Defective Vagal Innervation in Murine Tbx1 Mutant Hearts.

Haploinsufficiency of the T-box transcription factor TBX1 is responsible for many features of 22q11.2 deletion syndrome. Tbx1 is expressed dynamically in the pharyngeal apparatus during mouse development and Tbx1 homozygous mutants display numerous severe defects including abnormal cranial ganglion formation and neural crest cell defects. These abnormalities prompted us to investigate whether parasympathetic (vagal) innervation of the heart was affected in Tbx1 mutant embryos. In this report, we used an allelic series of Tbx1 mouse mutants, embryo tissue explants and cardiac electrophysiology to characterise, in detail, the function of Tbx1 in vagal innervation of the heart. We found that total nerve branch length was significantly reduced in Tbx1+/- and Tbx1neo2/- mutant hearts expressing 50% and 15% levels of Tbx1. We also found that neural crest cells migrated normally to the heart of Tbx1+/-, but not in Tbx1neo2 mutant embryos. In addition, we showed that cranial ganglia IXth and Xth were fused in Tbx1neo2/- but neuronal differentiation appeared intact. Finally, we used telemetry to monitor heart response to carbachol, a cholinergic receptor agonist, and found that heart rate recovered more quickly in Tbx1+/- animals versus controls. We speculate that this condition of decreased parasympathetic drive could result in a pro-arrhythmic substrate in some 22q11.2DS patients.This research was funded by a British Heart Foundation grant number RG/15/13/28570 to P.J. Scambler and A. Calmont and number RG/15/15/31742 to A Tinker and was facilitated by the NIHR Biomedical Research Centre at Barts. R.A. was supported by a Medical Research Council Clinical Research Training Fellowship. This research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centr

Assessing Zika Virus Transmission Within Households During an Outbreak in Martinique, 2015-2016.

Since 2015, Zika virus (ZIKV) has caused large epidemics in the Americas. Households are natural targets for control interventions, but quantification of the contribution of household transmission to overall spread is needed to guide policy. We developed a modeling framework to evaluate this contribution and key epidemic features of the ZIKV epidemic in Martinique in 2015-2016 from the joint analysis of a household transmission study (n = 68 households), a study among symptomatic pregnant women (n = 281), and seroprevalence surveys of blood donors (n = 457). We estimated that the probability of mosquito-mediated within-household transmission (from an infected member to a susceptible one) was 21% (95% credible interval (CrI): 5, 51), and the overall probability of infection from outside the household (i.e., in the community) was 39% (95% CrI: 27, 50). Overall, 50% (95% CrI: 43, 58) of the population was infected, with 22% (95% CrI: 5, 46) of infections acquired in households and 40% (95% CrI: 23, 56) being asymptomatic. The probability of presenting with Zika-like symptoms due to another cause was 16% (95% CrI: 10, 23). This study characterized the contribution of household transmission in ZIKV epidemics, demonstrating the benefits of integrating multiple data sets to gain more insight into epidemic dynamics

Comparison of Hepatic-like Cell Production from Human Embryonic Stem Cells and Adult Liver Progenitor Cells: CAR Transduction Activates a Battery of Detoxification Genes

In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Riedel, Rodrigo Nicolas. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pérez Alcázar, Germán Antonio. Hospital Universitario Virgen Macarena;Fil: Magatti, Marta. Istituto Ospedaliero;Fil: Maskin, Bernardo. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Dueñas, José Luis. Hospital Universitario Virgen Macarena;Fil: Parolini, Ornella. Istituto Ospedaliero;Fil: Sánchez-Margalet, Víctor. Hospital Universitario Virgen Macarena;Fil: Varone, Cecilia Laura. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Endothelial Neuropilin Disruption in Mice Causes DiGeorge Syndrome-Like Malformations via Mechanisms Distinct to Those Caused by Loss of Tbx1

The spectrum of human congenital malformations known as DiGeorge syndrome (DGS) is replicated in mice by mutation of Tbx1. Vegfa has been proposed as a modifier of DGS, based in part on the occurrence of comparable phenotypes in Tbx1 and Vegfa mutant mice. Many additional genes have been shown to cause DGS-like phenotypes in mice when mutated; these generally intersect in some manner with Tbx1, and therefore impact the same developmental processes in which Tbx1 itself is involved. In this study, using Tie2Cre, we show that endothelial-specific mutation of the gene encoding the VEGFA coreceptor neuropilin-1 (Nrp1) also replicates the most prominent terminal phenotypes that typify DGS. However, the developmental etiologies of these defects are fundamentally different from those caused by absence of TBX1. In Tie2Cre/Nrp1 mutants, initial pharyngeal organization is normal but subsequent pharyngeal organ growth is impaired, second heart field differentiation is normal but cardiac outflow tract cushion organization is distorted, neural crest cell migration is normal, and palatal mesenchyme proliferation is impaired with no change in apoptosis. Our results demonstrate that impairment of VEGF-dependent endothelial pathways leads to a spectrum of DiGeorge syndrome-type malformations, through processes that are distinguishable from those controlled by Tbx1

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field

Genetic inactivation of Semaphorin 3C protects mice from acute kidney injury

To guide the development of therapeutic interventions for acute kidney injury, elucidating the deleterious pathways of this global health problem is highly warranted. Emerging evidence has indicated a pivotal role of endothelial dysfunction in the etiology of this disease. We found that the class III semaphorin SEMA3C was ectopically upregulated with full length protein excreted into the blood and truncated protein secreted into the urine upon kidney injury and hypothesized a role for SEAM3C in acute kidney injury. Sema3c was genetically abrogated during acute kidney injury and subsequent kidney morphological and functional defects in two well-characterized models of acute kidney injury; warm ischemia/reperfusion and folic acid injection were analyzed. Employing a beta actin-dependent, inducible knockout of Sema3c, we demonstrate that in acute kidney injury SEMA3C promotes interstitial edema, leucocyte infiltration and tubular injury. Additionally, intravital microscopy combined with Evans Blue dye extravasation and primary culture of magnetically sorted peritubular endothelial cells identified a novel role for SEMA3C in promoting vascular permeability. Thus, our study points to microvascular permeability as an important driver of injury in acute kidney injury, and to SEMA3C as a novel permeability factor and potential target for therapeutic intervention