Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction.

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-ß and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

Src family tyrosine kinase regulates intracellular pH in cardiomyocytes.

The Anion Cl-/HCO3- Exchangers AE1, AE2, and AE3 are membrane pH regulatory ion transporters ubiquitously expressed in vertebrate tissues. Besides relieving intracellular alkaline and CO2 loads, the AEs have an important function during development and cell death and play a central role in such cellular properties as cell shape, metabolism, and contractility. The activity of AE(s) are regulated by neurohormones. However, little is known as to the intracellular signal transduction pathways that underlie this modulation. We show here that, in cardiomyocytes that express both AE1 and AE3, the purinergic agonist, ATP, triggers activation of anion exchange. The AE activation is observed in cells in which AE3 expression was blocked but not in cells microinjected with neutralizing anti-AE1 antibodies. ATP induces tyrosine phosphorylation of AE1, activation of the tyrosine kinase Fyn, and association of both Fyn and FAK with AE1. Inhibition of Src family kinases in vivo by genistein, herbimycin A, or ST638 prevents purinergic activation of AE1. Microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent activation of Src family kinase, significantly decreases ATP-induced activation of AE. Microinjection of an anti-FAK antibody as well as expression in cardiomyocytes of Phe397 FAK dominant negative mutant, also prevents purinergic activation of AE. Therefore, tyrosine kinases play a key role in acute regulation of intracellular pH and thus in cell function including excitation-contraction coupling of the myocardium.

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

Embryonic stem cells: from bench to bedside.

As early as their derivation, embryonic stem (ES) cells have attracted a great attention to clinicians. Derived from early embryos, these cells remain pluripotent in culture while they can be expanded in principle without limit. They give rise to most progenies and differentiate to all major somatic lineages of potential use in regenerative medicine. The great therapeutic promises put forward almost 10 years ago to cure or relieve degenerative diseases are still up to date. However, cell therapy is a complex process that significantly differs from drug-based medicine. Although a clinical trial has been announced by GERON for next year to cure spinal cord injury, many issues remain to be addressed at the bench before these cells can be used in clinics.

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking.

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.

Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells.

Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte-like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors.

A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production.

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.

Modulation of myocardial activity by extracellular ATP.

Extracellular ATP, at micromolar concentration, induces multiple functional changes in cardiac cells. Stimulation of P(2) purinoceptors is associated with Ca current increase and positive inotropic effect. On rapid application, ATP triggers membrane depolarization by activating a Cl conductance and by inducing an acidification following Cl-HCO(3) exchanger stimulation. Both effects might lead to cardiac arrhythmias following ATP release under pathophysiologic conditions.

Acute simulated ischaemia produces both inhibition and activation of K+ currents in isolated ventricular myocytes.

OBJECTIVE: The aim was to investigate the effects of acute ischaemia on cardiac repolarizing K+ currents. METHODS: We developed a model of acute ischaemia in isolated rat ventricular myocytes transiently surrounded with a mineral oil droplet. During ischaemic challenges, we recorded intracellular pH using the fluorescent probe seminaphthorhodafluor-1 (SNARF-1) and whole-cell K+ currents using the patch-clamp technique. RESULTS: Decrease in intracellular pH (pH1) during simulated ischaemia was dependent upon the extracellular proton buffer used (pH1 decreased from 7.44 +/- 0.02 to 7.16 +/- 0.04 in a Hepes-buffered medium and from 7.08 +/- 0.04 to 6.56 +/- 0.07 with bicarbonate buffer). In Hepes, action potential duration initially lengthened and then shortened under the effects of ischaemia. Initial action potential duration lengthening was concomitant with a block of the inward rectifier K+ current, whereas late shortening corresponded with the activation of the ATP-sensitive K+ current. Similar changes occurred in bicarbonate buffer although with different amplitudes and kinetics. Patch-clamp experiments also showed inhibition of the transient outward K+ current. Brief transient episodes of ischaemia activated ATP-sensitive K+ current in only 20% of control cells (n = 21) but in 100% of cells treated with 15 microM cromakalim (n = 9). CONCLUSIONS: (i) Simulated ischaemia produces complex effects on repolarizing K+ currents including both inhibition and activation; (ii) cromakalim accelerates activation of ATP-sensitive K+ current during simulated ischaemia.

Specific activation of adenylyl cyclase V by a purinergic agonist.

The present study was designed to investigate whether and how the purinergic stimulation of rat ventricular myocytes modulates the cAMP-dependent pathway. Stimulation of cardiomyocytes with ATPgammaS in the presence of the phosphodiesterase inhibitor IBMX increases by 3-fold intracellular cAMP content. In contrast to beta-adrenergic stimulation, the purinergic stimulation of adenylyl cyclase was not inhibited by activation or enhanced by inhibition of a Gi protein. Forskolin did not potentiate the effect of extracellular ATPgammaS on intracellular cAMP content but the effect of isoproterenol did. Like isoproterenol, the purinergic agonist decreased subsequent ADP-ribosylation of a 45 kDa G(alpha s) by cholera toxin. ATPgammaS also increased cAMP content in neonatal rat cardiomyocytes, a cell type that expresses a long form of Gs protein (alpha(s), 52 kDa) in contrast to adult rat cardiomyocytes that express mostly a short form of Gs protein (alpha(s), 45 kDa). Both purinergic and beta-adrenergic agonists increased cAMP in HEK 293 cells expressing type V adenylyl cyclase while cAMP was only increased by beta-adrenergic stimulation of HEK expressing type IV or VI adenylyl cyclases. Thus, we propose that the purinergic and beta-adrenergic stimulations differentially activate adenylyl cyclase isoforms in rat cardiomyocytes and that adenylyl cyclase V is the specific target of the purinergic stimulation.

A fluorescent reporter gene as a marker for ventricular specification in ES-derived cardiac cells.

We have established a CGR8 embryonic stem (ES) cell clone (MLC2ECFP) which expresses the enhanced cyan variant of Aequorea victoria green fluorescent protein (ECFP) under the transcriptional control of the ventricular myosin light chain 2 (MLC2v) promoter. Using epifluorescence imaging of vital embryoid bodies (EB) and reverse transcription-polymerase chain reaction (RT-PCR), we found that the MLC2v promoter is switched on as early as day 7 and is accompanied by formation of cell clusters featuring a bright ECFP blue fluorescence. The fluorescent areas within the EBs were all beating on day 8. MLC2ECFP ES cells showed the same time course of cardiac differentiation as mock ES cells as assessed by RT-PCR of genes encoding cardiac-specific transcription factors and contractile proteins. The MLC2v promoter conferred ventricular specificity to ECFP expression within the EB as revealed by MLC2v co-staining of ECFP fluorescent cells. MLC2ECFP-derived cardiac cells still undergo cell division on day 12 after isolation from EBs but withdraw from the cell cycle on day 16. This ES cell clone provides a powerful cell model to study the signalling roads of factors regulating cardiac cell proliferation and terminal differentiation with a view to using them for experimental cell therapy.

Phenylephrine and ATP enhance an amiloride insensitive bicarbonate-dependent alkalinizing mechanism in rat single cardiomyocytes.

To expel the excess protons generated during a cellular acidification and to fully recover basal intracellular pH (pHi), cardiac cells rely on the amiloride-sensitive Na/H antiport. We report that rat single ventricular cardiomyocytes, loaded with the fluorescent pH indicator Snarf-1 and treated with inhibitors of the Na/H antiport, amiloride or its analogues, partially restored their pHi through a bicarbonate-dependent mechanism following an acidosis (imposed by the ammonia-pulse technique). In the presence of ethylisopropylamiloride (10 microM) or amiloride (1 mM) and 25 mM bicarbonate in the extracellular solution, the average time that cells needed to recover half of their pHi, following the removal of 20 mM NH4Cl, was 3.4 min, while the rate of proton efflux was calculated to be 2.0 mM/min. The stilbene derivative, 4-4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS 200 microM), a known blocker of anion transporters, inhibited this recovery. Both phenylephrine (100 microM, 3 microM propranolol present), an alpha 1-adrenoceptor agonist, and ATP (10 microM), a purinergic agonist, significantly enhanced the rate of proton efflux that was due to this HCO3-dependent alkalinizing mechanism. Phenylephrine and ATP also shortened by three-fold the time that a myocyte needed to recover half of its initial pHi. This bicarbonate-dependent alkalinizing mechanism could provide an additional means by which cardiac cells recover their pHi from acidosis, especially under conditions in which the Na/H antiport is inhibited. Furthermore, catecholamines and ATP, which are released under various pathophysiological conditions often associated with intracellular acidosis, could play an important role in the modulation of pHi under these conditions.

[Fetal and umbilical blood cord stem cells: a room for the obstetrician and gynaecologist. Part two]. / Cellules souches foetales et du sang de cordon ombilical: une place pour le gynécologue-obstétricien. Deuxième partie.

Stem cells are undifferentiated cells, with the ability to self renew and to differentiate into specialised cells. Besides embryonic stem cells, adult, fetal and umbilical cord blood (UB) stem cells are to be distinguished. These cells are multipotent. Embryonic germ cells (EG) that also are fetal stem cells have proven to be truly pluripotent, since they are able to give derivatives of the three primitive embryonic layers. EG cells have a normal karyotype, and exhibit remarkable long-term proliferative potential. Fetal stem cells and UB cells have already been used in cell therapy trials (e.g., Parkinson's disease, congenital immunodeficiencies and hemopathies). The applications in the field of reproductive biology will lead to a better understanding of genomic imprinting with EG cells. The obstetrician and gynaecologist could act a central part in the production and study of fetal stem cells, using tissues from aborted fetuses or collecting cord blood stem cells.

[Embryonic stem cells: a position for the obstetrician and gynaecologist. Part one]. / Cellules souches embryonnaires: une place pour le gynécologue-obstétricien. Première partie.

Stem cells are undifferentiated cells, with the ability to self renew and to differentiate into specialised cells. Embryonic stem cells (ES) have proven to be truly pluripotent, since they are able to give derivatives of the three primitive embryonic layers. Human ES have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Though human ES cell-based transplantation therapy holds great promises to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials. Less spectacular, the applications in the field of reproductive biology are also outstanding: stem cell biology will lead us to a better understanding of the cellular and molecular mechanisms of events such as infertility, failure of implantation, genomic imprinting and meiosis. The obstetrician and gynaecologist could act an important part in the production and study of embryonic stem cells. However, these data have to be integrated in the ethical and juridical background of embryonic stem cell research in France.

Regulation of skin aging and heart development by TAp63.

Since the discovery of the TP63 gene in 1998, many studies have demonstrated that ΔNp63, a p63 isoform of the p53 gene family, is involved in multiple functions during skin development and in adult stem/progenitor cell regulation. In contrast, TAp63 studies have been mostly restricted to its apoptotic function and more recently as the guardian of oocyte integrity. TAp63 endogenous expression is barely detectable in embryos and adult (except in oocytes), presumably because of its rapid degradation and the lack of antibodies able to detect weak expression. Nevertheless, two recent independent studies have demonstrated novel functions for TAp63 that could have potential implications to human pathologies. The first discovery is related to the protective role of TAp63 on premature aging. TAp63 controls skin homeostasis by maintaining dermal and epidermal progenitor/stem cell pool and protecting them from senescence, DNA damage and genomic instability. The second study is related to the role of TAp63, expressed by the primitive endoderm, on heart development. This unexpected role for TAp63 has been discovered by manipulation of embryonic stem cells in vitro and confirmed by the severe cardiomyopathy observed in brdm2 p63-null embryonic hearts. Interestingly, in both cases, TAp63 acts in a cell-nonautonomous manner on adjacent cells. Here, we discuss these findings and their potential connection during development.

Direct myocardial implantation of human embryonic stem cells in a dog model of Duchenne cardiomyopathy reveals poor cell survival in dystrophic tissue.

Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Spontaneous canine muscular disorders are interesting settings to evaluate the relevance of innovative therapies in human using pre-clinical trials.

pHi regulatory ion transporters: an update on structure, regulation and cell function.

Intracellular pH (pHi) is a major regulator of various and critical cellular functions. A close regulation of pHi is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H+ or equivalent H+ into and out of the cell. Besides pHi, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters.