Intracoronary E-/L-selectin Blockade Reduces Neutrophil Infiltration in Heart Transplantation

The Annals of Thoracic Surgery. Dec, 2002  |  Pubmed ID: 12643396

This study examined the effect of local intracoronary delivery of a unique monoclonal antibody (mAb) to both E- and L-selectin (EL-246) on neutrophil infiltration after global ischemia during cardiac transplantation.

Skeletal Muscle Stem Cells Do Not Transdifferentiate into Cardiomyocytes After Cardiac Grafting

Journal of Molecular and Cellular Cardiology. Feb, 2002  |  Pubmed ID: 11851363

Skeletal muscle cell-derived grafts in the heart may benefit myocardial performance after infarction. Several studies have suggested that skeletal muscle stem cells (satellite cells) from adult muscle undergo transdifferentiation into cardiomyocytes after grafting into the heart, but expression of cardiac markers in graft cells has not been rigorously confirmed. To determine the fate of satellite cell-derived grafts in the heart, adult rat satellite cells were tagged in vitro with bromodeoxyuridine (BrdU) and grafted into normal hearts of syngeneic rats. At 4 and 12 weeks the graft cells formed multinucleated, cross-striated myofibers that expressed fast skeletal myosin heavy chain (MHC), thus indicating a mature skeletal muscle phenotype. Double staining for the BrdU tag and cardiac-specific markers was employed to identify transdifferentiation. Aside from four questionable cells, none of the 11 grafts examined expressed alpha-MHC, cardiac troponin I, or atrial natriuretic peptide. At 4 weeks, grafts expressed beta -MHC, a hallmark of slow twitch myofibers. By 12 weeks, however, the myofibers had atrophied and downregulated beta-MHC. Grafts never expressed the intercalated disk proteins N-cadherin or connexin43, hence electromechanical coupling did not occur. In conclusion, satellite cells differentiate into mature skeletal muscle and do not express cardiac-specific genes after grafting into the heart. Thus, transdifferentiation into cardiomyocytes did not occur.

In Vitro Generation of Differentiated Cardiac Myofibers on Micropatterned Laminin Surfaces

Journal of Biomedical Materials Research. Jun, 2002  |  Pubmed ID: 11920672

Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.

Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts

Circulation Research. Apr, 2002  |  Pubmed ID: 11934829

Human myocardium has long been considered to have essentially no intrinsic regenerative capacity. Recent studies in rodent models, however, have suggested the presence of an extracardiac stem cell population, perhaps in bone marrow, that is capable of some reconstitution of cardiomyocytes after injury. To determine whether similar mechanisms exist in the human heart, we evaluated human female allograft hearts transplanted into male patients. The presence of Y chromosomes in cardiomyocytes would indicate these cells arose from the recipient, rather than the donor heart. We identified 5 male patients who had retained a female heart at least 9 months before death and necropsy. Remarkably, in each case, the transplanted heart contained a minute but readily detectable fraction of Y chromosome-positive cardiomyocytes. The mean percentage of cardiomyocytes arising from the host was estimated to be 0.04% with a median of 0.016%. Most Y-positive cardiomyocytes were associated with regions of acute rejection, suggesting such chimerism involves an injury event. Furthermore, the sole patient whose immediate cause of death was allograft rejection showed a much higher percentage of host-derived cardiomyocytes, up to 29% in local, 1-mm(2) "hot spots." Thus, adult humans have extracardiac progenitor cells capable of migrating to and repopulating damaged myocardium, but this process occurs at very low levels.

Taking the Death Toll After Cardiomyocyte Grafting: a Reminder of the Importance of Quantitative Biology

Journal of Molecular and Cellular Cardiology. Mar, 2002  |  Pubmed ID: 11945017

Muscle Cell Grafting for the Treatment and Prevention of Heart Failure

Journal of Cardiac Failure. Dec, 2002  |  Pubmed ID: 12555170

The review aims to highlight recent advances in cardiac and skeletal muscle cell grafting for myocardial infarct repair.

Cell Grafting for Cardiac Repair

Methods in Molecular Biology (Clifton, N.J.). 2003  |  Pubmed ID: 12597001

Skeletal Muscle Meets Cardiac Muscle. Friends or Foes?

Journal of the American College of Cardiology. Apr, 2003  |  Pubmed ID: 12679205

Immunohistologic Labeling of Murine Endothelium

Cardiovascular Pathology : the Official Journal of the Society for Cardiovascular Pathology. Mar-Apr, 2003  |  Pubmed ID: 12684163

Reliable identification of endothelial cells is a prerequisite for understanding vascularity changes in many cardiovascular diseases and therapeutic interventions. With the rising use of mouse models of disease and genetic manipulation, a consistent system to label murine endothelial cells in normal and diseased tissues would be an invaluable tool.

Spatially Organized Layers of Cardiomyocytes on Biodegradable Polyurethane Films for Myocardial Repair

Journal of Biomedical Materials Research. Part A. Sep, 2003  |  Pubmed ID: 12918042

Tissue engineering constructs should match the physical and mechanical properties of the native tissue. This implies that pliable scaffolds might be better suited for soft-tissue applications than rigid polymeric materials. In this study, we examined spatially organized cardiomyocyte cultures on biodegradable, elastomeric polyurethane films patterned by microcontact printing of laminin lanes. The resulting cardiomyocyte patterns on polyurethane displayed a similar morphology to those previously achieved for up to 7-10 days on other substrates, such as polystyrene dishes. However, the integrity of the cardiomyocyte patterns on thin, spin-cast or solvent-cast polyurethane films was retained for up to 4 weeks in culture. When additional cardiomyocytes (labeled with Cell Tracker reagents) were seeded onto the patterned cultures, secondary and tertiary cell populations aligned between and on top of the primary patterned cells to form a multilayered, organized tissue construct approximately 2-3 cell layers thick. In addition, dense, highly aligned monolayers of patterned cardiomyocytes were able to contract the thin, solvent-cast polyurethane films. These results indicate that elastomeric, biodegradable polyurethane films can serve as an appropriate scaffold material to support stably the engineering of spatially organized layers of cardiomyocytes in vitro. This approach may serve as a novel method for transplantation of organized cardiac tissue constructs to the heart for myocardial repair.

Myofibroblast and Endothelial Cell Proliferation During Murine Myocardial Infarct Repair

The American Journal of Pathology. Dec, 2003  |  Pubmed ID: 14633615

Granulation tissue formation is a critical step in infarct repair, however, the kinetics of cell replication and the molecules that regulate this process are poorly understood. In uninjured mouse hearts and at 2 days post-infarction, very little DNA synthesis (measured by incorporation of a BrdU pulse) was detected in any cell type. Four days after permanent coronary occlusion, the rates of myofibroblast (smooth muscle alpha-actin and BrdU double-positive) and endothelial cell (CD31 and BrdU double-positive) proliferation were 15.4 +/- 1.1% and 2.9 +/- 0.5%, respectively. Most proliferating cells were located at the interface of the infarct and viable tissue. By 1 week, fibroblast and endothelial cell proliferation declined to 4.1 +/- 0.6% and 0.7 +/- 0.1%, respectively. In the 2-week infarct, the remaining necrosis had been phagocytosed, and fibroblast and endothelial cell proliferation were <0.5%. Although leukocytes were abundant throughout infarct repair, no significant proliferation was detected at any time in cells expressing CD45 or mac-3. Infarct size at 4 days was 38 +/- 5% of the left ventricle and contracted to 20 +/- 4% by 4 weeks. After 4 days, the chamber dilated to four times that of the control hearts and remained so for the duration of the time course. The vascular density (per mm(2)) declined from 3643 +/- 82 in control hearts to 2716 +/- 197 at 1 week and 1010 +/- 47 at 4 weeks post-myocardial infarction (MI). The average percent area occupied by vessels did not change significantly between the groups but the area/vessel ( micro m(2)) increased from 14.1 +/- 0.3 in control hearts to 16.9 +/- 1.9 at 1 week and 38.7 +/- 7.9 at 4 weeks post-MI. These data indicate that mitogens for fibroblasts and endothelial cells peak within 4 days of infarction in the mouse heart. This provides the basis for identifying the responsible molecules and developing strategies to alter wound repair and improve cardiac function.

Evidence for Fusion Between Cardiac and Skeletal Muscle Cells

Circulation Research. Apr, 2004  |  Pubmed ID: 15001531

Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are "fusigenic" cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from -myosin heavy chain promoter (-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of -MHC-Cre+ and -MHC-Cre- mice (n=5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of -MHC-Cre+ mice, but not after implantation into -MHC-Cre- mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org.

Myogenic Fusion of Human Bone Marrow Stromal Cells, but Not Hematopoietic Cells

Blood. Jul, 2004  |  Pubmed ID: 15010375

Following marrow transplantation in both patients and animals, cells containing donor nuclei have been detected in a variety of nonhematopoietic tissue. Whether this phenomenon represents transdifferentiation of marrow-derived cells, infiltration of blood cells, or cell fusion is still controversial. In muscle, where cell fusion occurs during normal myogenesis, fusion of marrow-derived cells with resident myotubes is a likely explanation. We tested 8 subpopulations of human bone marrow for their ability to fuse with mouse C2C12 myoblast cells. Relatively high fusion efficiency was observed with marrow stromal cells whereas hematopoietic cells, including populations enriched for stem cells, progenitor cells, and monocytes were refractory to fusion. Mouse myotubes containing human nuclei also contained transcripts for human muscle-specific genes. Injection in vivo of human stromal cells expressing green fluorescent protein (GFP) into the regenerating tibialis anterior muscle of nonobese diabetic-severe combined immunodeficient (NOD/SCID) beta 2m(-/-) mice resulted in regenerating mouse muscle fibers expressing GFP. These data suggest that marrow-derived cells contribute to myogenesis through fusion and that stromal cells are better fusion partners than hematopoietic cells.

Haematopoietic Stem Cells Do Not Transdifferentiate into Cardiac Myocytes in Myocardial Infarcts

Nature. Apr, 2004  |  Pubmed ID: 15034593

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

NFATc3-induced Reductions in Voltage-gated K+ Currents After Myocardial Infarction

Circulation Research. May, 2004  |  Pubmed ID: 15087419

Reductions in voltage-activated K+ (Kv) currents may underlie arrhythmias after myocardial infarction (MI). We investigated the role of beta-adrenergic signaling and the calcineurin/NFAT pathway in mediating the reductions in Kv currents observed after MI in mouse ventricular myocytes. Kv currents were produced by the summation of 3 distinct currents: I(to), I(Kslow1), and I(Kslow2). At 48 hours after MI, we found a 4-fold increase in NFAT activity, which coincided with a decrease in the amplitudes of I(to), I(Kslow1), and I(Kslow2). Consistent with this, mRNA and protein levels of Kv1.5, 2.1, 4.2, and 4.3, which underlie I(Kslow1), I(Kslow2), and I(to), were decreased after MI. Administration of the beta-blocker metoprolol prevented the activation of NFAT and the reductions in I(to), I(Kslow1), and I(Kslow2) after MI. Cyclosporine, an inhibitor of calcineurin, also prevented the reductions in these currents after MI. Importantly, Kv currents did not change after MI in ventricular myocytes from NFATc3 knockout mice. Conversely, chronic beta-adrenergic stimulation or expression of an activated NFATc3 decreased Kv currents to a similar extent as MI. Taken together, these data indicate that NFATc3 plays an essential role in the signaling pathway leading to reduced I(to), I(Kslow1), and I(Kslow2) after MI. We propose that increased beta-adrenergic signaling after MI activates calcineurin and NFATc3, which decreases I(to), I(Kslow1), and I(Kslow2) via a reduction in Kv1.5, Kv2.1, Kv4.2, and Kv4.3 expression.

Gene Transfer of Connexin43 into Skeletal Muscle

Human Gene Therapy. Jul, 2004  |  Pubmed ID: 15242523

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.

Regenerating the Heart

Nature Biotechnology. Jul, 2005  |  Pubmed ID: 16003373

Cell-based cardiac repair offers the promise of rebuilding the injured heart from its component parts. Work began with committed cells such as skeletal myoblasts, but recently the field has expanded to explore an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and both mouse and human embryonic stem cells. A related strategy for cardiac repair involves cell mobilization with factors such as cytokines. Translation of cell-based approaches to the clinic has progressed rapidly, and clinical trials using autologous skeletal myoblasts and bone marrow cells are under way. Many challenges remain before the vision of healing an infarct by muscle regeneration can be realized. Future research is likely to focus on improving our ability to guide the differentiation of stem cells, control their survival and proliferation, identify factors that mediate their homing and modulate the heart's innate inflammatory and fibrotic responses.

Formation of Human Myocardium in the Rat Heart from Human Embryonic Stem Cells

The American Journal of Pathology. Sep, 2005  |  Pubmed ID: 16127147

Human embryonic stem cells (hESCs) offer the opportunity to replenish cells lost in the postinfarct heart. We explored whether human myocardium could be generated in rat hearts by injecting differentiated cardiac-enriched hESC progeny into the left ventricular wall of athymic rats. Although initial grafts were predominantly epithelial, noncardiac elements were lost over time, and grafts consisted predominantly of cardiomyocytes by 4 weeks. No teratomatous elements were observed. Engrafted cardiomyocytes were glycogen-rich and expressed expected cardiac markers including beta-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. Heat-shock treatment improved graft size approximately threefold. The cardiac implants exhibited substantial angiogenesis, both recipient and graft derived. Importantly, there was greater proliferation in human cardiomyocytes than previously seen in rodent-derived cardiomyocytes: 14.4% of graft cardiomyocytes expressed the proliferation marker Ki-67, and 2.7% incorporated the thymidine analog BrdU 4 weeks after transplantation. This proliferation was associated with a sevenfold increase in graft size over the 4-week interval. Thus, hESCs can form human myocardium in the rat heart, permitting studies of human myocardial development and physiology and supporting the feasibility of their use in myocardial repair.

Proliferation of Cardiomyocytes Derived from Human Embryonic Stem Cells is Mediated Via the IGF/PI 3-kinase/Akt Signaling Pathway

Journal of Molecular and Cellular Cardiology. Dec, 2005  |  Pubmed ID: 16242146

Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse embryonic stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (15-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing differentiation of the cells. Neither cell density nor different matrix attachment factors affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like growth factor-1 (IGF-1) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-1 or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-1 receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.

Extracardiac Progenitor Cells Repopulate Most Major Cell Types in the Transplanted Human Heart

Circulation. Nov, 2005  |  Pubmed ID: 16275883

Extracardiac progenitor cells are capable of repopulating cardiomyocytes at very low levels in the human heart after injury. Here, we explored the extent of endothelial, smooth muscle, and Schwann cell chimerism in patients with sex-mismatched (female-to-male) heart transplants.

Cell-based Cardiac Repair: Reflections at the 10-year Point

Circulation. Nov, 2005  |  Pubmed ID: 16286608

Regeneration Gaps: Observations on Stem Cells and Cardiac Repair

Journal of the American College of Cardiology. May, 2006  |  Pubmed ID: 16682301

Substantial evidence indicates that cell transplantation can improve function of the infarcted heart. A surprisingly wide range of non-myogenic cell types improves ventricular function, suggesting that benefit may result in part from mechanisms that are distinct from true myocardial regeneration. While clinical trials explore cells derived from skeletal muscle and bone marrow, basic researchers are investigating sources of new cardiomyocytes, such as resident myocardial progenitors and embryonic stem cells. In this commentary, we briefly review the evolution of cell-based cardiac repair, discuss the current state of clinical research, and offer some thoughts on how newcomers can critically evaluate this emerging field.

RAAV6-microdystrophin Preserves Muscle Function and Extends Lifespan in Severely Dystrophic Mice

Nature Medicine. Jul, 2006  |  Pubmed ID: 16819550

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

Transplantation of Undifferentiated Murine Embryonic Stem Cells in the Heart: Teratoma Formation and Immune Response

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. May, 2007  |  Pubmed ID: 17284483

Embryonic stem (ES) cells are promising for cardiac repair, but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind-limb teratomas, suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts, indicating injury-related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas, but these were immunologically rejected after several weeks, in association with increased inflammation and up-regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo, but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence, undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts, and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation, avoid introducing undifferentiated cells, and will likely require immune modulation to avoid rejection.

Chemical Dimerization of Fibroblast Growth Factor Receptor-1 Induces Myoblast Proliferation, Increases Intracardiac Graft Size, and Reduces Ventricular Dilation in Infarcted Hearts

Human Gene Therapy. May, 2007  |  Pubmed ID: 17518610

The ability to control proliferation of grafted cells in the heart and consequent graft size could dramatically improve the efficacy of cell therapies for cardiac repair. To achieve targeted graft cell proliferation, we created a chimeric receptor (F36Vfgfr-1) composed of a modified FK506-binding protein (F36V) fused with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR-1). We retrovirally transduced mouse C2C12 and MM14 skeletal myoblasts with this construct and treated them with AP20187, a dimeric F36V ligand ("dimerizer"), in vitro and in vivo to induce receptor dimerization. Dimerizer treatment in vitro activated the mitogen-activated protein kinase pathway and induced proliferation in myoblasts expressing F36Vfgfr-1 comparable with the effects of basic FGF. Wild-type myoblasts did not respond to dimerizer. Subcutaneous grafts composed of myoblasts expressing F36Vfgfr-1 showed a dose-dependent increase in DNA synthesis with dimerizer treatment. When myoblasts expressing F36Vfgfr-1 were injected into infarcted hearts of nude mice, dimerizer treatment resulted in a dose-dependent increase in graft size, from 20 +/- 3 to 42.9 +/- 4.3% of the left ventricle. Blinded echocardiographic analysis demonstrated that larger graft size was associated with a dose-dependent reduction in ventricular dilation after myocardial infarction, although animals with the largest grafts showed an increased incidence of ventricular tachycardia. Thus, selective proliferation of genetically modified graft cells can be induced with a systemically administered synthetic molecule in vitro or in vivo. Control of intramyocardial graft size by this approach may allow optimization of cell-based therapy to obtain desired cardiac function postinfarction.

Biphasic Role for Wnt/beta-catenin Signaling in Cardiac Specification in Zebrafish and Embryonic Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2007  |  Pubmed ID: 17522258

Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/beta-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/beta-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/beta-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/beta-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of beta-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the beta-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/beta-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.

Selective Control of Endothelial Cell Proliferation with a Synthetic Dimerizer of FGF Receptor-1

Laboratory Investigation; a Journal of Technical Methods and Pathology. Aug, 2007  |  Pubmed ID: 17572688

Basic fibroblast growth factor (bFGF) is a potent angiogenic molecule, but its therapeutic use is limited by mitogenic effects on multiple cell types. To specifically activate FGF signaling in endothelial cells, a chimeric FGF receptor was generated that contained a modified FK506 drug-binding domain (F36V) fused to the FGF receptor-1 (FGFR1) cytoplasmic domain. Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells were retrovirally transduced with this chimeric receptor, and the effects of administering synthetic receptor-dimerizing ligands were studied. As expected, both control and transduced cells proliferated in response to bFGF treatment; however, only transduced endothelial cells exhibited dose-dependent proliferative responses to dimerizer treatment. Dimerizer-induced proliferation was MEK-dependent and was accompanied by MAP kinase phosphorylation, indicating that the chimeric receptor utilizes signaling pathways similar to endogenous FGFR1. Although bFGF stimulated wound re-epithelialization in HUVECs (which natively express FGFR1 and FGFR4), chemical dimerization of FGFR1 did not; this suggests FGFR4 may control migration in these cells. The ability to selectively activate receptor subtypes should facilitate the study of signaling pathways in vitro and in vivo beyond what can be accomplished with nonselective natural ligands, and it may eventually permit stimulation of graft cell angiogenesis without driving overgrowth of host cells.

Cardiac Aid to the Injured but Not the Elderly?

Nature Medicine. Aug, 2007  |  Pubmed ID: 17679999

Cardiomyocytes Derived from Human Embryonic Stem Cells in Pro-survival Factors Enhance Function of Infarcted Rat Hearts

Nature Biotechnology. Sep, 2007  |  Pubmed ID: 17721512

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

Fibroblast Growth Factor-2 Regulates Myocardial Infarct Repair: Effects on Cell Proliferation, Scar Contraction, and Ventricular Function

The American Journal of Pathology. Nov, 2007  |  Pubmed ID: 17872976

Fibroblast growth factor-2 (FGF2, bFGF) has been proposed to regulate wound healing and angiogenesis, but skin wound healing in FGF2-knockout (FGF2-KO) animals is only slightly delayed. To determine the role of FGF2 in myocardial infarct repair, we studied the evolution of left ventricular geometry, cell proliferation, matrix content, and cardiac function in mice lacking or overexpressing (FGF2-Tg) FGF2. Despite having no effect on initial infarct size, deletion of FGF2 resulted in reduced fibroblast proliferation and interstitial collagen deposition, decreased endothelial proliferation and vascular density, and decreased cardiomyocyte hypertrophy. Furthermore, FGF2-KO mice demonstrated a complete absence of scar contraction, resulting in increased final infarct size and marked increases in chamber size and infarct expansion. These deficits ultimately impaired left ventricular dP/dt compared with wild-type infarcted mice. Conversely, overexpression of FGF2 increased fibroblast proliferation and collagen deposition, accelerated endothelial proliferation, and enhanced cardiomyocyte hypertrophy after infarction. These changes curbed infarct expansion and preserved left ventricular function. Thus, FGF2 is an important regulator of cell proliferation, angiogenesis, collagen synthesis, myocyte hypertrophy, scar contraction, and, ultimately, left ventricular contractile function during infarct repair. FGF2 may be more important in healing of infarcts compared with skin wounds because of the mechanical stress under which infarcts heal.

Cell-based Therapy for Myocardial Ischemia and Infarction: Pathophysiological Mechanisms

Annual Review of Pathology. 2007  |  Pubmed ID: 18039102

Cell-based cardiac repair has emerged as an attractive approach to preventing or reversing heart failure resulting from myocyte dysfunction-e.g., due to infarction-and to enhancing the development of collaterals in patients with symptoms of myocardial ischemia. These two problems involve both overlapping and differing mechanisms, and these differences must be considered in cell-based therapies. In terms of myocardial dysfunction due to infarction, only committed cardiomyocytes have been shown to form new myocardium that is electrically coupled with the host heart. Despite this, multiple cell populations appear to improve function of the infarcted heart, including many that are clearly nonmyogenic. In terms of myocardial ischemia, although cell-based strategies improve ischemia in animal models, clinical trials to date have not shown robustly beneficial results. We review the evidence for potential mechanisms underlying the benefits of cell transplantation in the heart and discuss the clinical contexts in which they may be relevant.

Absence of Regeneration in the MRL/MpJ Mouse Heart Following Infarction or Cryoinjury

Cardiovascular Pathology : the Official Journal of the Society for Cardiovascular Pathology. Jan-Feb, 2008  |  Pubmed ID: 18160055

Myocardial infarcts in mammals heal by scar formation rather than formation of new muscle tissue. The MRL/MpJ [Murphy Roths large (MRL) derived by the Murphy group of the Jackson Laboratory (MpJ)] mouse, however, has been reported to exhibit minimal scarring and subsequent cardiac regeneration after cryoinjury of the right ventricle. Other groups have reported that permanent and temporary ligation of the coronary artery resulted in scarring without regeneration.

Differentiation of Embryonic Stem Cells to Clinically Relevant Populations: Lessons from Embryonic Development

Cell. Feb, 2008  |  Pubmed ID: 18295582

The potential to generate virtually any differentiated cell type from embryonic stem cells (ESCs) offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Embryology has offered important insights into key pathways regulating ESC differentiation, resulting in advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, and ectoderm and many of their downstream derivatives. This has led to the identification of new multipotential progenitors for the hematopoietic, neural, and cardiovascular lineages and to the development of protocols for the efficient generation of a broad spectrum of cell types including hematopoietic cells, cardiomyocytes, oligodendrocytes, dopamine neurons, and immature pancreatic beta cells. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of human disease.

Chromatin Remodeling During Mouse and Human Embryonic Stem Cell Differentiation

Developmental Dynamics : an Official Publication of the American Association of Anatomists. May, 2008  |  Pubmed ID: 18425849

Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome-wide acetylation (euchromatic) and tri-methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm-specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has "bivalent" tri-methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre-cardiac mesoderm, the activated brachyury locus has high levels of tri-methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome-wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation.

Bones of Contention: Marrow-derived Cells in Myocardial Regeneration

Journal of Molecular and Cellular Cardiology. Jun, 2008  |  Pubmed ID: 18440020

Almost 7 years have passed since the initial publication reporting that bone marrow cells regenerate infarcted myocardium. The subsequent years produced hundreds of investigations that ran the gamut of findings from validation to disproof. Undeterred by the concurrent debate, clinical trials ensued to test the safety and efficacy of bone marrow-derived cell population for autologous therapy in clinical treatment of myocardial disease. In the following conversational exchange, two scientists with distinct perspectives weigh the pros and cons of pursuing bone marrow stem cell therapy and look toward finding a consensus of where the future lies for regenerative medicine and the heart. The conclusion is that the two camps may not be as far apart as it may seem from the rancor in literature and at meetings, and the potential of one day achieving regenerative therapy is indeed a vision that both parties enthusiastically share.

A Hierarchical Network Controls Protein Translation During Murine Embryonic Stem Cell Self-renewal and Differentiation

Cell Stem Cell. May, 2008  |  Pubmed ID: 18462695

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

Systems Approaches to Preventing Transplanted Cell Death in Cardiac Repair

Journal of Molecular and Cellular Cardiology. Oct, 2008  |  Pubmed ID: 18466917

Stem cell transplantation may repair the injured heart, but tissue regeneration is limited by death of transplanted cells. Most cell death occurs in the first few days post-transplantation, likely from a combination of ischemia, anoikis and inflammation. Interventions known to enhance transplanted cell survival include heat shock, over-expressing anti-apoptotic proteins, free radical scavengers, anti-inflammatory therapy and co-delivery of extracellular matrix molecules. Combinatorial use of such interventions markedly enhances graft cell survival, but death still remains a significant problem. We review these challenges to cardiac cell transplantation and present an approach to systematically address them. Most anti-death studies use histology to assess engraftment, which is time- and labor-intensive. To increase throughput, we developed two biochemical approaches to follow graft viability in the mouse heart. The first relies on LacZ enzymatic activity to track genetically modified cells, and the second quantifies human genomic DNA content using repetitive Alu sequences. Both show linear relationships between input cell number and biochemical signal, but require correction for the time lag between cell death and loss of signal. Once optimized, they permit detection of as few as 1 graft cell in 40,000 host cells. Pro-survival effects measured biochemically at three days predict long-term histological engraftment benefits. These methods permitted identification of carbamylated erythropoietin (CEPO) as a pro-survival factor for human embryonic stem cell-derived cardiomyocyte grafts. CEPO's effects were additive to heat shock, implying independent survival pathways. This system should permit combinatorial approaches to enhance graft viability in a fraction of the time required for conventional histology.

Get with the (re)program: Cardiovascular Potential of Skin-derived Induced Pluripotent Stem Cells

Circulation. Jul, 2008  |  Pubmed ID: 18663099

Scaffold-free Human Cardiac Tissue Patch Created from Embryonic Stem Cells

Tissue Engineering. Part A. Jun, 2009  |  Pubmed ID: 19063661

Progress in cardiac tissue engineering has been limited by (1) unfavorable cell and host responses to biomaterial scaffolds, (2) lack of suitable human cardiomyocyte sources, and (3) lack of fabrication techniques for scalable production of engineered tissue constructs. Here we report a novel and scalable method to generate scaffold-free human cardiac tissue patches. Human embryonic stem cells were differentiated to cardiomyocytes using activin A and BMP4 and placed into suspension on a rotating orbital shaker. Cells aggregated to form macroscopic disc-shaped patches of beating tissue after 2 days. Patch diameter was directly proportional to input cell number (approximately 11 mm with 12 million cells), and patches were 300-600 mum thick. Cardiomyocytes were concentrated around the patch edges and exhibited increased purity and maturation with time, comprising approximately 80% of total cells after 11 days. Noncardiac cell elements, primarily epithelium, were present at day 2 but were diminished markedly at later time points. Cardiomyocyte proliferation occurred throughout the patches at day 2 but declined by day 8. Patches exhibited automaticity and synchronous calcium transients, indicating electromechanical coupling. These novel scaffold-free human myocardial patches address critical challenges related to human cell sourcing and tissue fabrication that previously inhibited progress in cardiac tissue engineering.

Development Biology. Turnover After the Fallout

Science (New York, N.Y.). Apr, 2009  |  Pubmed ID: 19342577

Cell Therapy Enhances Function of Remote Non-infarcted Myocardium

Journal of Molecular and Cellular Cardiology. Nov, 2009  |  Pubmed ID: 19683533

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts 1 week after infarction would, after 8-10 weeks: 1) improve global myocardial function, 2) contract in a Ca2+ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca2+, whereas scar tissue did not. Furthermore, the Ca2+ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca2+ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.

Myostatin Represses Physiological Hypertrophy of the Heart and Excitation-contraction Coupling

The Journal of Physiology. Oct, 2009  |  Pubmed ID: 19736304

Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF-stimulated proliferation and differentiation and also modulates cardiac excitation-contraction (EC) coupling. Loss of myostatin induced eccentric hypertrophy and enhanced cardiac responsiveness to beta-adrenergic stimulation in vivo. This was due to myostatin null ventricular myocytes having larger [Ca(2+)](i) transients and contractions and responding more strongly to beta-adrenergic stimulation than wild-type cells. Enhanced cardiac output and beta-adrenergic responsiveness of myostatin null mice was therefore due to increased SR Ca(2+) release during EC coupling and to physiological hypertrophy, but not to enhanced myofilament function as determined by simultaneous measurement of force and ATPase activity. Our studies support the novel concept that myostatin is a repressor of physiological cardiac muscle growth and function. Thus, the controlled inhibition of myostatin action could potentially help repair damaged cardiac muscle by inducing physiological hypertrophy.

Retention and Biodistribution of Microspheres Injected into Ischemic Myocardium

Journal of Biomedical Materials Research. Part A. Mar, 2009  |  Pubmed ID: 18335529

Intramyocardial injection of therapeutic agents may enhance heart repair after infarction. Incomplete retention of intramyocardial injections has been reported, but modes of loss are undefined. We determined the fate of neutron-activated microspheres injected into acutely ischemic rat myocardium using saline, Pluronic F127, or Matrigel as vehicle. Twenty minutes after injection in saline, 63% +/- 12% of 10-mum microspheres was retained in the heart. Similar retention was observed after 6 days. Injection site leakage accounted for 14% +/- 5% of the microspheres, whereas exit via coronary veins resulted in 11.2% +/- 9.5% collecting in the lungs. Microspheres distribution to other organs was minimal. Retention of 40-mum microspheres was similar to that observed with the 10-mum microspheres. Pluronic F127 and Matrigel reduced immediate leakage to 4% +/- 1% and 2% +/- 1%, respectively. Surprisingly, microsphere retention in the heart was not improved at 20 min using either gelling vehicle, suggesting that leakage occurs over a prolonged period. Thus, most injected particles are retained in the ischemic rat heart following direct injection, but significant fractions are lost from the injection site and through coronary veins. Gelling agents reduced short-term leakage, but failed to enhance longer-term retention. Hydrogels with stiffer mechanical properties might enhance retention and reduce variability.

VEGF Induces Differentiation of Functional Endothelium from Human Embryonic Stem Cells: Implications for Tissue Engineering

Arteriosclerosis, Thrombosis, and Vascular Biology. Jan, 2010  |  Pubmed ID: 19875721

Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs.

Taking a Load Off: Nuclear Remodeling After Mechanically Supporting the Failing Human Heart

Circulation. Mar, 2010  |  Pubmed ID: 20159836

The Role of Macrophage-derived Urokinase Plasminogen Activator in Myocardial Infarct Repair: Urokinase Attenuates Ventricular Remodeling

Journal of Molecular and Cellular Cardiology. Sep, 2010  |  Pubmed ID: 20380835

Cardiac plasmin activity is increased following myocardial ischemia. To test the hypothesis that macrophage-derived uPA is a key mediator of repair following myocardial infarction, we performed myocardial infarction on mice with macrophage-specific over-expression of uPA (SR-uPA mice). SR-uPA(+/0) mice and wild-type littermates were sacrificed at 5 days or 4 weeks after infarction and cardiac content of macrophages, collagen, and myofibroblasts was quantified. Cardiac function and dimensions were assessed by echocardiography at baseline and at 4 weeks post-infarction. At 4 weeks after myocardial infarction, macrophage counts were increased in SR-uPA(+/0) mice in the infarct (13.1 vs. 4.9%, P<0.001) and distant uninfarcted regions (5.9 vs. 2.4%, P<0.001). Infarct scar was thicker in SR-uPA(+/0) mice (0.54+/-0.03 mm vs. 0.45+/-0.03 mm, P<0.05) and infarct cardiac collagen content was increased (72.4+/-3.3% vs. 63.0+/-3.6%, P<0.06). Functionally, these changes resulted in mildly improved fractional shortening in SR-uPA(+/0) mice compared to controls (24.6+/-1.68 vs. 19.8+/-1.3%, P=0.03). At 5 days after infarction there was increased collagen content in the scar without increases in macrophages or myofibroblasts. To understand the mechanisms by which macrophage-derived uPA increases collagen, cardiac fibroblasts were treated with macrophage-conditioned medium or plasmin and expression of ColIalpha1 measured by qPCR. Conditioned media from SR-uPA(+/0) or plasmin-treated non-transgenic macrophages but not plasmin alone increased collagen expression in isolated cardiac fibroblasts. We hypothesize that plasmin generation in the heart in response to injury may induce activation of macrophages to a profibrotic phenotype to allow rapid formation of collagenous scar.

Endogenous Wnt/beta-catenin Signaling is Required for Cardiac Differentiation in Human Embryonic Stem Cells

PloS One. 2010  |  Pubmed ID: 20559569

Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Differentiation of Cardiomyocytes from Human Embryonic Stem Cells is Accompanied by Changes in the Extracellular Matrix Production of Versican and Hyaluronan

Journal of Cellular Biochemistry. Oct, 2010  |  Pubmed ID: 20564236

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.

Ferritin Overexpression for Noninvasive Magnetic Resonance Imaging-based Tracking of Stem Cells Transplanted into the Heart

Molecular Imaging. Aug, 2010  |  Pubmed ID: 20643023

An unmet need in cardiac cell therapy is a noninvasive imaging technique capable of tracking changes in graft size over time and monitoring cell dynamics such as replication and death, factors to which commonly used superparamagnetic nanoparticles are insensitive. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart. Mouse skeletal myoblasts (C2C12 cells) were engineered to overexpress ferritin. Ferritin overexpression did not interfere with cell viability, proliferation, or differentiation into multinucleated myotubes. Ferritin overexpression caused a 25% decrease in T2 relaxation time in vitro compared to wild-type cells. Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Graft size evaluation by MRI correlated tighly with histologic measurements (R2 = .8). Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart. These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart.

Proangiogenic Scaffolds As Functional Templates for Cardiac Tissue Engineering

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2010  |  Pubmed ID: 20696917

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30-40 microm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.

Cardiogenesis from Human Embryonic Stem Cells

Circulation Journal : Official Journal of the Japanese Circulation Society. Nov, 2010  |  Pubmed ID: 21084757

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

Delivery of Basic Fibroblast Growth Factor with a PH-responsive, Injectable Hydrogel to Improve Angiogenesis in Infarcted Myocardium

Biomaterials. Mar, 2011  |  Pubmed ID: 21186056

A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly(N-isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) (p[NIPAAm-co-PAA-co-BA]), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37 °C but formed a physical gel at pH 6.8 and 37 °C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo, capillary and arteriolar densities were increased 30-40% by polymer + bFGF treatment versus saline + bFGF or polymer-only controls. Treatment with polymer + bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline + bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer + bFGF (30 ± 1.4%) versus saline (25 ± 1.2%) and polymer alone (25 ± 1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.

Developing Vasculature and Stroma in Engineered Human Myocardium

Tissue Engineering. Part A. May, 2011  |  Pubmed ID: 21187004

We recently developed a scaffold-free patch of human myocardium with human embryonic stem cell-derived cardiomyocytes and showed that stromal and endothelial cells form vascular networks in vitro and improve cardiomyocyte engraftment. Here, we hypothesize that stromal cells regulate the angiogenic phenotype by modulating the extracellular matrix (ECM). Human marrow stromal cells (hMSCs) support the greatest degree of endothelial cell organization, at 1.3- to 2.4-fold higher than other stromal cells tested. Stromal cells produce abundant ECM components in patches, including fibrillar collagen, hyaluronan, and versican. We identified two clonal hMSC lines that supported endothelial networks poorly and robustly. Interestingly, the pro-angiogenic hMSCs express high levels of versican, a chondroitin sulfate proteglycan that modulates angiogenesis and wound healing, whereas poorly angiogenic hMSCs produce little versican. When transplanted onto uninjured athymic rat hearts, patches with proangiogenic hMSCs develop ~ 50-fold more human vessels and form anastomoses with the host circulation, resulting in chimeric vessels containing erythrocytes. Thus, stromal cells play a key role in supporting vascularization of engineered human myocardium. Different stromal cell types vary widely in their proangiogenic ability, likely due in part to differences in ECM synthesis. Comparison of these cells defines an in vitro predictive platform for studying vascular development.

Reprogramming Fibroblasts into Cardiomyocytes

The New England Journal of Medicine. Jan, 2011  |  Pubmed ID: 21226585

Engineered Human Cardiac Tissue

Pediatric Cardiology. Mar, 2011  |  Pubmed ID: 21293854

The human heart is the first organ to develop during embryogenesis and is arguably the most essential organ for life. However, after birth, the heart has very little capacity to repair malformations such as congenital heart defects or to regenerate after an injury such as myocardial infarction. Cardiac tissue engineering addresses the need for a therapeutic biologic implant to restore cardiac structure and muscle mass. This review highlights current research in cardiac tissue engineering that uses human cardiomyocytes derived from embryonic stem cells. Other human cell sources are discussed because future human therapies will benefit from novel techniques using human-induced pluripotent stem cells and cardiomyocytes derived from direct reprogramming of somatic cells. Furthermore, this review examines the main approaches to creating engineered cardiac tissue with synthetic scaffolds, natural scaffolds, or no exogenous scaffold (i.e., "scaffold free"). The choice of scaffold and cells ultimately depends on the goals of the therapy, so the review considers how congenital heart defects define the design parameters for cardiac tissue engineering needed for surgical repair in pediatric cardiac patients.

A Repair "kit" for the Infarcted Heart

Cell Stem Cell. Apr, 2011  |  Pubmed ID: 21474095

Transplanted, c-kit expressing marrow-derived progenitors can enhance the function of an infarcted heart, but the mechanism remains unclear. In this issue of Cell Stem Cell, Loffredo et al. (2011) provide evidence that hematopoietic precursors do not differentiate into new cardiomyocytes but, rather, stimulate production of new cardiomyocytes from endogenous progenitors.

Heart Regeneration

Nature. May, 2011  |  Pubmed ID: 21593865

Heart failure plagues industrialized nations, killing more people than any other disease. It usually results from a deficiency of specialized cardiac muscle cells known as cardiomyocytes, and a robust therapy to regenerate lost myocardium could help millions of patients every year. Heart regeneration is well documented in amphibia and fish and in developing mammals. After birth, however, human heart regeneration becomes limited to very slow cardiomyocyte replacement. Several experimental strategies to remuscularize the injured heart using adult stem cells and pluripotent stem cells, cellular reprogramming and tissue engineering are in progress. Although many challenges remain, these interventions may eventually lead to better approaches to treat or prevent heart failure.

Growth of Engineered Human Myocardium with Mechanical Loading and Vascular Coculture

Circulation Research. Jun, 2011  |  Pubmed ID: 21597009

The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood.

Evidence That Gene Activation and Silencing During Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State

PloS One. 2011  |  Pubmed ID: 21886766

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3' transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98-99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.

Upregulation of Cardiomyocyte Ribonucleotide Reductase Increases Intracellular 2 Deoxy-ATP, Contractility, and Relaxation

Journal of Molecular and Cellular Cardiology. Dec, 2011  |  Pubmed ID: 21925507

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca(2+)-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca(2+) fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca(2+)], indicating increased contractility is primarily due to increased myofilament activity without altering Ca(2+) release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca(2+) activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin-myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca(2+). This article is part of a Special issue entitled "Possible Editorial".

Synthetic Matrices to Serve As Niches for Muscle Cell Transplantation

Cells, Tissues, Organs. 2012  |  Pubmed ID: 22005610

Poor cell retention and limited cell survival after grafting are major limitations of cell therapy. Recent studies showed that the use of matrices as vehicles at the time of cell injection can significantly improve cell engraftment by providing an appropriate structure and physical support for the injected cells. Properly designed matrices can also promote the organization of the cells into a functioning cardiac-like tissue and enhance integration between the host and the engrafted tissue. Furthermore, the use of an injectable biomaterial provides an opportunity to release in situ bioactive molecules that can further enhance the beneficial effects of cell transplantation. In this article we review a large variety of biologically derived synthetic and hybrid materials that have been tested as matrices for cardiac repair. We summarize the optimal parameters required for an ideal matrix including biocompatibility, injectability, degradation rate, and mechanical properties. Using an in vivo subcutaneous grafting model, we also provide novel data involving a side-by-side comparison of six synthetic matrices derived from maltodextrin. By systematically varying polymer molecular weight, cross-link density, and availability of cell adhesion motifs, a synthetic matrix was identified that supported skeletal myotube formation similar to Matrigel™. Our results emphasize not only the need to have a range of tunable matrices for cardiac cell therapy but also the importance of further characterizing the physical properties required for an ideal injectable matrix.

Truncations of Titin Causing Dilated Cardiomyopathy

The New England Journal of Medicine. Feb, 2012  |  Pubmed ID: 22335739

Dilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size.