G-Protein coupled receptor signalling in pluripotent stem cell-derived cardiovascular cells: Implications for disease modelling

Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement.

Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution

A Study to Improve the Differentiation of Human Embryonic Stem Cells to Functional Hepatocytes

Human embryonic stem cells (hESCs) possess 2 unique properties (1) pluripotency and (2) self-renewal, and therefore, hold great promise for biomedical application and regenerative medicine. In vitro differentiation of hESCs is a vital tool to generate unlimited human hepatocytes. To date, several multi-step protocols have been established to generate hepatocyte-like cells from undifferentiated hESCs via definitive endoderm (DE) formation. However, hESC derived-hepatocytes in these systems exhibit some immature characteristics, thus it remains a challenge on how to further improve hepatic differentiation. In addition, the molecular mechanisms regulating the differentiation are still ambiguous, making in vitro differentiation a difficult task. The ultimate aim of this project is to improve the differentiation of hESCs to functional hepatocytes. In this thesis, the work includes two main parts: (1) modulating signalling pathways to explore molecular mechanisms controlling DE differentiation; (2) developing a 3-dimensional (3D) culture system to improve the functionality of hESC-derived hepatocytes. DE formation is a critical step for the production of hepatocytes. In the first part of this thesis, I showed that suppression of PI3K signalling using the LY 294002 inhibitor (LY) during hESC differentiation significantly improves Activin A (AA)-induced DE generation, which subsequently augments hepatocyte production. Further mechanistic interrogation of this phenomenon has revealed that dual treatment of hESCs with AALY enhances the Activin downstream signalling, Smad2/3 phosphorylation and their nuclear translocation with Smad4. Furthermore, dual treatment with AALY also affects the disruption of β-catenin/E-cadherin complexes, which cooperatively contribute to distinctive morphological changes that may signify the occurrence of EMT and hence improved specification of DE. These findings suggest that suppression of PI3K/Akt modulates both Nodal/Activin and β-catenin pathways, the two most important signalling involved in mesendoderm and DE cell fate specification, therefore improved DE differentiation of hESCs. Liver development in vivo is regulated by cell–cell contacts in a 3D environment and the absences of this may account for, at least partly, some of the immature features of hESC-derived hepatocytes. In the second part of my thesis, based on initial encouraging results obtained from HepG2 cells, I applied alginate based 3D culture system to hESCs after they are differentiated into DE cells and optimised culture conditions. The results confirmed that 3D culture microenvironment enhanced hepatic differentiation and functionality of hESC-derived hepatocytes in compared to the monolayer format. Collectively, this study has demonstrated a significant cornerstone in the strategies to improve hepatic differentiation of hESCs by addressing the molecular signalling and micro-niche cues that govern hepatocyte lineage commitment. Hence, this will pave the way for the use of these hepatocytes in future regenerative therapies and biomedical applications

MED12 regulates a transcriptional network of calcium-handling genes in the heart

The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart

Basic leucine zipper and W2 domain-containing protein 2 (BZW2): A novel cardiac WNT component

Die Regulation des Wnt/β-catenin Signalwegs ist nicht nur entscheidend für alle Stadien der kardialen Entwicklung, sondern auch für die Homöostase im adulten Herzen. Tatsächlich ist die Aktivierung des Wnt/β-catenin Signalwegs mit dem pathologischen Herz-Remodeling assoziiert. Ein besseres Verständnis der Regulation des Wnt/β-catenin Signalwegs bei Herzinsuffizienz könnte die Identifikation potentieller Faktoren zum Blockieren des pathologischen Herz-Remodelings und/oder das Aktivieren der endogenen Regeneration ermöglichen. Diese Arbeit konzentriert sich auf die Identifikation von gewebespezifischen Regulatoren des Wnt/β-catenin Signalwegs im Herzen. Frühere Arbeiten unserer Gruppe identifizierten basic leucine zipper and W2 domains containing protein (BZW) 2 als einen kardialen Interaktionspartner von β-catenin und KLF15. Die Rolle von BZW2 im Wnt/β-catenin Signalweg bei der Kardiogenese und der Homöostase des adulten Herzens war das Thema dieser Studie. Eine Analyse des BZW2-Proteins zeigte, dass die mutmaßliche ZIP und bZIP umfassende Domäne wichtig für die nukleare Lokalisation ist. Die Expression von BZW2 hat sich als bedeutsam im adulten Herzen, aber auch während der embryonalen Kardiogenese erwiesen. Eine niedrige BZW2-Expression ist für die effektive Bildung des kardialen Mesoderms notwendig, denn es kam unter BZW2-Überexpression zu einem Abbruch der Kardiomyozytenbildung in einem Modell der in vitro Kardiogenese. Dennoch war die BZW2-Expression nicht entscheidend für die Embryogenese, was auf eine kompensatorische Funktion verwandter Proteine hindeuten könnte. Interessanterweise war BZW2 für den Erhalt der normalen Herzfunktion und während der Reaktion auf Stress notwendig. Obwohl BZW2 die Wnt-Transkriptionsaktivität in vitro nicht signifikant inhibiert hat, resultierte das Fehlen von BZW2 in de novo Synthese von β-catenin spezifisch im adulten Herzgewebe in vivo. Außerdem wurde BZW2 selbst durch den Wnt/β-catenin Signalweg reguliert, was auf seine Rolle als regulatorischer Rückkopplungsfaktor in vivo hindeutet. Zusammenfassend identifizierte diese Arbeit einen neuen herzgewebespezifischen Faktor des Wnt/β-catenin Signalwegs auf einer neuen Regulationsebene und demonstrierte seine Relevanz für die normale Herzhomöostase. Angesichts der ubiquitären Expression und der vielfältigen Funktionen von β-catenin könnte die gewebespezifische Modulation neue Therapieoptionen darstellen