Yasuo Miyagi

Differentiation of Allogeneic Mesenchymal Stem Cells Induces Immunogenicity and Limits Their Long-Term Benefits for Myocardial Repair

Background— Cardiac cell therapy for older patients who experience a myocardial infarction may require highly regenerative cells from young, healthy (allogeneic) donors. Bone marrow mesenchymal stem cells (MSCs) are currently under clinical investigation because they can induce cardiac repair and may also be immunoprivileged (suitable for allogeneic applications). However, it is unclear whether allogeneic MSCs retain their immunoprivilege or functional efficacy late after myocardial implantation. We evaluated the effects of MSC differentiation on the immune characteristics of cells in vitro and in vivo and monitored cardiac function for 6 months after post–myocardial infarction MSC therapy. Methods and Results— In the in vitro experiments, inducing MSCs to acquire myogenic, endothelial, or smooth muscle characteristics (via 5-azacytidine or cytokine treatment) increased major histocompatibility complex-Ia and -II (immunogenic) expression and reduced major histocompatibility complex-Ib (immunosuppressive) expression, in association with increased cytotoxicity in coculture with allogeneic leukocytes. In the in vivo experiments, we implanted allogeneic or syngeneic MSCs into infarcted rat myocardia. We measured cell differentiation and survival (immunohistochemistry, real-time polymerase chain reaction) and cardiac function (echocardiography, pressure-volume catheter) for 6 months. MSCs (versus media) significantly improved ventricular function for at least 3 months after implantation. Allogeneic (but not syngeneic) cells were eliminated from the heart by 5 weeks after implantation, and their functional benefits were lost within 5 months. Conclusions— The long-term ability of allogeneic MSCs to preserve function in the infarcted heart is limited by a biphasic immune response whereby they transition from an immunoprivileged to an immunogenic state after differentiation, which is associated with an alteration in major histocompatibility complex–immune antigen profile.

Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair.

Vascularization of engineered tissues in vitro and in vivo remains a key problem in translation of engineered tissues to clinical practice. Growth factor signalling can be prolonged by covalent tethering, thus we hypothesized that covalent immobilization of vascular endothelial growth factor (VEGF-165) to a porous collagen scaffold will enable rapid vascularization in vivo. Covalent immobilization may be preferred over controlled release or cell transfection if the effects are desired within the biomaterial rather than the surrounding tissue. Scaffolds were prepared with 14.5 ± 1.4 ng (Low) or 97.2 ± 8.0 ng (High) immobilized VEGF, or left untreated (control), and used to replace a full right ventricular free wall defect in rat hearts. In addition to rapid vascularization, an effective cardiac patch should exhibit neither thinning nor dilatation upon implantation. In vitro, VEGF enhanced the growth of endothelial and bone marrow cells seeded onto scaffolds. In vivo, High VEGF patches had greater blood vessel density (p < 0.01) than control at Day 7 and 28 due to increased cell recruitment and proliferation (p < 0.05 vs. control). At Day 28, VEGF-treated patches were significantly thicker (p < 0.05) than control, and thickness correlated positively with neovascularization (r = 0.67, p = 0.023). Importantly, angiogenesis in VEGF scaffolds contributed to improved cell survival and tissue formation.

The effect of cyclic stretch on maturation and 3D tissue formation of human embryonic stem cell-derived cardiomyocytes

The goal of cardiac tissue engineering is to restore function to the damaged myocardium with regenerative constructs. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can produce viable, contractile, three-dimensional grafts that function in vivo. We sought to enhance the viability and functional maturation of cardiac tissue constructs by cyclical stretch. hESC-CMs seeded onto gelatin-based scaffolds underwent cyclical stretching. Histological analysis demonstrated a greater proportion of cardiac troponin T-expressing cells in stretched than non-stretched constructs, and flow sorting demonstrated a higher proportion of cardiomyocytes. Ultrastructural assessment showed that cells in stretched constructs had a more mature phenotype, characterized by greater cell elongation, increased gap junction expression, and better contractile elements. Real-time PCR revealed enhanced mRNA expression of genes associated with cardiac maturation as well as genes encoding cardiac ion channels. Calcium imaging confirmed that stretched constructs contracted more frequently, with shorter calcium cycle duration. Epicardial implantation of constructs onto ischemic rat hearts demonstrated the feasibility of this platform, with enhanced survival and engraftment of transplanted cells in the stretched constructs. This uniaxial stretching system may serve as a platform for the production of cardiac tissue-engineered constructs for translational applications.

Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair

AIMS Ultrasound-targeted microbubble destruction (UTMD) uses ultrasound energy to selectively deliver genes into the myocardium using plasmids conjugated to microbubbles. We hypothesized that repeated delivery of stem cell-mobilizing genes could boost the ability of this therapy to enhance cardiac repair and ventricular function after a myocardial infarction. METHODS AND RESULTS Beginning 7 days after coronary artery ligation, stem cell factor (SCF) and stromal cell-derived factor (SDF)-1α genes were administered to adult rats using 1, 3, or 6 UTMD treatments (repeat 1, 3, and 6 groups) at 2-day intervals (control=6 treatments with empty plasmid). Cardiac function (echocardiography) and myocardial perfusion (myocardial contrast echocardiography) were assessed on Days -7, 0, and 24 relative to the first treatment. Histological and biochemical assessments were performed on Day 24. Multiple UTMD treatments were associated with an increased presence of myocardial SCF and SDF-1α proteins and their receptors (vs. control and Repeat 1). All UTMD recipients exhibited increased vascular densities and smaller infarct regions (vs. control), with the highest ventricular densities in response to multiple treatments. Myocardial perfusion and ventricular function at Day 24 also improved progressively (vs. control) with the number of UTMD treatments. CONCLUSIONS Targeted ultrasound delivery of SCF and SDF-1α genes to the infarcted myocardium recruited progenitor cells and increased vascular density. Multiple UTMD treatments enhanced tissue repair, perfusion, and cardiac function. Repeated UTMD therapy may be applied to tailor the number of interventions required to optimize cardiac regeneration after an infarction.

Surgical ventricular restoration with a cell- and cytokine-seeded biodegradable scaffold

Late after a myocardial infarction (MI), surgical ventricular restoration (SVR) can reduce left ventricular volumes, but an enhanced cardiac patch may be required to restore function. We developed a new, biodegradable patch (modified gelfoam, MGF) consisting of a spongy inner core (gelfoam) to encourage cell engraftment and an outer coating (poly epsilon-caprolactone) to provide sufficient strength to permit ventricular repair. Two weeks after coronary ligation in rats, SVR was performed using one of the following: gelfoam, MGF, MGF patches with hydrogel alone, or with hydrogel and cytokines (stem cell factor, stromal cell-derived factor-1alpha), bone marrow mesenchymal stem cells, or both. Cardiac function and morphology were evaluated by echocardiography, conduction catheterization, magnetic resonance imaging, and histology. Animals whose hearts were repaired with untreated gelfoam died of ventricular rupture. The MGF groups had significantly improved myocardial systolic function vs. MI controls. Enhancement with cytokines and/or cells promoted more alpha-smooth muscle actin-positive cells, more capillaries, greater wall thickness, a more ellipsoid shape, greater fractional shortening, and better-preserved systolic elastance than MGF alone. This combination of the new, reinforced, biodegradable biomaterial and cytokine/cell treatment created a viable tissue after SVR and produced better functional outcomes than un-reinforced gelfoam or MGF alone.

A Conductive Polymer Hydrogel Supports Cell Electrical Signaling and Improves Cardiac Function After Implantation into Myocardial Infarct

Background— Efficient cardiac function requires synchronous ventricular contraction. After myocardial infarction, the nonconductive nature of scar tissue contributes to ventricular dysfunction by electrically uncoupling viable cardiomyocytes in the infarct region. Injection of a conductive biomaterial polymer that restores impulse propagation could synchronize contraction and restore ventricular function by electrically connecting isolated cardiomyocytes to intact tissue, allowing them to contribute to global heart function. Methods and Results— We created a conductive polymer by grafting pyrrole to the clinically tested biomaterial chitosan to create a polypyrrole (PPy)-chitosan hydrogel. Cyclic voltammetry showed that PPy-chitosan had semiconductive properties lacking in chitosan alone. PPy-chitosan did not reduce cell attachment, metabolism, or proliferation in vitro. Neonatal rat cardiomyocytes plated on PPy-chitosan showed enhanced Ca2+ signal conduction in comparison with chitosan alone. PPy-chitosan plating also improved electric coupling between skeletal muscles placed 25 mm apart in comparison with chitosan alone, demonstrating that PPy-chitosan can electrically connect contracting cells at a distance. In rats, injection of PPy-chitosan 1 week after myocardial infarction decreased the QRS interval and increased the transverse activation velocity in comparison with saline or chitosan, suggesting improved electric conduction. Optical mapping showed increased activation in the border zone of PPy-chitosan–treated rats. Echocardiography and pressure–volume analysis showed improvement in load-dependent (ejection fraction, fractional shortening) and load-independent (preload recruitable stroke work) indices of heart function 8 weeks after injection. Conclusions— We synthesized a biocompatible conductive biomaterial (PPy-chitosan) that enhances biological conduction in vitro and in vivo. Injection of PPy-chitosan better maintained heart function after myocardial infarction than a nonconductive polymer.

VEGF-loaded microsphere patch for local protein delivery to the ischemic heart

BACKGROUND Revascularization of the heart after myocardial infarction (MI) using growth factors delivered by hydrogel-based microspheres represents a promising therapeutic approach for cardiac regeneration. Microspheres have tuneable degradation properties and support the prolonged release of soluble factors. Cardiac patches provide mechanical restraint, preventing dilatation associated with ventricular remodelling. METHODS We combined these approaches and produced a compacted calcium-alginate microsphere patch, restrained by a chitosan sheet, to deliver vascular endothelial growth factor (VEGF) to the heart after myocardial injury in rats. RESULTS Microspheres had an average diameter of 3.2μm, were nonporous, and characterized by a smooth dimpled surface. Microsphere patches demonstrated prolonged in vitro release characteristics compared to non-compacted microspheres and VEGF supernatants obtained from patches maintained their bioactivity for the 5day duration of the study in vitro. In vivo, patches were assessed with magnetic resonance imaging following MI, and demonstrated 50% degradation 25.6days after implantation. Both VEGF(-) and VEGF(+) microsphere patch-treated hearts had better cardiac function than unpatched (chitosan sheet only) controls. However, VEGF(+) microsphere-patched hearts had thicker scars characterized by higher capillary density in the border zone than did those treated with VEGF(-) patches. VEGF was detected in the patches 4weeks post-implantation. CONCLUSION The condensed microsphere patch represents a new therapeutic platform for cytokine delivery and could be used as an adjuvant to current biomaterial and cell-based therapies to promote localized angiogenesis in the infarcted heart. STATEMENT OF SIGNIFICANCE Following a heart attack, a lack of blood flow to the heart results in loss of heart cells. Growth factors may facilitate growth of blood vessels and heart tissue repair and prevent the onset of heart failure. Determining a way to deliver these growth factors directly to the heart is vital. Here, we combined two biomaterial-based approaches to deliver vascular endothelial growth factor (VEGF) to rat hearts after heart attack: a microsphere for prolonged release of VEGF, and a cardiac patch for mechanical restraint to prevent heart dysfunction. The feasibility of this microsphere patch was demonstrated by surgically implanting it over the infarct region of the heart post-injury. VEGF-patched hearts had better blood vessel growth, tissue repair, and heart function.

Fate of modular cardiac tissue constructs in a syngeneic rat model

Modular cardiac tissues developed both vascular and cardiac structures in vivo, provided that the host response was attenuated by omitting xenoproteins from the modules. Collagen gel modules (with MatrigelTM) containing cardiomyocytes (CMs) alone or CMs with surface‐seeded endothelial cells (ECs; CM/EC modules) were injected into the peri‐infarct zone of the heart in syngeneic Lewis rats. After 3 weeks, donor ECs developed into blood vessel‐like structures that also contained erythrocytes. However, no donor CMs were found within the implant sites, presumably because host cells including macrophages and T cells infiltrated extensively into the injection sites. To lessen the host response, Matrigel was omitted from the matrix and the modules were rinsed with serum‐free medium prior to implantation. Host cell infiltration was attenuated, resulting in a higher degree of vascularization with CM/EC modules than with CM modules without ECs. Most importantly, donor CMs matured into striated muscle‐like structures in Matrigel‐free implants. Copyright