Cell Delivery Techniques Research Articles

Stem cells in articular cartilage regeneration.

Mesenchymal stem cells (MSCs) have emerged as a promising option to treat articular defects and early osteoarthritis (OA) stages. However, both their potential and limitations for a clinical use remain controversial. Thus, the aim of this systematic review was to examine MSCs treatment strategies in clinical settings, in order to summarize the current evidence of their efficacy for the treatment of cartilage lesions and OA.Among the 60 selected studies, 7 were randomized, 13 comparative, 31 case series, and 9 case reports; 26 studies reported the results after injective administration, whereas 33 used surgical implantation. One study compared the two different modalities. With regard to the cell source, 20 studies concerned BMSCs, 17 ADSCs, 16 BMC, 5 PBSCs, 1 SDSCs, and 1 compared BMC versus PBSCs. Overall, despite the increasing literature on this topic, the evidence is still limited, in particular for high-level studies. On the other hand, the available studies allow to draw some indications. First, no major adverse events related to the treatment or to the cell harvest have been reported. Second, a clinical benefit of using MSCs therapies has been reported in most of the studies, regardless of cell source, indication, or administration method. This effectiveness has been reflected by clinical improvements and also positive MRI and macroscopic findings, whereas histologic features gave more controversial results among different studies. Third, young age, lower BMI, smaller lesion size for focal lesions, and earlier stages of OA joints have been shown to correlate with better outcomes, even though the available data strength does not allow to define clear cutoff values. Finally, definite trends can be observed with regard to the delivery method: currently cultured cells are mostly being administered by i.a. injection, while one-step surgical implantation is preferred for cell concentrates. In conclusion, while promising results have been shown, the potential of these treatments should be confirmed by reliable clinical data through double-blind, controlled, prospective and multicenter studies with longer follow-up, and specific studies should be designed to identify the best cell sources, manipulation, and delivery techniques, as well as pathology and disease phase indications.

Aerosol-Based Cell Therapy for Treatment of Lung Diseases.

Aerosol-based cell delivery technique via intratracheal is an effective route for delivering transplant cells directly into the lungs. An aerosol device known as the MicroSprayer(®) Aerosolizer is invented to transform liquid into an aerosol form, which then can be applied via intratracheal administration for drug delivery. The device produces a uniform and concentrated distribution of aerosolized liquid. Using the capability of MicroSprayer(®) Aerosolizer to transform liquid into aerosol form, our group has designed a novel method of cell delivery using an aerosol-based technique. We have successfully delivered skin-derived fibroblast cells and airway epithelial cells into the airway of a rabbit with minimum risk of cell loss and have uniformly distributed the cells into the airway. This chapter illustrates the application of aerosol device to deliver any type of cells for future treatment of lung diseases.

Progenitor Cell Therapy to Treat Acute Myocardial Infarction: The Promise of High-Dose Autologous CD34+Bone Marrow Mononuclear Cells

ST elevation myocardial infarction (STEMI) is associated with an increased risk for congestive heart failure and long-term mortality despite the widespread use of thrombolysis and catheter-based revascularization. The need for improved post-STEMI therapies has led to a surge of novel therapeutics, especially regenerative approaches using autologous mononuclear cells. Indeed, the past decade has been marked by a number of human trials studying the safety and efficacy of progenitor cell delivery in the post-STEMI setting. While a variety of cell types and delivery techniques have been utilized, directed therapy to the infarct-related artery has been the most widely used approach. From over 1300 subjects randomized in these studies, there is sufficient evidence to conclude that cell therapy after STEMI is uniformly safe, while the efficacy of this intervention for improving outcomes is less clear. Recent meta-analyses have highlighted the importance of both timing of cell delivery, as well as the type, quantity, and mobility of delivered cells as determinants of response. Here, we show the case in which higher doses of CD34+ cells, which are more potent in terms of their migratory capacity, offer the best hope for preserving cardiac function following STEMI.

Optimization of an Intra-Bone Hematopoietic Stem Cell Delivery Technique in a Swine Model.

AbstractAbstract 2990 IntroductionTransplantation of allogeneic hematopoietic progenitor cells (HPC) is an effective treatment for a variety of hematological diseases. Intravenous (IV) injection is the routine method for HPC transplantation, based on the concept of “homing” to the bone marrow. To date, intrabone (IB) HPC transplantation has largely been investigated in the preclinical setting with only limited application in humans. Furthermore, an optimized method for the direct IB infusion of HPC in humans which maximizes cellular retention in the bone has not yet been developed. In this study, we aimed to optimize the IB transplantation procedure using a large animal model with the goal of retaining HPCs within the pelvic bone and to explore the feasibility of radionuclide labeling to assess the trafficking of HPCs using PET/CT imaging early after IB transplantation. MethodsHPC collection: a) Human HPCswere mobilized from healthy volunteers using G-CSF then were positively selected for CD34+ cells using immuno-magnetic beads (MiltenyiBiotec, MA) then were cryopreserved. b) Porcine bone marrow (BM) cells were aspirated (approximately 40 ml) from the iliac crest of swine then were filtered and mononuclear cells (MNCs) were isolated using Ficoll-Paque™ with density gradient separation. All animal procedures were conducted using domestic swine (Susscrofadomesticus) on NHLBI Animal Use Committee approved protocols. IB access in animals was initially achieved using the OnControl driver (Vidacare Corp. TX). To evaluate flow through the marrow and venous drainage, direct IB injection into the hemipelvis with iopamidol-370 contrast was performed under anesthesia with dynamic CT images acquired using a 320-detector row scanner (Aquilion One, Toshiba Medical, Japan). Human CD34+ and swine BM MNCs were labeled with Zirconium-89 (89Zr) then were assessed for viability, cell number, and the level of cellular radioactivity. Radiolabeled cells were then injected into pigs either IV or directly IB into the porcine pelvis at different infusion rates. Intramarrow(IM) pressures were measured continuously during IB injection using Millarcatheters and acquired simultaneously with intra-arterial pressure and electrocardiography on a PowerLab data acquisition system (ADInstruments, CO) and analyzed using LabChart 7. After injection of labeled cells, positron emission tomography (PET) images were acquired for up to 180 minutes with a clinical PET/CT system (Gemini TF, Philips Medical Systems, MA) to assess cellular distribution and homing. ResultsPeak IM pressures during bolus hand IB injection were high, substantially exceeding systemic systolic arterial pressures. In contrast, IM pressures during slow IB infusion were significantly lower, remaining well below diastolic arterial pressures. During manual sequential hand IB injection of 5 ml aliquots of contrast at two different sites in the ipsilateral iliac crest, dynamic CT images revealed leakage from the initial access site after the first injection as well as immediate drainage into the ipsilateral iliac vein. Following manual hand injection of 89Zr labeled human CD34+ cells (89Zr-hCD34+) given IV in swine via the external jugular vein, there was persistent PET activity noted in the lungs for up to 3 hrs. Bolus hand IB injection of 89Zr labeled swine BM MNCs or 89Zr-hCD34+ cells revealed PET activity in the iliac bone as well as activity in the lungs. Furthermore, PET activity following bolus hand IB injection was also noted in surrounding tissues outside the bone when more than a single ipsilateral injection site was used. In contrast, slow infusion of 89Zr labeled swine BMMNCs or 89Zr-hCD34+cells resulted in PET activity that was limited to the iliac bone, indicating retention of cells within the marrow space with no leakage of cells to the lungs. ConclusionRapid hand infusion of HPCs into the pelvic bone results in cellular leakage out of the marrow space into the lungs. In contrast, slow IB infusion of HPCs localizes cells to the bone marrow without leakage to the lungs. These data suggest maintaining low IM pressures may be critical to maximize cellular trapping in the marrow space following IB HPC transplantation in humans. Further study will be required to determine whether this optimized IB transplantation approach can be used to improve engraftment in recipients of transplants containing low HPC numbers. Disclosures:No relevant conflicts of interest to declare.

New cell therapies in cardiology

Even today, cardiovascular disease remains the leading cause of death globally. Cardiological conditions such as myocardial infarction, ischemic injury and chronic cardiomyopathy result in permanent cardiac tissue damage followed by heart failure. Current therapies primarily aim to trigger the pathological remodeling that occurs after cardiac injury and also to reduce risk factors involved in cardiovascular diseases. Animal model studies over the last decade indicate that the systematic administration of autologous and allogeneic stem cells possesses therapeutic potential to improve overall cardiac functions. This evidence robustly indicates that cardiac tissue has the potential to undergo a systematic repair process. The past few years have also witnessed a splurge in clinical research that particularly aims to explore the regenerative properties of the naive stem cells to restore cardiac functions. The mechanisms involved in stem cell therapy remain unclear. The magnitude of benefit demonstrated in animal models is yet to be completely translated into humans. The future of cardiac research will require tangible synchrony between clinicians and basic scientists to unravel the precise mechanism of stem cell therapy. It is also pivotal to define an ideal cell type and a suitable cell delivery technique that provide maximum benefit, while eliminating the grey areas in translational cardiology research. In this article, the authors review the properties and therapeutic potential of the stem cell plethora reported for cardiac repair and regeneration, recent stem cell therapies, mode of action, their delivery techniques, recent clinical developments and the future for these stem cell therapies in cardiology.

Heart to heart: Cardiospheres for myocardial regeneration

Cardiac regenerative therapies seek to grow new myocardium after "irreversible" injury such as myocardial infarction. Various cell types and delivery techniques have been used in experimental models of human disease and clinical trials. When selecting a candidate stem cell type for clinical use, multiple factors need to be taken into consideration. The ability to regenerate myocardium without potentiating arrhythmogenesis is a critical property. Skeletal myoblasts engraft, differentiate, and are arrhythmogenic; in contrast, bone marrow-derived cells do not engraft long-term and have not been associated with excess arrhythmias. Neither cell type, however, achieves true myocardial regeneration. Recognition of the existence of cardiac stem cells and of the ability of mature myocytes to reenter the cell cycle and proliferate has motivated the development of new approaches to cardiac regenerative medicine. Cardiosphere-derived cells decrease scar mass and regenerate viable myocardium both in animal models and in the CADUCEUS (Cardiosphere-Derived Cells For Heart Regeneration After Myocardial Infarction) clinical trial. Although cardiosphere-derived cells fulfill the criteria for stem cells, their stemness appears not to mediate the therapeutic benefit; instead, indirect mechanisms lead to proliferation of the host myocardium. Being of endogenous origin, the newly grown heart muscle is electrically and mechanically well integrated with preexisting myocardial tissue. We hypothesize that cardiac arrhythmias are less likely to complicate cell therapy when the mechanisms of benefit involve secondary proliferation of endogenous myocardium. Conversely, arrhythmias will more likely bedevil therapeutic approaches (such as transplantation of skeletal myoblasts or pluripotent stem cells) that lead to exogenous grafts within the heart, with the degree of coupling and the extent of inhomogeneity being critical determinants of the net effect.

Rapid magnetic cell delivery for large tubular bioengineered constructs.

Delivery of cells into tubular tissue constructs with large diameters poses significant spatial and temporal challenges. This study describes preliminary findings for a novel process for rapid and uniform seeding of cells onto the luminal surface of large tubular constructs. Fibroblasts, tagged with superparamagnetic iron oxide nanoparticles (SPION), were directed onto the luminal surface of tubular constructs by a magnetic field generated by a k4-type Halbach cylinder device. The spatial distribution of attached cells, as measured by the mean number of cells, was compared with a conventional, dynamic, rotational cell-delivery technique. Cell loading onto the constructs was measured by microscopy and magnetic resonance imaging. The different seeding techniques employed had a significant effect on the spatial distribution of the cells (p < 0.0001). The number of attached cells at defined positions within the same construct was significantly different for the dynamic rotation technique (p < 0.05). In contrast, no significant differences in the number of cells attached to the luminal surface were found between the defined positions on the construct loaded with the Halbach cylinder. The technique described overcomes limitations associated with existing cell-delivery techniques and is amenable to a variety of tubular organs where rapid loading and uniform distribution of cells for therapeutic applications are required.

Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy

Although there is tremendous interest in stem cell (SC)-based therapies for cardiomyopathy caused by chronic myocardial infarction, many unanswered questions regarding the best approach remain. The TAC-HFT study is a phase I/II randomized, double-blind, placebo-controlled trial designed to address several of these questions, including the optimal cell type, delivery technique, and population. This trial compares autologous mesenchymal SCs (MSCs) and whole bone marrow mononuclear cells (BMCs). In addition, the study will use a novel helical catheter to deliver cells transendocardially. Although most trials have used intracoronary delivery, the optimal method is unknown and data suggest that the transendocardial approach may have important advantages. Several trials support the benefit of SCs in patients with chronic ischemic cardiomyopathy (ICMP), although the sample sizes have been small and the number of trials sparse. After a pilot phase of 8 patients, 60 patients with ICMP (left ventricular ejection fraction 15%-50%) will be randomized to group A (30 patients further randomized to receive MSC injection or placebo in a 2:1 fashion) or group B (30 patients further randomized to BMCs or placebo in a 2:1 fashion). All patients will undergo bone marrow aspiration and transendocardial injection of SCs or placebo. The primary and secondary objectives are, respectively, to demonstrate the safety and efficacy (determined primarily by cardiac magnetic resonance imaging) of BMCs and MSCs administered transendocardially in patients with ICMP.

Non-surgical stem cell delivery strategies and in vivo cell tracking to injured myocardium

Heart failure is a major economic and public health problem. Despite the recent advances in drug therapy and coronary revascularization, the lost cardiomyocytes due to necrosis and apoptosis are not replaced by new myocardial tissue. Cell therapy is an interesting therapeutic option as it potentially improves contractility and restores regional ventricular function. Early clinical data demonstrated that cell transplantation, mainly delivered through non-surgical methods, is safe and feasible. However, several important issues need to be elucidated. This includes, next to determining the best cell type, the optimal delivery strategy, the biodistribution and the survival of implanted stem cells after transplantation. In this view, pre-clinical animal experiments are indispensable. Reporter genes, magnetic or radioactive labeling of stem cells have been developed to observe the fate and the distribution of transplanted cells using non-invasive imaging techniques. Several studies have demonstrated that these direct and non-direct labeling techniques may become an important tool in cell therapy. Integration of cell delivery and cell tracking will probably be a key for the success of cell therapy in patients. This review will provide a comprehensive overview on the various cell tracking and non-surgical cell delivery techniques, which are highly important in view of experimental and clinical studies.

Bio‐electrospraying: A potentially safe technique for delivering progenitor cells

Bio-electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their "stemness" is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6 mL/h and voltages of 7.5-15 kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5 kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio-electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications.

Labeling human embryonic stem cell-derived cardiomyocytes with indocyanine green for noninvasive tracking with optical imaging: an FDA-compatible alternative to firefly luciferase.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37 degrees C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data (p < 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Labeling Human Embryonic Stem Cell-Derived Cardiomyocytes with Indocyanine Green for Noninvasive Tracking with Optical Imaging: An FDA-Compatible Alternative to Firefly Luciferase

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37°C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data ( p &lt; 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Invited Commentary

In recent years, stem cell therapy has received considerable attention as a novel therapeutic option for refractory congestive heart failure. Although the mechanisms through which these cells elicit functional improvement are yet to be fully determined, recent studies suggest that paracrine effects, rather than direct trans-differentiation of donor cells into cardiomyocytes, may predominate in augmenting postinfarction cardiac function. Progress in cell therapy, however, is hampered by suboptimal engraftment of donor cells into the myocardium, related to the extensive cell death that ensues from the hostile milieu into which the donor cells are introduced. This problem has stimulated further investigation of the use of bioengineered scaffolds as cell delivery vehicles, with the hope that these constructs may enhance cell retention and functional recovery. In this study, Hamdi and colleagues [1Hamdi H. Furuta A. Bellamy V. et al.Cell delivery: intramyocardial injections or epicardial deposition? A head-to-head comparison.Ann Thorac Surg. 2009; 87: 1196-1204Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar] evaluated the effect of differing cell delivery techniques on postinfarct heart function. Using skeletal myoblasts as donor cells in a rat infarct model, the authors compared the conventional intramyocardial injection technique with epicardial placement of two cell-seeded constructs, one made of a double-layered myoblast cell sheet and the other constructed from a cell-seeded Gelfoam (Pharmacia & Upjohn/Pfizer, New York, NY) patch. The authors concluded that the two epicardially delivered tissue scaffolds led to significant improvements in left ventricular ejection fraction (LVEF) compared with the controls injected with culture medium, whereas intramyocardial injection prevented further deterioration of ventricular function but did not improve LVEF. Histologic analysis also showed greater angiogenesis in the two epicardially treated groups. The major quandary posed by these findings is that the effects of the varying techniques of cell transfer were unrelated to the detectable persistence of the myoblasts at the studied time point. Analysis of cell engraftment revealed minimal donor cell retention, with residual human cells detected in only 2 of 7 hearts in each of the two groups treated with the epicardial scaffold. The authors attribute the between-group differences in LVEF to donor cell effects at earlier, unevaluated time points (ie, earlier than 1 month). Their analysis of cytokines 1 month after cell delivery showed no differences between groups that might explain differences in functional recovery, but this, like the absence of persistent donor cells, may merely be a reflection of the time point chosen. Somewhat surprisingly, end-systolic and end-diastolic volumes did not differ between groups. Thus, although the cell sheets and Gelfoam patch improved LVEF, they did not do so by reducing infarct expansion compared with intramyocardial injection. Ultimately, the lack of a demonstrable mechanism to explain the differences observed between groups somewhat limits the strength of the conclusions that can be drawn about the relative merits of these different cell delivery techniques. Regardless, this is a topical study that compares the conventional intramyocardial delivery method to two potentially attractive methods of epicardial delivery. The results are provocative, and should spur further investigation to determine whether there are intrinsic benefits in using biomaterials in the setting of a recent or remote myocardial infarction. If there are, what are the mechanisms through which these constructs exert synergistic effects with the seeded cells? The optimal cell type or mixture of cell types that is best suited to a particular scaffold material has not been determined, nor have the physical, electrical, or surface characteristics that render one cell type more appropriate for seeding than another. Those studies may eventually lead to the development of off-the-shelf bioengineered cell-seeded constructs that may sidestep the issues of cell harvesting, expansion, and delivery that complicate current applications of cell therapy. Cell Delivery: Intramyocardial Injections or Epicardial Deposition? A Head-to-Head ComparisonThe Annals of Thoracic SurgeryVol. 87Issue 4PreviewMultiple needle-based injections of cells in the myocardium are associated with a low engraftment rate, which may limit the benefits of the procedure. This study used skeletal myoblasts to perform a head-to-head comparison of conventional injections with epicardial deposition of scaffold-embedded cells. Full-Text PDF

Stem Cell Implantation for Myocardial Disorders

Cell therapy is currently attracting growing interest as a potential new means of improving the prognosis of patients with heart failure. For practical reasons, autologous skeletal myoblasts have been the first to be tested in clinical trials, but recently cardiovascular researchers has explored many other cell types, including bone marrow cells, endothelial progenitor cells, mesenchymal stem cells, embryonic stem cells, and resident cardiac stem cells. While recent experimental studies and early-phase clinical trials seem to support the concept that cell therapy may enhance cardiac repair, many challenges remain before achieving this goal. Further studies should focus on finding the optimal donor cells for transplantation, the mechanism by which engrafted cells improve cardiac function, controlling the survival and proliferation of transplanted cells, and the development of more efficient cell delivery techniques.

A Tissue Engineering Approach to Progenitor Cell Delivery Results in Significant Cell Engraftment and Improved Myocardial Remodeling

Cell replacement therapy has become an attractive solution for myocardial repair. Typical cell delivery techniques, however, suffer from poor cell engraftment and inhomogeneous cell distributions. Therefore, we assessed the hypothesis that an epicardially applied, tissue-engineered cardiac patch containing progenitor cells would result in enhanced exogenous cell engraftment. Human mesenchymal stem cells (hMSCs) were embedded into a rat tail type I collagen matrix to form the cardiac patch. Myocardial infarction was induced by left anterior descending coronary artery ligation in immunocompetent male cesarean-derived fischer rats, and patches with or without cells were secured to hearts with fibrin sealant. After patch formation, hMSCs retained a viability of >90% over 5 days in culture. In addition, >75% of hMSCs maintained a high degree of potency prior to patch implantation. After 4 days in culture, patches were applied to the epicardial surface of the infarct area and resulted in 23% +/- 4% engraftment of hMSCs at 1 week (n = 6). Patch application resulted in a reduction in left ventricle interior diameter at systole, increased anterior wall thickness, and a 30% increase in fractional shortening. Despite this improvement in myocardial remodeling, hMSCs were not detectable at 4 weeks after patch application, implying that improvement did not require long-term cell engraftment. Patches devoid of progenitor cells showed no improvement in remodeling. In conclusion, pluripotent hMSCs can be efficiently delivered to a site of myocardial injury using an epicardial cardiac patch, and such delivery results in improved myocardial remodeling after infarction. Disclosure of potential conflicts of interest is found at the end of this article.

Percutaneous transplantation of genetically-modified autologous fibroblasts in the rabbit femoral artery: a novel approach for cardiovascular gene therapy

Arterial cell and gene therapies are promising strategies for the treatment of cardiovascular diseases; however, the optimal cell type and delivery technique for such treatment remain to be determined. The aim of the present study was to design a new approach for arterial cell and gene therapy in which genetically modified autologous skin fibroblasts are percutaneously delivered in stented rabbit femoral arteries in vivo. Autologous skin fibroblasts underwent in vitro transfection with the cationic lipid FuGene and plasmids expressing the human form of the tissue inhibitor of metalloproteinase (hTIMP-1) or nls-LacZ reporter genes. Transfection efficiency was about 50% and there were high levels of hTIMP-1 secretion up to 14 days after gene transfer. We demonstrated the feasibility of in vivo percutaneous transplantation of fluorescent fibroblasts in the rabbit femoral artery. Results were confirmed by scanning electron microscopy. In vivo local delivery of hTIMP-1-expressing fibroblasts in stented femoral arteries also resulted in high-levels of hTIMP-1 secretion ex vivo for 7 days. Fibroblast transplantation resulted in a modest increase in intimal hyperplasia at the target site, which was reversed with hTIMP-1-transfected fibroblasts. Percutaneous transplantation of genetically modified autologous fibroblasts could be used as a cellular platform for locoregional secretion of therapeutic proteins to treat either specific arterial diseases or the diseased organ (eg, the heart) supplied by the target artery. Cell and gene therapies are potential new treatments for cardiovascular diseases. We demonstrated that autologous fibroblasts could be easily harvested from a skin biopsy specimen, genetically modified in vitro with nonviral vectors, and percutaneously seeded in vivo in rabbit femoral arteries, leading to locoregional secretion of abundant amounts of recombinant proteins. This new approach has important advantages over alternative approaches that use endothelial cells, viral vectors, and intraoperative cell delivery. Clinical applications may include local treatment of atherosclerotic plaques or aneurysms and also treatment of the diseased organs supplied by the target artery (eg, ischemic or failing heart).

Myogenic Potential of Muscle Side and Main Population Cells after Intravenous Injection into Sub-lethally IrradiatedmdxMice

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.

Intramyocardial microdepot injection increases the efficacy of skeletal myoblast transplantation

Recent progress in the field of cellular cardiomyoplasty has opened new prospects for the treatment of ischemic heart disease and currently moves from bench to bedside. The aim of the present study was to develop a novel cell delivery technique, reducing target tissue damage and improving cell dispersion and engraftment. In 30 male Fischer F344 rats an infarction of the left ventricle was generated by ligation of the left anterior descendent artery. Seven days after infarction, either 15 microdepots of 10 microl myoblast cell suspension (microdepot group) or culture medium (control group) were injected into the infarcted region using an automatic pressure injection device, or three depots of 50 microl myoblast cell suspension (macrodepot group) were injected using the standard surgical technique. Echocardiography was performed in all rats before and 6 weeks after cell injection. In all groups the perioperative mortality was below 20%. Six weeks after cell transplantation, a significant improvement of ejection fraction was seen in both myoblast treated groups compared to controls (macrodepot, microdepot, control; 53.7+/-11.9, 70.7+/-2.0, 39.1+/-6.4; P=0.026, P<0.001). The microdepot group showed a more decent improvement than the macrodepot group (70.7+/-2.0 vs. 53.7+/-11.9, P=0.013). In both treated groups, grafted myoblasts differentiated into multinucleated myotubes within host myocardium, however, the engraftment pattern was different and angiogenesis was enhanced in the microdepot group. Intramyocardial multisite pressure injection allows the safe and reliable transplantation of several myoblast microdepots into an infarcted myocardium and improves the efficacy of myoblast transplantation compared to the standard technique.

Stem cells for clinical use in cardiovascular medicine

Cell transplantation is currently gaining a growing interest as a potential new means of improving the prognosis of patients with cardiac failure. The basic assumption is that left ventricular dysfunction is largely due to the loss of a critical number of cardiomyocytes and that it can be partly reversed by implantation of new contractile cells into the postinfarction scars. Primarily for practical reasons, autologous skeletal myoblasts have been the first to undergo clinical trials and now that the feasibility of the procedure is well established, efficacy data are expected from the ongoing randomized studies. Bone marrow stem cells are also generating a great deal of interest, particularly in patients with acute myocardial infarction, and are currently undergoing extensive clinical testing although recent data have raised a cautionary note about the transdifferentiation potential of these cells. While experimental studies and early-phase clinical trials tend to support the concept that cell therapy may enhance cardiac repair, several key issues still need to be addressed including (1) the optimal type of donor cells in relation to the clinical profile of the patients, (2) the mechanism by which cell engraftment improves cardiac function, (3) the optimization of cell survival, (4) the development of less invasive cell delivery techniques and (5) the potential benefits of cell transplantation in nonischemic heart failure. Current evidence suggests, however, that adult stem cells (myogenic or marrow-derived) fail to electromechanically integrate within the recipient heart, thereby mandating the search for second generation cell types able to achieve this goal which is the prerequisite for an effective enhancement of contractile function. Preliminary data suggest that cells that feature a true cardiomyogenic phenotype such as cardiac stem cells and cardiac-precommitted embryonic stem cells may fall in this category and carry the potential for ensuring a true regeneration of dead myocardium.