Immune-modulatory alginate protects mesenchymal stem cells for sustained delivery of reparative factors to ischemic myocardium.

Paracrine factors secreted by mesenchymal stem cells (MSCs) have been previously shown to improve cardiac function following acute myocardial infarction (MI). However, cell therapy activates the innate immune response, leading to the rapid elimination of transplanted cells and only short-term therapeutic delivery. Herein, we describe a new strategy to deliver sustained paracrine-mediated MSC therapy to ischemic myocardium. Using an immune evasive, small molecule modified alginate, we encapsulated rat MSC cells in a core-shell hydrogel capsule and implanted them in the pericardial sac of post-MI rats. Encapsulated cells allowed diffusion of reparative paracrine factors at levels similar to non-encapsulated cells in vitro. Encapsulation enabled sustained cell survival with localization over the heart for 2 weeks. The effect of the experimental group on ventricular function and fibrosis was compared with blank (cell free) capsules and unencapsulated MSCs injected into infarcted myocardium. MSC capsules improved post-MI ventricular function ∼2.5× greater than MSC injection. After 4 weeks, post-MI fibrosis was reduced ∼2/3 with MSC capsules, but unchanged with MSC injection. MSC encapsulation with alginate core-shell capsules sustains cell survival and potentiates efficacy of therapy.

p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

OBJECTIVE: Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. METHODS: p63 Knockout (-/-) and knockdown murine embryonic fibroblasts (MEFs), p63-/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM). RESULTS: Flow cytometry revealed that a significantly greater number of p63-/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63-/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63-/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. CONCLUSIONS: Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.

The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis.

BACKGROUND: The stem cell factor spalt-like transcription factor 4 (SALL4) plays important roles in normal hematopoiesis and also in leukemogenesis. We previously reported that SALL4 exerts its effect by recruiting important epigenetic factors such as DNA methyltransferases DNMT1 and lysine-specific demethylase 1 (LSD1/KDM1A). Both of these proteins are critically involved in mixed lineage leukemia (MLL)-rearranged (MLL-r) leukemia, which has a very poor clinical prognosis. Recently, SALL4 has been further linked to the functions of MLL and its target gene homeobox A9 (HOXA9). However, it remains unclear whether SALL4 is indeed a key player in MLL-r leukemia pathogenesis. METHODS: Using a mouse bone marrow retroviral transduction/ transplantation approach combined with tamoxifen-inducible, CreERT2-mediated Sall4 gene deletion, we studied SALL4 functions in leukemic transformation that was induced by MLL-AF9-one of the most common MLL-r oncoproteins found in patients. In addition, the underlying transcriptional and epigenetic mechanisms were explored using chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq), mRNA microarray, qRT-PCR, histone modification, co-immunoprecipitation (co-IP), cell cycle, and apoptosis assays. The effects of SALL4 loss on normal hematopoiesis in mice were also investigated. RESULTS: In vitro and in vivo studies revealed that SALL4 expression is critically required for MLL-AF9-induced leukemic transformation and disease progression in mice. Loss of SALL4 in MLL-AF9-transformed cells induced apoptosis and cell cycle arrest at G1. ChIP-Seq assay identified that Sall4 binds to key MLL-AF9 target genes and important MLL-r or non-MLL-r leukemia-related genes. ChIP-PCR assays indicated that SALL4 affects the levels of the histone modification markers H3K79me2/3 and H3K4me3 at MLL-AF9 target gene promoters by physically interacting with DOT1-like histone H3K79 methyltransferase (DOT1l) and LSD1/KDM1A, and thereby regulates transcript expression. Surprisingly, normal Sall4 f/f /CreERT2 mice treated with tamoxifen or vav-Cre-mediated (hematopoietic-specific) Sall4 -/- mice were healthy and displayed no significant hematopoietic defects. CONCLUSIONS: Our findings indicate that SALL4 critically contributes to MLL-AF9-induced leukemia, unraveling the underlying transcriptional and epigenetic mechanisms in this disease and suggesting that selectively targeting the SALL4 pathway may be a promising approach for managing human MLL-r leukemia.

Cardiac reprogramming factor Gata4 reduces postinfarct cardiac fibrosis through direct repression of the profibrotic mediator snail.

OBJECTIVE: The administration of a variety of reprogramming factor cocktails has now been shown to reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, reductions in ventricular fibrosis observed after reprogramming factor administration seem to far exceed the extent of induced cardiomyocyte-like cell generation in vivo. We investigated whether reprogramming factor administration might primarily play a role in activating antifibrotic molecular pathways. METHODS: Adult rat cardiac fibroblasts were infected with lentivirus encoding the transcription factors Gata4, Mef2c, or Tbx5, all 3 vectors, or a green fluorescent protein control vector. Gene and protein expression assays were performed to identify relevant antifibrotic targets of these factors. The antifibrotic effects of these factors were then investigated in a rat coronary ligation model. RESULTS: Gata4, Mef2c, or Tbx5 administration to rat cardiac fibroblasts in vitro significantly downregulated expression of Snail and the profibrotic factors connective tissue growth factor, collagen1a1, and fibronectin. Of these factors, Gata4 was shown to be the one responsible for the downregulation of the profibrotic factors and Snail (mRNA expression fold change relative to green fluorescent protein for Snail, Gata4: 0.5 ± 0.3, Mef2c: 1.3 ± 1.0, Tbx5: 0.9 ± 0.5, Gata4, Mef2c, or Tbx5: 0.6 ± 0.2, P < .05). Chromatin immunoprecipitation quantitative polymerase chain reaction identified Gata4 binding sites in the Snail promoter. In a rat coronary ligation model, only Gata4 administration alone improved postinfarct ventricular function and reduced the extent of postinfarct fibrosis. CONCLUSIONS: Gata4 administration reduces postinfarct ventricular fibrosis and improves ventricular function in a rat coronary ligation model, potentially as a result of Gata4-mediated downregulation of the profibrotic mediator Snail.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

OBJECTIVE: The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. METHODS: Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. RESULTS: Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). CONCLUSIONS: Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans.

"Triplet" polycistronic vectors encoding Gata4, Mef2c, and Tbx5 enhances postinfarct ventricular functional improvement compared with singlet vectors.

OBJECTIVE: The in situ reprogramming of cardiac fibroblasts into induced cardiomyocytes by the administration of gene transfer vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) has been shown to improve ventricular function in myocardial infarction models. The efficacy of this strategy could, however, be limited by the need for fibroblast targets to be infected 3 times--once by each of the 3 transgene vectors. We hypothesized that a polycistronic "triplet" vector encoding all 3 transgenes would enhance postinfarct ventricular function compared with use of "singlet" vectors. METHODS: After validation of the polycistronic vector expression in vitro, adult male Fischer 344 rats (n=6) underwent coronary ligation with or without intramyocardial administration of an adenovirus encoding all 3 major vascular endothelial growth factor (VEGF) isoforms (AdVEGF-All6A positive), followed 3 weeks later by the administration to AdVEGF-All6A-positive treated rats of singlet lentivirus encoding G, M, or T (1×10(5) transducing units each) or the same total dose of a GMT "triplet" lentivirus vector. RESULTS: Western blots demonstrated that triplet and singlet vectors yielded equivalent GMT transgene expression, and fluorescence activated cell sorting demonstrated that triplet vectors were nearly twice as potent as singlet vectors in generating induced cardiomyocytes from cardiac fibroblasts. Echocardiography demonstrated that GMT triplet vectors were more effective than the 3 combined singlet vectors in enhancing ventricular function from postinfarct baselines (triplet, 37%±10%; singlet, 13%±7%; negative control, 9%±5%; P<.05). CONCLUSIONS: These data have confirmed that the in situ administration of G, M, and T induces postinfarct ventricular functional improvement and that GMT polycistronic vectors enhance the efficacy of this strategy.

In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor.

BACKGROUND: In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. METHODS AND RESULTS: Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF-All6A(+)) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12 ± 2% vs 24 ± 3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4-fold. GMT-treated animals also demonstrated greater density of cardiomyocyte-specific marker beta myosin heavy chain 7(+) cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12 ± 3% vs -7 ± 3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4-fold greater in GMT/VEGF vs GMT/null animals (17 ± 2% vs 4 ± 1%, P<0.05). CONCLUSIONS: VEGF administration to infarcted myocardium enhances the efficacy of GMT-mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.

A clinically relevant large-animal model for evaluation of tissue-engineered cardiac surgical patch materials.

Extracellular matrix (ECM) scaffolds may be useful as a tissue engineering approach toward myocardial regeneration in the infarcted heart. An appropriate large-animal model for testing the utility of biologically derived ECM in this application is needed. The purpose of this study was to develop such a model for optimal procedural success during and after patch implantation surgery. Myocardial infarction (MI) was created by embolization of the diagonal artery (DA) branch of the left anterior descending coronary artery with collagen suspension. After 4 to 6 weeks, 14 pigs received patch implant (ECM or expanded polytetrafluoroethylene). Six pigs were infarcted in the first DA and seven pigs in the second DA. Electrophysiology study was performed within 3 days before surgery. During surgery, the size and location of the infarct were measured. Infarcted myocardium (1.5-cm diameter) was transmurally excised under partial cardiopulmonary bypass. Patches (3-cm diameter) were sutured to the endomyocardial defect. Four pigs died postoperatively. After 1 month, 10 pigs were euthanized and the locations of patches were examined. Success rate of patch implant in the second DA (85.7%) was higher than the first DA (50%) group. Infarct size in the second DA was smaller than in the first DA (4.6+/-1.2 vs. 10.8+/-2.4 cm(2), P<.05). The second DA was more anteriorly positioned, which enabled easier access from the midsternal thoracotomy. However, the first DA was more laterally located requiring more manipulation of the heart during surgery. Electrophysiology revealed no ventricular tachyarrhythmia in the second DA but 33.3% in the first DA group (P<.05). At necropsy, the endocardial position of the first DA-infarct patches was anteroapical, whereas the second DA-infarct patches were more basolateral and often involved the anterior papillary muscle. The success rate of patch implant was associated with infarction size and location, and may be related to arrhythmic substrate. Experimental MI created by the second DA embolization is a feasible model for investigation of tissue-engineered cardiac patch implantation. This large-animal model is also suitable for study of cell therapy via endocardial catheter-based approaches or open surgical methods.