Megumi Mathison

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.

MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte‐like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Methods and Results Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)‐590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte‐like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR‐590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT‐expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR‐590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR‐590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR‐590 in inducing cellular reprogramming. Conclusions These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR‐590‐mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming.

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

BackgroundThe 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.MethodsUsing 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.ResultsIn 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 Sall4f/f/CreERT2 mice treated with tamoxifen or vav-Cre-mediated (hematopoietic-specific) Sall4−/− mice were healthy and displayed no significant hematopoietic defects.ConclusionsOur 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.

Direct Cardiac Cellular Reprogramming for Cardiac Regeneration

Opinion statementDirect cardiac cellular reprogramming of endogenous cardiac fibroblasts directly into induced cardiomyocytes is a highly feasible, promising therapeutic option for patients with advanced heart failure. The most successful cardiac reprogramming strategy will likely be a multimodal approach involving an optimal combination of cardio-differentiating factors, suppression of fibroblast gene expression, and induction of angiogenic factors.

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 &agr;‐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. Graphical abstract Figure. No Caption available.

Cardiac Regenerative Strategies for Advanced Heart Failure

The premise of myocardial regeneration, once deemed scientifically improbable if not impossible, today represents a novel and excitingly promising therapeutic option for patients with advanced heart failure. Spurred by efforts over the past two decades to enhance myocardial vascularization (therapeutic angiogenesis) and to repopulate myocardial scar with exogenous stem cells, the current era of in situ direct cellular reprogramming is derived from new knowledge of cellular molecular biology and the astonishing Nobel Prize winning work of Yamanaka demonstrating that genetic manipulation could induce the dedifferentiation of adult somatic cells into induced pluripotent stem (iPS) cells. Unlike exogenous stem cell-based therapy, direct cardiac cellular reprogramming strategies capitalize on the presence of abundant endogenous cardiac fibroblasts in regions of myocardial infarction that can be targeted to generate induced cardiomyocytes (iCMs) following treatment with “reprogramming factors” as a means of improving post-infarct ventricular function. A multimodal regenerative strategy involving cardiac cellular reprogramming and induction of angiogenesis may represent a most promising therapeutic option for patients with heart failure.

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.