Intravenous xenogeneic transplantation of human adipose-derived stem cells improves left ventricular function and microvascular integrity in swine myocardial infarction model.

OBJECTIVES: The potential for beneficial effects of adipose-derived stem cells (ASCs) on myocardial perfusion and left ventricular dysfunction in myocardial ischemia (MI) has not been tested following intravenous delivery. METHODS: Surviving pigs following induction of MI were randomly assigned to 1 of 3 different groups: the placebo group (n = 7), the single bolus group (SB) (n = 7, 15 × 10(7) ASCs), or the divided dose group (DD) (n = 7, 5 × 10(7) ASCs/day for three consecutive days). Myocardial perfusion defect area and coronary flow reserve (CFR) were compared during the 28-day follow-up. Also, serial changes in the absolute number of circulating CD4(+) T and CD8(+) T cells were measured. RESULTS: The increases in ejection fraction were significantly greater in both the SB and the DD groups compared to the placebo group (5.4 ± 0.9%, 3.7 ± 0.7%, and -0.4 ± 0.6%, respectively), and the decrease in the perfusion defect area was significantly greater in the SB group than the placebo group (-36.3 ± 1.8 and -11.5 ± 2.8). CFR increased to a greater degree in the SB and the DD groups than in the placebo group (0.9 ± 0.2, 0.8 ± 0.1, and 0.2 ± 0.2, respectively). The circulating number of CD8(+) T cells was significantly greater in the SB and DD groups than the placebo group at day 7 (3,687 ± 317/µL, 3,454 ± 787/µL, and 1,928 ± 457/µL, respectively). The numbers of small vessels were significantly greater in the SB and the DD groups than the placebo group in the peri-infarct area. CONCLUSIONS: Both intravenous SB and DD delivery of ASCs are effective modalities for the treatment of MI in swine. Intravenous delivery of ASCs, with its immunomodulatory and angiogenic effects, is an attractive noninvasive approach for myocardial rescue.

Multimodality imaging methods for assessing retinoblastoma orthotopic xenograft growth and development.

Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux ([Formula: see text]) through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed [Formula: see text], as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, [Formula: see text] in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of [Formula: see text] modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future.

Development of a porcine delayed wound-healing model and its use in testing a novel cell-based therapy.

PURPOSE: A delayed full-thickness wound-healing model was developed and used for examining the capacity of adipose-derived stem cells (ASCs), either alone or in platelet-rich fibrin gels, to promote healing. METHODS AND MATERIALS: Four pigs received electron beam radiation to the dorsal skin surface. Five weeks after radiation, subcutaneous fat was harvested from nonirradiated areas and processed to yield ASCs. Two weeks later, 28 to 30 full-thickness 1.5-cm(2) wounds were made in irradiated and nonirradiated skin. Wounds were treated with either saline solution, ASCs in saline solution, platelet-rich plasma (PRP) fibrin gel, ASCs in PRP, or non-autologous green fluorescence protein-labeled ASCs. RESULTS: The single radiation dose produced a significant loss of dermal microvasculature density (75%) by 7 weeks. There was a significant difference in the rate of healing between irradiated and nonirradiated skin treated with saline solution. The ASCs in PRP-treated wounds exhibited a significant 11.2% improvement in wound healing compared with saline solution. Enhancement was dependent on the combination of ASCs and PRP, because neither ASCs nor PRP alone had an effect. CONCLUSIONS: We have created a model that simulates the clinically relevant late radiation effects of delayed wound healing. Using this model, we showed that a combination of ASCs and PRP improves the healing rates of perfusion-depleted tissues, possibly through enhancing local levels of growth factors.

LifeShirt acquisition system to monitor ECG from ambulatory swine and the implementation of an arrhythmia detection algorithm.

A wearable cardiopulmonary monitoring system, a LifeShirt, was used to acquire continuous electrocardiograms (ECGs) from ambulatory swine. The animals received intracoronary injections of autologous mesenchymal stem cells, and the LifeShirt was used for long-duration ECG monitoring in pre-defined periods post cell infusion. The system used here was developed for measurements from non-human primates and canines; however, we demonstrated that it could be used to non-invasively measure ECGs from swine without creating undue stress or restricting movement. A MATLAB-based analysis algorithm was developed to automatically detect premature ventricular contractions (PVCs) that arose 8-10 hours after cell delivery with spontaneous resolution 2-3 days post-infusion. Template based cross-correlation was used to detect the PVCs and identify regions of consecutive ventricular rhythm. The final algorithm was highly specific and sensitive when tested on records from the MIT-BIH arrhythmia database. The algorithm was subsequently used to automatically identify and quantify PVCs from over 200 hours of ECG data obtained from nine ambulatory swine.

Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing.

BACKGROUND: The authors examined the efficacy of adipose stem cells, when supplied either alone or in platelet-rich fibrin gels, to improve wound healing. METHODS: A porcine full-thickness wound model was used to compare six topical treatments: platelet-poor plasma; platelet-rich plasma; autologous adipose stem cells plus platelet-poor plasma; autologous adipose stem cells plus platelet-rich plasma; allogeneic adipose stem cells containing green fluorescent protein plus platelet-poor plasma; and saline (control). One week after isolation, adipose stem cells were applied to full-thickness wounds on the paraspinal and thoracic regions of three pigs (44 wounds per pig; each treatment was applied to eight separate wounds). Each wound was monitored over 21 days for closure, cosmesis, and histopathology. RESULTS: There was no significant difference in the reepithelialization rate, but treatments containing adipose stem cells demonstrated increased microvessel densities (31.75 +/- 5.73 vessels/cm2 versus 7.93 +/- 3.61 vessels/cm2) compared with groups without adipose stem cells. Wound cosmesis was improved in the adipose stem cell plus platelet-rich plasma group compared with other treatment groups (p < 0.05). Vascular endothelial growth factor levels detected in matrices containing adipose stem cells were approximately 7-fold higher compared with platelet-rich plasma or platelet-poor plasma (p < 0.05). Localization of transgenic green fluorescent protein plus adipose stem cells indicated incorporation near neovasculature. CONCLUSIONS: In normal healing wounds, adipose stem cells appear to enhance the healing process only when provided in a fibrin gel vehicle containing a number of complementary wound-healing trophic factors. Perivascular adipose stem cell localization suggests a function in enhancing blood supply through providing physical and paracrine support to newly forming vessels.

Enhancing myocardial plasmid expression by retrograde coronary venous delivery.

Myocardial delivery of genes holds great promise for treating many heart diseases; however, the optimal delivery technique, which maximizes safety and efficacy, has not been established. Two delivery techniques were evaluated in swine; percutaneous retrograde coronary venous delivery (RCVD) and direct intramyocardial injection (IM). RCVD was performed in the anterior interventricular vein (AIV) with an end-hole occlusion balloon catheter. The plasmid gWiz, encoding beta-galactosidase (10 ml; 1 mg/ml), was injected using either manual high pressure (HP-RCVD; n = 5) or pressure wire-guided low pressure (LP-RCVD; n = 4). For the IM group (n = 4), beta-Gal plasmid (5 mg/ml) was injected at 10 sites (200 microl/site) in the anterior left ventricular wall. Animals were euthanized after 5 days. The percentage of beta-Gal expressing cells in the delivered region was higher in the HP-RCVD (0.26% +/- 0.05%) than the LP-RCVD (0.05% +/- 0.03%; P = 0.07) and IM groups (0.02% +/- 0.01%; P = 0.01). Myocardium from the HP-RCVD group contained 7- and 17-fold higher levels of beta-Gal activity than either LP-RCVD and IM groups, respectively (P = 0.05 for both). The results of this study confirm the safety and efficacy of RCVD for myocardial gene delivery.

Widespread regional myocardial transfection by plasmid encoding Del-1 following retrograde coronary venous delivery.

This study quantifies myocardial transfection following percutaneous retrograde coronary venous delivery (RCVD) of a plasmid encoding human Del-1. RCVD of Del-1, GFP plasmid, or marker dye was conducted in 14 pigs. After selective cannulation of a coronary vein, a delivery site was confirmed by contrast injection and myocardial blush. Ten milliliters of plasmid hDel-1 or GFP was administered. Animals were euthanized 3 and 7 days post-RCVD. hDel-1 gene expression was evaluated by quantitative RT-PCR. An average myocardial expression of 4.5 x 10(5) copies hDel-1/microg total RNA was observed within the approximately 5 x 5 cm(2) target tissue of the left ventricle. GFP expression was detected by fluorescent microscopy. hDel-1 protein expression was confirmed by immunohistochemistry. Regionalized myocardial expression was found in all pigs. hDel-1 RNA was not found in distant tissues except in the three pigs with prominent venovenous washout (PVW). These levels were 3 to 4 log unites lower than those found in myocardium. Single retrograde coronary venous administration resulted in efficient regional myocyte transfection of hDel-1 and GFP. This method may be useful and clinically feasible for myocardial angiogenesis therapy.