PubMed Health⌕ Search

Biomedical subjects

Michel Pucéat

Publications and source records attributed to Michel Pucéat.

At least 19 recordsLinked to original sources
PubMed2006

Oct-3/4 dose dependently regulates specification of embryonic stem cells toward a cardiac lineage and early heart development.

The transcriptional mechanisms underlying lineage specification and differentiation of embryonic stem (ES) cells remain elusive. Oct-3/4 (POU5f1) is one of the earliest transcription factors expressed in the embryo. Both the pluripotency and the fate of ES cells depend upon a tight control of Oct-3/4 expression. We report that transgene- or TGFbeta-induced increase in Oct-3/4 mRNA and protein levels in undifferentiated ES cells and at early stages of differentiation triggers expression of mesodermal and cardiac specific genes through Smad2/4. cDNA antisense- and siRNA-mediated inhibition of upregulation of Oct-3/4 in ES cells prevent their specification toward the mesoderm and their differentiation into cardiomyocytes. Similarly, Oct-3/4 siRNA injected in the inner cell mass of blastocysts impairs cardiogenesis in early embryos. Thus, quantitative Oct-3/4 expression is regulated by a morphogen, pointing to a pivotal and physiological function of the POU factor in mesodermal and cardiac commitments of ES cells and of the epiblast.

Animals↗
PubMed2006

Stem cell therapy in heart failure: where do we stand and where are we heading?

Heart failure is a multifactorial and complex debilitating disease facing limitations in available pharmacological therapeutics. To cure such a disease, cardiac regeneration has been envisioned using stem cells. A few clinical trials using bone marrow-derived stem cells have been carried out without conclusive results. Stem cells of other origins should therefore be considered for future trials. In this article, the advantages and disadvantages of the most promising stem cells to be used in cell therapy are reviewed. The remaining challenges of cell therapy of heart failure are also presented.

Animals↗
PubMed2005

Rb and LEK1: a "pas de deux" in cardiogenesis.

Cardiogenesis results from activation of a complex transcriptional network. The transcription factors work in a combinatorial manner to transactivate expression of constitutive genes. Embryonic stem (ES) cells are capable to recapitulate the early stages of cardiac cell differentiation. So far only the Bone Morphogenetic Protein (BMP)-dependent pathway switching on expression of Nkx2.5, one of the earliest cardiac transcription factor, has been described as the canonical cardiogenic pathway. Recently, using Rb-/- and LEK1-downregulated ES cells, we described a novel BMP-independent cardiogenic pathway implicating both Rb and LEK1, a mammalian homolog of the chicken cardiomyogenic factor 1(CMF1). Here, I discuss the role of Rb pathways in cell differentiation and the potential mechanisms underlying the effect of Rb/LEK1 interaction on cardiac specification of stem cells.

Animals↗
PubMed2005

Interplay between the retinoblastoma protein and LEK1 specifies stem cells toward the cardiac lineage.

The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb-/- mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb-dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb-/- embryonic stem (ES) cells differentiating into cardiomyocytes. Rb-/- cells displayed a dramatic delay in expression of cardiac-specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb-/- ES cell-derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP-dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb-LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb-/- ES cells. Thus, we provide evidence for a novel Rb/LEK1-dependent and BMP-independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.

Animals↗
PubMed2005

Fine-tuning in Ca2+ homeostasis underlies progression of cardiomyopathy in myocytes derived from genetically modified embryonic stem cells.

Mutations of genes encoding contractile proteins are responsible for familial hypertrophic cardiomyopathies. Understanding the process of differentiation of cardiomyocytes carrying a mutated protein is a crucial step towards potential treatments of inherited cardiac disorders. Embryonic Stem (ES) cells which faithfully recapitulate in vitro the process of cardiac cell differentiation can be genetically modified to incorporate a mutation mimicking a cardiomyopathy. ES cell lines engineered to express a wild-type (MLC2vGFP) or a mutated form (R58QMLC2vGFP) of ventricular myosin light chain 2 (MLC2v) fused to GFP were differentiated into cardiomyocytes within embryoid bodies (EBs). Visualization of GFP combined with sarcomeric actinin immunofluorescence of EBs revealed that mutated MLC2v dramatically prevented myofibrillogenesis. Cardiomyocytes expressing wild-type MLC2v featured spontaneous Ca(2+) spiking, but not those harboring the mutation. Expression of cardiac transcription factors Mef2c, GATAs, myocardin and Nkx2.5 was not affected by cell expression of mutated MLC2v. A dramatic decrease in expression of mRNAs encoding alpha-actin, MLC2a and MLC2v was observed in R58QMLC2vGFP EBs. This event was attributed to a failure of Mef2c to translocate into the nucleus, a Ca(2+)-dependent process. Expression in mutated cells of a constitutively active Ca(2+)- and calmodulin-dependent kinase II or treating EBs with ionomycin fully restored translocation of Mef2c into the nucleus and expression of mRNAs encoding sarcomeric proteins partially rescued contractile activity of EBs. Alteration of Ca(2+) homeostasis in mutated cardioblasts affects the transcriptional program of cardiac cell differentiation leading to a defect in myofibrillogenesis, and, in turn, in contractility. Genetically modified ES cells provide a unique cell model to determine abnormalities in Ca(2+) homeostasis underlying progression of human cardiomyopathies.

Calcium↗
PubMed2005

Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling.

In the adult, the heart rate is driven by spontaneous and repetitive depolarizations of pacemaker cells to generate a firing of action potentials propagating along the conduction system and spreading into the ventricles. In the early embryo before E9.5, the pacemaker ionic channel responsible for the spontaneous depolarization of cells is not yet functional. Thus the mechanisms that initiate early heart rhythm during cardiogenesis are puzzling. In the absence of a functional pacemaker ionic channel, the oscillatory nature of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ signaling could provide an alternative pacemaking mechanism. To test this hypothesis, we have engineered pacemaker cells from embryonic stem (ES) cells, a model that faithfully recapitulates early stages of heart development. We show that InsP3-dependent shuttle of free Ca2+ in and out of the endoplasmic reticulum is essential for a proper generation of pacemaker activity during early cardiogenesis and fetal life.

Animals↗
PubMed2005

[Embryonic stem cells to study early myocardial development and potential regenerative medicine].

Embryonic stem cells are capable to recapitulate the first stages of myocardial development. Using mouse embryonic stem cells, transcriptional networks specifying the cardiac fate can be delineated. Furthermore, using members of the TGFbeta superfamily to commit mouse ES cells toward a cardiac lineage, recent studies showed that ESC-derived cardiomyocytes were capable to repair post-infarcted myocardium of small and large animals. The next challenges are to validate such results using human ESCs in order to better comprehend cardiac congenital diseases and to foresee a cell therapy of heart failure. double dagger.

Animals↗
PubMed2005

Administration of allogenic stem cells dosed to secure cardiogenesis and sustained infarct repair.

The mitotic capacity of heart muscle is too limited to fully substitute for cells lost following myocardial infarction. Emerging stem cell-based strategies have been proposed to overcome the self-renewal shortfall of native cardiomyocytes, yet there is limited evidence for their capability to achieve safe de novo cardiogenesis and repair. We present our recent experience in treating long-term, infarcted hearts with embryonic stem cells, a prototype source for allogenic cell therapy. The cardiogenic potential of the engrafted murine embryonic stem cell colony was pre-tested by in vitro differentiation, with derived cells positive for nuclear cardiac transcription factors, sarcomeric proteins and functional excitation-contraction coupling. Eight weeks after infarct, rats were randomized into sham- or embryonic stem cell-treated groups. Acellular sham controls or embryonic stem cells, engineered to express enhanced cyan fluorescent protein (ECFP) under control of the cardiac actin promoter, were injected through a 28-gauge needle at three sites into the peri-infarct zone for serial assessment of functional and structural impact. In contrast to results with sham-treated animals, stem cell therapy yielded, over the 5-month follow-up period, new ECFP-positive cardiomyocytes that integrated with the infarcted myocardium. The stem cell-treated group showed a stable contractile performance benefit with normalization of myocardial architecture post infarction. Transition of embryonic stem cells into cardiomyocytes required host signaling to support cardiac-specific differentiation and could result in tumorigenesis if the stem cell dose exceeded the heart's cardioinductive capacity. Supported by the host environment, proper dosing and administration of embryonic stem cells is thus here shown useful in the chronic management of cardiac injury promoting sustained repair.

Animals↗
PubMed2005

Cardiac commitment of embryonic stem cells for myocardial repair.

Embryonic stem (ES) cells represent a source for cell-based regenerative therapies of heart failure. The pluripotency and the plasticity of ES cells allow them to be committed to a cardiac lineage following treatment with growth factors of the transforming growth factor (TGF)-beta superfamily. We describe a protocol designed to turn on expression of cardiac-specific genes in undifferentiated murine ES cells stimulated with BMP2 and/or TGF-beta. Cell commitment results in a significant improvement in spontaneous cardiac differentiation of ES cells both in vitro and in vivo.

Animals↗
PubMed2004

Cardiac specification of embryonic stem cells.

Over the past decade, cell transplantation has been recognized as a mean of repairing infarcted myocardium. Both adult stem cells and differentiated cells have yielded encouraging results with regard to engraftment into postinfarction scars. However, these cells now feature serious restrictions. Asan alternative, embryonic stem (ES) cells are particularly attractive, because of their plasticity and the subsequent possibility to drive them towards a cardiomyogenic phenotype after exposure to appropriate growth factors. An additional theoretical advantage of ES cells is their expected immune privilege. In this article, we summarize the findings obtained in cell therapy using ES cells and discuss the molecular mechanisms of cardiac specification of the cells.

Animals↗
PubMed2004

Stable benefit of embryonic stem cell therapy in myocardial infarction.

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.

Animals↗
PubMed2004

[Confocal microscopy: a tool to visualise differentiation of stem cells into cardiomyocytes].

Confocal microscopy offers important advantages compared to conventional epifluorescence microscopy. It works as an "optical microtome" leading to a accurate image resolution of a defined focal plane. Furthermore, the addition of a Nipkow disk on the confocal microscope greatly accelerates the image acquisition, up to 30 frames per second. Nevertheless, the software-assisted mathematical restoration of images acquired using a wide-field microscope allows to get images with a resolution similar to the one obtained in confocal microscopy. These imaging technologies allowed us to monitor on line cardiac differentiation of murine embryonic stem (ES) cells within 3D structures called embryoid bodies. The high rate acquisition of images using the confocal microscope equipped with a Nipkow disk allows to monitor calcium spiking in differentiating cardiomyocytes within embryoid bodies.

Animals↗
PubMed2003

A dual role of the GTPase Rac in cardiac differentiation of stem cells.

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

Animals↗
PubMed2003

Structural adaptation of the nuclear pore complex in stem cell-derived cardiomyocytes.

Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell-derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle-isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell-derived cardiomyocytes. Stem cell-derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell-derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell-derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.

Animals↗
PubMed2002

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca(2+)-binding protein that regulates diverse vital cell functions, including Ca(2+) storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt(-/-) embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt(-/-) EBs, despite normal expression levels of cardiac transcription factors. Crt(-/-) EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt(-/-) phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca(2+) with EGTA or BAPTA, or by inhibiting Ca(2+)/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca(2+) concentration ([Ca(2+)](c)) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt(-/-) cardiac cells in the absence of ionomycin-triggered [Ca(2+)](c) increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca(2+)-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

Animals↗
PubMed2002

Stem cell differentiation requires a paracrine pathway in the heart.

Members of the transforming growth factor beta1 (TGF-beta) superfamily--namely, TGF-beta and BMP2--applied to undifferentiated murine embryonic stem cells up-regulated mRNA of mesodermal (Brachyury) and cardiac specific transcription factors (Nkx2.5, MEF2C). Embryoid bodies generated from stem cells primed with these growth factors demonstrated an increased potential for cardiac differentiation with a significant increase in beating areas and enhanced myofibrillogenesis. In an environment of postmitotic cardiomyocytes, stem cells engineered to express a fluorescent protein under the control of a cardiac promoter differentiated into fluorescent ventricular myocytes beating in synchrony with host cells, a process significantly enhanced by TGF-beta or BMP2. In vitro, disruption of the TGF-beta/BMP signaling pathways by latency-associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF-beta family member induced a cardiac phenotype in stem cells. In vivo, transplantation of stem cells into heart also resulted in cardiac differentiation provided that TGF-beta/BMP2 signaling was intact. In infarcted myocardium, grafted stem cells differentiated into functional cardiomyocytes integrated with surrounding tissue, improving contractile performance. Thus, embryonic stem cells are directed to differentiate into cardiomyocytes by signaling mediated through TGF-beta/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart.

Animals↗
PubMedDate not supplied

Ca2+ signalling in cardiogenesis.

A variety of InsP3-dependent Ca2+ signals coded in time, amplitude and space occur during the process of cardiac cell differentiation and heart development. Studies performed in both embryos and embryonic stem cell differentiating in cardiomyocytes have uncovered that Ca2+ regulates multiple steps of these biological processes in vertebrates. These include secretion of cardiogenic factors, cardiac transcriptional cascades and in turn gene expression, myofibrillogenesis and initiation of embryonic pacemaker activity. Evidence is accumulating to foresee Ca2+ as a major second messenger in directing the fate of stem cells and patterning the heart in the embryo.

Animals↗