Changes in Cardiomyocyte Cell Cycle and Hypertrophic Growth During Fetal to Adult in Mammals.

The failure of adult cardiomyocytes to reproduce themselves to repair an injury results in the development of severe cardiac disability leading to death in many cases. The quest for an understanding of the inability of cardiac myocytes to repair an injury has been ongoing for decades with the identification of various factors which have a temporary effect on cell-cycle activity. Fetal cardiac myocytes are continuously replicating until the time that the developing fetus reaches a stage of maturity sufficient for postnatal life around the time of birth. Recent reports of the ability for early neonatal mice and pigs to completely repair after the severe injury has stimulated further study of the regulators of the cardiomyocyte cell cycle to promote replication for the remuscularization of injured heart. In all mammals just before or after birth, single-nucleated hyperplastically growing cardiomyocytes, 1X2N, undergo ≥1 additional DNA replications not followed by cytokinesis, resulting in cells with ≥2 nuclei or as in primates, multiple DNA replications (polyploidy) of 1 nucleus, 2X2(+)N or 1X4(+)N. All further growth of the heart is attributable to hypertrophy of cardiomyocytes. Animal studies ranging from zebrafish with 100% 1X2N cells in the adult to some strains of mice with up to 98% 2X2N cells in the adult and other species with variable ratios of 1X2N and 2X2N cells are reviewed relative to the time of conversion. Various structural, physiologic, metabolic, genetic, hormonal, oxygenation, and other factors that play a key role in the inability of post-neonatal and adult myocytes to undergo additional cytokinesis are also reviewed.

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

RATIONALE: Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration. OBJECTIVE: To examine the contribution of EMPs to intraembryonic endothelial cells. METHODS AND RESULTS: We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc)+ circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development. CONCLUSIONS: Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.

Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

Fetal cardiomyocytes shift from glycolysis to oxidative phosphorylation around the time of birth. Myeloid ecotropic viral integration site 1 (MEIS1) is a transcription factor that promotes glycolysis in hematopoietic stem cells. We reasoned that MEIS1 could have a similar role in the developing heart. We hypothesized that suppression of MEIS1 expression in fetal sheep cardiomyocytes leads to a metabolic switch as found at birth. Expression of MEIS1 was assayed in left ventricular cardiac tissue and primary cultures of cardiomyocytes from fetal (100- and 135-d gestation, term = 145 d), neonatal, and adult sheep. Cultured cells were treated with short interfering RNA (siRNA) to suppress MEIS1. Oxygen consumption rate was assessed with the Seahorse metabolic flux analyzer, and mitochondrial activity was assessed by staining cells with MitoTracker Orange. Cardiomyocyte respiratory capacity increased with advancing age concurrently with decreased expression of MEIS1. MEIS1 suppression with siRNA increased maximal oxygen consumption in fetal cells but not in postnatal cells. Mitochondrial activity was increased and expression of glycolytic genes decreased when MEIS1 expression was suppressed. Thus, we conclude that MEIS1 is a key regulator of cardiomyocyte metabolism and that the normal down-regulation of MEIS1 with age underlies a gradual switch to oxidative metabolism.-Lindgren, I. M., Drake, R. R., Chattergoon, N. N., Thornburg, K. L. Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

KEY POINTS: Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRß1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRß1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. ABSTRACT: We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102 ± 3 and 135 ± 1 days of gestational age (dGA) sheep (n = 7 per age; term ∼145 dGA). Cells were treated with T3 (1.5 nm), insulin-like growth factor (IGF)-1 (1 µg mL-1 ) or a combination in the presence of TR antagonist NH3 (100 nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10 µm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/ß1 and p21. Age specific levels of TRα1/ß1 were measured in normal hearts from fetuses [95 dGA (n = 8), 135 dGA (n = 7)], neonates (n = 8) and adult ewes (n = 7). TRα1 protein levels were consistently >50% more than TRß1 at each gestational age (P < 0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100 dGA and by ∼75% in 135 dGA cardiomyocytes (P < 0.05). NH3 blocked the T3  + IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100 dGA cardiomyocytes in 135 dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P < 0.05), as well as T3 -induced p21 (-75%, P < 0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3  + IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135 dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

Developmental changes in the balance of glycolytic ATP production and oxidative phosphorylation in ventricular cells: A simulation study.

The developmental program of the heart requires accurate regulation to ensure continuous circulation and simultaneous cardiac morphogenesis, because any functional abnormalities may progress to congenital heart malformation. Notably, energy metabolism in fetal ventricular cells is regulated in a manner that differs from adult ventricular cells: fetal cardiomyocytes generally have immature mitochondria and fetal ventricular cells show greater dependence on glycolytic ATP production. However, although various characteristics of energy metabolism in fetal ventricular cells have been reported, to our knowledge, a quantitative description of the contributions of these factors to fetal ventricular cell functions has not yet been established. Here, we constructed a mathematical model to integrate various characteristics of fetal ventricular cells and predicted the contribution of each characteristic to the maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results demonstrated that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped prolong the time for which ventricular cell contraction was maintained under anoxic conditions. The integrated model also enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells. Because fetal cardiomyocytes exhibit similar energy metabolic profiles to stem cell-derived cardiomyocytes and those in the failing heart, an improved understanding of these fetal ventricular cells will contribute to a better comprehension of the processes in stem cell-derived cardiomyocytes or under pathological conditions.

Wnt/ß-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/ß-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

Sca-1+ cells from fetal heart with high aldehyde dehydrogenase activity exhibit enhanced gene expression for self-renewal, proliferation, and survival.

Stem/progenitor cells from multiple tissues have been isolated based on enhanced activity of cytosolic aldehyde dehydrogenase (ALDH) enzyme. ALDH activity has emerged as a reliable marker for stem/progenitor cells, such that ALDH(bright/high) cells from multiple tissues have been shown to possess enhanced stemness properties (self-renewal and multipotency). So far though, not much is known about ALDH activity in specific fetal organs. In this study, we sought to analyze the presence and activity of the ALDH enzyme in the stem cell antigen-1-positive (Sca-1+) cells of fetal human heart. Biochemical assays showed that a subpopulation of Sca-1+ cells (15%) possess significantly high ALDH1 activity. This subpopulation showed increased expression of self-renewal markers compared to the ALDH(low) fraction. The ALDH(high) fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the ALDH(high) and not the ALDH(low) fraction could give rise to all the cell types of the original population, demonstrating multipotency. ALDH(high) cells showed increased resistance against aldehyde challenge compared to ALDH(low) cells. These results indicate that ALDH(high) subpopulation of the cultured human fetal cells has enhanced self-renewal, multipotency, high proliferation, and survival, indicating that this might represent a primitive stem cell population within the fetal human heart.

Unexpected maturation of PI3K and MAPK-ERK signaling in fetal ovine cardiomyocytes.

In the first two-thirds of gestation, ovine fetal cardiomyocytes undergo mitosis to increase cardiac mass and accommodate fetal growth. Thereafter, some myocytes continue to proliferate while others mature and terminally differentiate into binucleated cells. At term (145 days gestational age; dGA) about 60% of cardiomyocytes become binucleated and exit the cell cycle under hormonal control. Rising thyroid hormone (T3) levels near term (135 dGA) inhibit proliferation and stimulate maturation. However, the degree to which intracellular signaling patterns change with age in response to T3 is unknown. We hypothesized that in vitro activation of ERK, Akt, and p70(S6K) by two regulators of cardiomyocyte cell cycle activity, T3 and insulin like growth factor-1 (IGF-1), would be similar in cardiomyocytes at gestational ages 100 and 135 dGA. IGF-1 and T3 each independently stimulated phosphorylation of ERK, Akt, and p70(S6K) in cells at both ages. In the younger mononucleated myocytes, the phosphorylation of ERK and Akt was reduced in the presence of IGF-1 and T3. However, the same hormone combination led to a dramatic twofold increase in the phosphorylation of these signaling proteins in the 135 dGA cardiomyocytes-even in cells that were not proliferating. In the older cells, both mono- and binucleated cells were affected. In conclusion, fetal ovine cardiomyocytes undergo profound maturation-related changes in signaling in response to T3 and IGF-1, but not to either factor alone. Differences in age-related response are likely to be related to milestones in fetal cardiac development as the myocardium prepares for ex utero life.

Mesenchymal stem cells from fetal heart attenuate myocardial injury after infarction: an in vivo serial pinhole gated SPECT-CT study in rats.

Mesenchymal stem cells (MSC) have emerged as a potential stem cell type for cardiac regeneration after myocardial infarction (MI). Recently, we isolated and characterized mesenchymal stem cells derived from rat fetal heart (fC-MSC), which exhibited potential to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. In the present study, we investigated the therapeutic efficacy of intravenously injected fC-MSC in a rat model of MI using multi-pinhole gated SPECT-CT system. fC-MSC were isolated from the hearts of Sprague Dawley (SD) rat fetuses at gestation day 16 and expanded ex vivo. One week after induction of MI, 2×10(6) fC-MSC labeled with PKH26 dye (n = 6) or saline alone (n = 6) were injected through the tail vein of the rats. Initial in vivo tracking of 99mTc-labeled fC-MSC revealed a focal uptake of cells in the anterior mid-ventricular region of the heart. At 4 weeks of fC-MSC administration, the cells labeled with PKH26 were located in abundance in infarct/peri-infarct region and the fC-MSC treated hearts showed a significant increase in left ventricular ejection fraction and a significant decrease in the end diastolic volume, end systolic volume and left ventricular myo-mass in comparison to the saline treated group. In addition, fC-MSC treated hearts had a significantly better myocardial perfusion and attenuation in the infarct size, in comparison to the saline treated hearts. The engrafted PKH26-fC-MSC expressed cardiac troponin T, endothelial CD31 and smooth muscle sm-MHC, suggesting their differentiation into all major cells of cardiovascular lineage. The fC-MSC treated hearts demonstrated an up-regulation of cardio-protective growth factors, anti-fibrotic and anti-apoptotic molecules, highlighting that the observed left ventricular functional recovery may be due to secretion of paracrine factors by fC-MSC. Taken together, our results suggest that fC-MSC therapy may be a new therapeutic strategy for MI and multi-pinhole gated SPECT-CT system may be a useful tool to evaluate cardiac perfusion, function and cell tracking after stem cell therapy in acute myocardial injury setting.

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

BACKGROUND: Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. METHODS AND RESULTS: We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. CONCLUSIONS: The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Mid-gestation ovine cardiomyocytes are vulnerable to mitotic suppression by thyroid hormone.

Circulating fetal 3,3',5-tri-iodo-l-thyronine (T(3) ) is maintained at very low levels until a dramatic prepartum surge. 3,3',5-Tri-iodo-l-thyronine inhibits serum-stimulated proliferation in near-term ovine cardiomyocytes, but it is not known whether midgestation myocytes are also inhibited. Because early cessation of cardiomyocyte mitosis would result in an underendowed heart, we hypothesized that 0.67 gestation (100 of 145 days gestation) ovine cardiomyocytes would be insensitive to suppressive growth effects of T(3) . These younger cardiomyocytes were grown with T(3) in 10% serum-enriched media for 24 hours. Physiological (0.37, 0.75, and 1.5 nmol/L) concentrations of T(3) dramatically suppressed mitotic activity in cardiomyocytes (P < .001). 3,3',5-Tri-iodo-l-thyronine stimulated phosphorylation of extracellular signal-regulated kinase and AKT (also known as Protein Kinase B [PKB]) signaling pathways. Nevertheless, the protein content of the cell cycle suppressor, p21, increased 2-fold (P < .05), and promoter, cyclin D1, decreased by 50%. Contrary to our hypothesis, elevated levels of T(3) powerfully inhibit proliferation of midgestation fetal cardiomyocytes. Thus, midgestation maternal hyperthyroidism might lead to an underendowed fetal myocardium.

Isolation of cardiovascular precursor cells from the human fetal heart.

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium.

AIMS: Recent clinical studies revealed that positive results of cell transplantation on cardiac function are limited to the short- and mid-term restoration phase following myocardial infarction (MI), emphasizing the need for long-term follow-up. These transient effects may depend on the transplanted cell-type or its differentiation state. We have identified a population of cardiomyocyte progenitor cells (CMPCs) capable of differentiating efficiently into beating cardiomyocytes, endothelial cells, and smooth muscle cells in vitro. We investigated whether CMPCs or pre-differentiated CMPC-derived cardiomyocytes (CMPC-CM) are able to restore the injured myocardium after MI in mice. METHODS AND RESULTS: MI was induced in immunodeficient mice and was followed by intra-myocardial injection of CMPCs, CMPC-CM, or vehicle. Cardiac function was measured longitudinally up to 3 months post-MI using 9.4 Tesla magnetic resonance imaging. The fate of the human cells was determined by immunohistochemistry. Transplantation of CMPCs or CMPC-CM resulted in a higher ejection fraction and reduced the extent of left ventricular remodelling up to 3 months after MI when compared with vehicle-injected animals. CMPCs and CMPC-CM generated new cardiac tissue consisting of human cardiomyocytes and blood vessels. Fusion of human nuclei with murine nuclei was not observed. CONCLUSION: CMPCs differentiated into the same cell types in situ as can be obtained in vitro. This excludes the need for in vitro pre-differentiation, making CMPCs a promising source for (autologous) cell-based therapy.

Agrin regulation of alpha3 sodium-potassium ATPase activity modulates cardiac myocyte contraction.

Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of alpha3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the alpha3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the alpha3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na(+) in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-alpha3 Na,K-ATPase interaction and suggest that agrin modulation of the alpha3 Na,K-ATPase is important in regulating heart function.

Human embryonic stem cells and cardiac repair.

The muscle lost after a myocardial infarction is replaced with noncontractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. Human embryonic stem cell-derived cardiomyocytes exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain in the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-derived cardiomyocytes and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges.

Cardioinductive network guiding stem cell differentiation revealed by proteomic cartography of tumor necrosis factor alpha-primed endodermal secretome.

In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.

Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications.

The discovery of human pluripotent stem cells has laid the foundation for an emerging new field of biomedical research that holds promise to develop models of human development and disease, establish new strategies for discovering and testing drugs, and provide systems for the generation of cells and tissues for transplantation for the treatment of disease. The remarkable potential of pluripotent stem cells has sparked interest and excitement in academia, the biotechnology and pharmaceutical industries, as well as the lay public. Although the potential of human pluripotent stem cells is truly outstanding, fulfilling this potential is solely dependent on our ability to efficiently generate functional cell types from them. Some of the most successful approaches in this area to date are those that have applied the principles of developmental biology to stem cell differentiation. In this chapter, we review these concepts and highlight specific examples demonstrating that pluripotent stem cell differentiation in culture recapitulates the key aspects of early embryonic development. By continuing to translate insights from embryology to stem cell biology, progress in our ability to generate specific cell types from pluripotent stem cells will advance, yielding enriched populations of human cell types, including cardiomyocytes, hematopoietic cells, hepatocytes, pancreatic beta cells, and neural cells, for drug discovery, functional evaluation in preclinical models of human disease, and ultimately clinical applications.

Second-harmonic generation and two-photon-excited autofluorescence microscopy of cardiomyocytes: quantification of cell volume and myosin filaments.

The ability to quantify changes in cardiomyocyte and myosin volume across gestation and in response to intrauterine insults will lead to a better understanding of the link between low birth weight and an increased risk of heart disease in adult life. We present the use of second-harmonic generation (SHG) and two-photon excitation autofluorescence (TPEF) microscopy to image unstained isolated fetal cardiomyocytes. The simultaneous collection of these two images provides a wealth of information on the morphology of cardiomyocytes. The SHG signal provides high-contrast images of myosin filaments and the TPEF signal can be used to clearly visualize cell morphology. A potential issue may arise if SHG microscopy is performed exclusively due to the lack of sensitivity to distinguish between mononucleated and binucleated cardiomyocytes. However, TPEF microscopy has the ability to efficiently separate the two types of cardiomyocytes. In addition, quantitative analysis of the SHG and TPEF images enables quantification of myosin filament level and accurate determination of cell volume. In short, we demonstrate that advanced nonlinear optical microscopy can be used to answer key physiological questions in the early origins of adult health with increased accuracy and speed compared to previously used methods.

Identification of myocardial and vascular precursor cells in human and mouse epicardium.

During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit(+) and CD34(+) cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit(+) and CD34(+) cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit(+) cells 3 days after coronary ligation; at this time point, epicardial c-kit(+) cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit(+), together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

Identification and characterization of a novel Schwann and outflow tract endocardial cushion lineage-restricted periostin enhancer.

Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.