurol. 199, 179-190 (2006).</li><li>Green, J. J., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+polymer+library+approach+yields+insight+into+nonviral+gene+delivery.">A combinatorial polymer library approach yields insight into nonviral gene delivery.</a> Acc Chem Res. 41, 749-759 (2008).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+human+adult+and+embryonic+cell-derived+stem+cells+using+biodegradable+nanoparticulate+polymeric+vectors.">Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors.</a> Gene Ther. 16, 533-546 (2009).</li><li>Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> J. Vis. Exp. , e1035 (2009).</li><li>Ceradini, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+cell+trafficking+is+regulated+by+hypoxic+gradients+through+HIF-1+induction+of+SDF-1.">Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.</a> Nat. Med. 10, 858-864 (2004).</li><li>Kidd, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+mesenchymal+stem+cell+tropism+for+tumor+and+wounding+microenvironments+using+in+vivo+bioluminescent+imaging.">Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging.</a> Stem Cells. 27, 2614-2623 (2009).</li><li>Sunshine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+end-groups+of+linear+polymer+determine+cell-type+gene-delivery+efficacy.">Small-molecule end-groups of linear polymer determine cell-type gene-delivery efficacy.</a> Adv. Mater. 21, 4947-4951 (2009).</li><li>Sunshine, J. C., Akanda, M. I., Li, D., Kozielski, K. L., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+base+polymer+hydrophobicity+and+end-group+modification+on+polymeric+gene+delivery.">Effects of base polymer hydrophobicity and end-group modification on polymeric gene delivery.</a> Biomacromolecules. 12, 3592-3600 (2011).</li><li>Lynn, D. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradable+poly(&#946;-amino+esters):+Synthesis,+characterization,+and+self-assembly+with+plasmid+DNA.">Degradable poly(&#946;-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA.</a> J. Am. Chem. Soc. 122, 10761-10768 (2000).</li><li>Eltoukhy, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+molecular+weight+of+amine+end-modified+poly(beta-amino+ester)s+on+gene+delivery+efficiency+and+toxicity.">Effect of molecular weight of amine end-modified poly(beta-amino ester)s on gene delivery efficiency and toxicity.</a> Biomaterials. 33, 3594-3603 (2012).</li><li>Glover, D. J., Lipps, H. J., Jans, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+safe,+non-viral+therapeutic+gene+expression+in+humans.">Towards safe, non-viral therapeutic gene expression in humans.</a> Nat. Rev. Genet. 6, 299-310 (2005).</li><li>Dave, U. P., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+insertional+mutagenesis+insights.">Gene therapy insertional mutagenesis insights.</a> Science. 303, 333 (2004).</li></ol>

The authors declare that they have no competing financial interests.

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 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(&beta;-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.

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

	&nbsp;

programming stem cells for therapeutic angiogenesis using biodegradable polymeric nanoparticles

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.

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...

Watch this Scientific Journal Video about Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles at JoVE.com

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

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, ...

programming-stem-cells-for-therapeutic-angiogenesis-using

Research

JoVE Journal

Bioengineering

11.0K Views.  Stanford University. 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.

Video: Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...