Yingli Fu

X‐Ray‐Visible Microcapsules Containing Mesenchymal Stem Cells Improve Hind Limb Perfusion in a Rabbit Model of Peripheral Arterial Disease

The therapeutic goal in peripheral arterial disease (PAD) patients is to restore blood flow to ischemic tissue. Stem cell transplantation offers a new avenue to enhance arteriogenesis and angiogenesis. Two major problems with cell therapies are poor cell survival and the lack of visualization of cell delivery and distribution. To address these therapeutic barriers, allogeneic bone marrow‐derived mesenchymal stem cells (MSCs) were encapsulated in alginate impregnated with a radiopaque contrast agent (MSC‐Xcaps.) In vitro MSC‐Xcap viability by a fluorometric assay was high (96.9% ± 2.7% at 30 days postencapsulation) and as few as 10 Xcaps were visible on clinical x‐ray fluoroscopic systems. Using an endovascular PAD model, rabbits (n = 21) were randomized to receive MSC‐Xcaps (n = 6), empty Xcaps (n = 5), unencapsulated MSCs (n = 5), or sham intramuscular injections (n = 5) in the ischemic thigh 24 hours postocclusion. Immediately after MSC transplantation and 14 days later, digital radiographs acquired on a clinical angiographic system demonstrated persistent visualization of the Xcap injection sites with retained contrast‐to‐noise. Using a modified TIMI frame count, quantitative angiography demonstrated a 65% improvement in hind limb perfusion or arteriogenesis in MSC‐Xcap‐treated animals versus empty Xcaps. Post‐mortem immunohistopathology of vessel density by anti‐CD31 staining demonstrated an 87% enhancement in angiogenesis in Xcap‐MSC‐treated animals versus empty Xcaps. MSC‐Xcaps represent the first x‐ray‐visible cellular therapeutic with enhanced efficacy for PAD treatment. STEM CELLS2012;30:1286–1296

Microencapsulated cell tracking

Microencapsulation of therapeutic cells has been widely pursued to achieve cellular immunoprotection following transplantation. Initial clinical studies have shown the potential of microencapsulation using semi‐permeable alginate layers, but much needs to be learned about the optimal delivery route, in vivo pattern of engraftment, and microcapsule stability over time. In parallel with noninvasive imaging techniques for ‘naked’ (i.e. unencapsulated) cell tracking, microcapsules have now been endowed with contrast agents that can be visualized by 1H MRI, 19F MRI, X‐ray/computed tomography and ultrasound imaging. By placing the contrast agent formulation in the extracellular space of the hydrogel, large amounts of contrast agents can be incorporated with negligible toxicity. This has led to a new generation of imaging biomaterials that can render cells visible with multiple imaging modalities. Copyright

Stem cell labeling for noninvasive delivery and tracking in cardiovascular regenerative therapy

Clinical and basic scientific studies of stem cell-based therapies have shown promising results for cardiovascular diseases. Despite a rapid transition from animal studies to clinical trials, the mechanisms by which stem cells improve heart function are yet to be fully elucidated. To optimize cell therapies in patients will require a noninvasive means to evaluate cell survival, biodistribution and fate in the same subject over time. Cell labeling offers the ability to image distinct cell lineages in vivo and investigate the efficacy of these therapies using standard noninvasive imaging techniques. In this article, we will discuss the most promising cell labeling techniques for translation to clinical cardiovascular applications.

Fused X-ray and MR Imaging Guidance of Intrapericardial Delivery of Microencapsulated Human Mesenchymal Stem Cells in Immunocompetent Swine

PURPOSEnTo assess intrapericardial delivery of microencapsulated, xenogeneic human mesenchymal stem cells (hMSCs) by using x-ray fused with magnetic resonance (MR) imaging (x-ray/MR imaging) guidance as a potential treatment for ischemic cardiovascular disease in an immunocompetent swine model.nnnMATERIALS AND METHODSnAll animal experiments were approved by the institutional animal care and use committee. Stem cell microencapsulation was performed by using a modified alginate-poly-l-lysine-alginate encapsulation method to include 10% (wt/vol) barium sulfate to create barium-alginate microcapsules (BaCaps) that contained hMSCs. With x-ray/MR imaging guidance, eight female pigs (approximately 25 kg) were randomized to receive either BaCaps with hMSCs, empty BaCaps, naked hMSCs, or saline by using a percutaneous subxiphoid approach and were compared with animals that received empty BaCaps (n = 1) or BaCaps with hMSCs (n = 2) by using standard fluoroscopic delivery only. MR images and C-arm computed tomographic (CT) images were acquired before injection and 1 week after delivery. Animals were sacrificed immediately or at 1 week for histopathologic validation. Cardiac function between baseline and 1 week after delivery was evaluated by using a paired Student t test.nnnRESULTSnhMSCs remained highly viable (94.8% ± 6) 2 days after encapsulation in vitro. With x-ray/MR imaging, successful intrapericardial access and delivery were achieved in all animals. BaCaps were visible fluoroscopically and at C-arm CT immediately and 1 week after delivery. Whereas BaCaps were free floating immediately after delivery, they consolidated into a pseudoepicardial tissue patch at 1 week, with hMSCs remaining highly viable within BaCaps; naked hMSCs were poorly retained. Follow-up imaging 1 week after x-ray/MR imaging-guided intrapericardial delivery showed no evidence of pericardial adhesion and/or effusion or adverse effect on cardiac function. In contradistinction, BaCaps delivery with x-ray fluoroscopy without x-ray/MR imaging (n = 3) resulted in pericardial adhesions and poor hMSC viability after 1 week.nnnCONCLUSIONnIntrapericardial delivery of BaCaps with hMSCs leads to high cell retention and survival. With x-ray/MR imaging guidance, intrapericardial delivery can be performed safely in the absence of preexisting pericardial effusion to provide a novel route for cardiac cellular regenerative therapy.

Using C-Arm X-Ray Imaging to Guide Local Reporter Probe Delivery for Tracking Stem Cell Engraftment

Poor cell survival and difficulties with visualization of cell delivery are major problems with current cell transplantation methods. To protect cells from early destruction, microencapsulation methods have been developed. The addition of a contrast agent to the microcapsule also could enable tracking by MR, ultrasound, and X-ray imaging. However, determining the cell viability within the microcapsule still remains an issue. Reporter gene imaging provides a way to determine cell viability, but delivery of the reporter probe by systemic injection may be hindered in ischemic diseases. In the present study, mesenchymal stem cells (MSCs) were transfected with triple fusion reporter gene containing red fluorescent protein, truncated thymidine kinase (SPECT/PET reporter) and firefly luciferase (bioluminescence reporter). Transfected cells were microencapsulated in either unlabeled or perfluorooctylbromide (PFOB) impregnated alginate. The addition of PFOB provided radiopacity to enable visualization of the microcapsules by X-ray imaging. Before intramuscular transplantation in rabbit thigh muscle, the microcapsules were incubated with D-luciferin, and bioluminescence imaging (BLI) was performed immediately. Twenty-four and forty-eight hours post transplantation, c-arm CT was used to target the luciferin to the X-ray-visible microcapsules for BLI cell viability assessment, rather than systemic reporter probe injections. Not only was the bioluminescent signal emission from the PFOB-encapsulated MSCs confirmed as compared to non-encapsulated, naked MSCs, but over 90% of injection sites of PFOB-encapsulated MSCs were visible on c-arm CT. The latter aided in successful targeting of the reporter probe to injection sites using conventional X-ray imaging to determine cell viability at 1-2 days post transplantation. Blind luciferin injections to the approximate location of unlabeled microcapsules resulted in successful BLI signal detection in only 18% of injections. In conclusion, reporter gene probes can be more precisely targeted using c-arm CT for in vivo transplant viability assessment, thereby avoiding large and costly systemic injections of a reporter probe.

Intrapericardial delivery of visible microcapsules containing stem cells using xfm (x-ray fused with magnetic resonance imaging)

Previously, we have demonstrated a technique to enhance survival of transplanted cells using XFM-visible microcapsules. However, theoretical concerns exist about the induction of arrhythmias if microcapsules are delivered to the heart by transendocardial injection. Delivery of these microcapsules to the pericardial space may provide an alternative approach with less potential for arrhythmia. Pericardial approaches to the epicardium have been used for the delivery of cardioactive drugs, cardiac ablation techniques and the implantation of cardiac pacemakers. However, there are no reports involving the utilization of the pericardial space for stem cell delivery.

Bariatric Radioembolization: A Pilot Study on Technical Feasibility and Safety in a Porcine Model

PURPOSEnTo evaluate feasibility of left gastric artery (LGA) yttrium-90 ((90)Y) radioembolization as potential treatment for obesity in a porcine model.nnnMATERIALS AND METHODSnThis study included 8 young female pigs (12-13 weeks, 21.8-28.1 kg). Six animals received infusions of (90)Y resin microspheres (46.3-105.1 MBq) into the main LGA and the gastric artery arising from the splenic artery. Animal weight and serum ghrelin were measured before treatment and weekly thereafter. Animals were euthanized 69-74 days after treatment, and histologic analyses of mucosal integrity and ghrelin immunoreactive cell density were performed.nnnRESULTSnSuperficial mucosal ulcerations < 3.0 cm(2) were noted in 5 of 6 treated animals. Ghrelin immunoreactive cell density was significantly lower in treated versus untreated animals in the stomach fundus (13.5 vs 34.8, P < .05) and stomach body (11.2 vs 19.8, P < .05). Treated animals gained less weight than untreated animals over the study duration (40.2 kg ± 5.4 vs 54.7 kg ± 6.5, P = .053). Average fundic parietal area (165 cm(2) vs 282 cm(2), P = .067) and average stomach weight (297.2 g vs 397.0 g, P = .067) were decreased in treated versus untreated animals. Trichrome staining revealed significantly more fibrosis in treatment animals compared with control animals (13.0 vs 8.6, P < .05). No significant differences were identified in plasma ghrelin concentrations (P = .24).nnnCONCLUSIONSnLGA (90)Y radioembolization is promising as a potential treatment for obesity. A larger preclinical study is needed to evaluate the safety and efficacy of this procedure further.

Bariatric Arterial Embolization: Effect of Microsphere Size on the Suppression of Fundal Ghrelin Expression and Weight Change in a Swine Model

Purpose To determine whether microsphere size effects ghrelin expression and weight gain after selective bariatric arterial embolization (BAE) in swine. Materials and Methods BAE was performed in 10 swine by using smaller (100-300 μm; n = 5) or larger (300-500 μm; n = 5) calibrated microspheres into gastric arteries. Nine control pigs underwent a sham procedure. Weight and fasting plasma ghrelin levels were measured at baseline and weekly for 16 weeks. Ghrelin-expressing cells (GECs) in the stomach were assessed by using immunohistochemical staining and analyzed by using the Wilcoxon rank-sum test. Results In pigs treated with smaller microspheres, mean weight gain at 16 weeks (106.9% ± 15.0) was less than in control pigs (131.9% ± 11.6) (P < .001). Mean GEC density was lower in the gastric fundus (14.8 ± 6.3 vs 25.0 ± 6.9, P < .001) and body (27.5 ± 12.3 vs 37.9 ± 11.8, P = .004) but was not significantly different in the gastric antrum (28.2 ± 16.3 vs 24.3 ± 11.6, P = .84) and duodenum (9.2 ± 3.8 vs 8.7 ± 2.9, P = .66) versus in control pigs. BAE with larger microspheres failed to suppress weight gain or GECs in any stomach part compared with results in control swine. Plasma ghrelin levels were similar between BAE pigs and control pigs, regardless of microsphere size. Week 1 endoscopic evaluation for gastric ulcers revealed none in control pigs, five ulcers in five pigs embolized by using smaller microspheres, and three ulcers in five pigs embolized by using larger microspheres. Conclusion In bariatric arterial embolization, smaller microspheres rather than larger microspheres showed greater weight gain suppression and fundal ghrelin expression with more gastric ulceration in a swine model.