The significance of blood vessels in organogenesis and cadherins in exploratory behaviour

Organogenesis of epithelial organs requires interaction between epithelial andmesenchymal tissues. In pancreas development three different mesenchymal derivedstructures, the notochord, the endothelial cells and the splanchnic mesenchyme inducespecification, growth and further differentiation of the pancreatic epithelium.Previously, N-cadherin deficient embryos were shown to suffer from agenesis ofthe dorsal pancreas due to apoptosis of the pancreatic mesenchyme. However, byexpressing N- and E-cadherin selectively in the heart, the pancreatic phenotype was shownto be secondary to the non-functional cardiac- and vascular-system in N-cadherin-/-embryos. In addition, plasma from wild-type embryos rescued dorsal pancreas formationin N-cadherin-/- explants, suggesting factors from the circulation to be involved inpancreatic ontogeny. Recently, sphingosine-1-phosphate receptor-1 was shown to berequired for proper recruitment of vascular smooth muscle cells to the aortic wall.Vascular smooth muscle cells and dorsal pancreatic mesenchyme may originate from acommon cellular source, the splanchnic mesenchyme, and migrate to endothelial andepithelial cells, respectively, in close vicinity, suggesting that they may be regulated bysimilar developmental regulatory pathways. Consequently, the ligand, a blood bornesphingolipid metabolite, sphingosine-1-phosphate, may be involved in pancreas ontogenyas well. Indeed, the sphingosine-1-phosphate rescued dorsal pancreas in N-cadherin-/-explants by inducing proliferation of the mesenchyme. To clarify the requirement forsphingosine-1-phosphate in pancreas development, embryos deficient in sphingosine-1-phosphate receptor-1 were analysed. Whereas initial development of dorsal and ventralpancreas proceeded normally, obvious morphological changes of the dorsal pancreaticepithelium were observed at later stages, indicating defective mesenchymal-to-epithelialinteractions. The sphingosine-1-phosphate receptor-1 was mainly expressed in endothelialcells, suggesting that sphingosine-1-phosphate signals to the mesenchyme via endothelialcells. Altogether, we show for the first time that vascular function and spingosine-1-phosphate-mediating signalling regulate pancreas ontogeny by inducing mesenchymal-toepithelialsignalling.Cadherins are cell-to-cell adhesion molecules localised at the synaptic junctions,mediating synaptogenesis and neuronal path finding during development. By using theCre/loxP-system, an in vivo model was established where a dominant negative cadherinwas expressed selectively in the neurons of the central nervous system in the adult mouse.Cadherins role in neural plasticity and behaviour was analysed and demonstrated normalsynaptic transmission, long term potentiation, spatial learning and anxiety responses, whilerearing behaviour, a component in exploration was significantly reduced. Datademonstrate, for the first time, a functional role for cadherins in modifying rearing andexploratory behaviour in vivo

Signals From the Embryonic Mouse Pancreas Induce Differentiation of Human Embryonic Stem Cells Into Insulin-Producing {beta}-Cell-Like Cells.

The recent success in restoring normoglycemia in type 1 diabetes by islet cell transplantation indicates that cell replacement therapy of this severe disease is achievable. However, the severe lack of donor islets has increased the demand for alternative sources of beta-cells, such as adult and embryonic stem cells. Here, we investigate the potential of human embryonic stem cells (hESCs) to differentiate into beta-cells. Spontaneous differentiation of hESCs under two-dimensional growth conditions resulted in differentiation of Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but no insulin-producing cells. However, cotransplantation of differentiated hESCs with the dorsal pancreas, but not with the liver or telencephalon, from mouse embryos resulted in differentiation of beta-cell-like cell clusters. Comparative analysis of the basic characteristics of hESC-derived insulin(+) cell clusters with human adult islets demonstrated that the insulin(+) cells share important features with normal beta-cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta-cell transcription factors, including Foxa2, Pdx1, and Isl1

Growth-limiting role of endothelial cells in endoderm development.

Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs

High Content Analysis of Human Pluripotent Stem Cell Derived Hepatocytes Reveals Drug Induced Steatosis and Phospholipidosis

Hepatotoxicity is one of the most cited reasons for withdrawal of approved drugs from the market. The use of nonclinically relevant in vitro and in vivo testing systems contributes to the high attrition rates. Recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into pure cultures of hepatocyte-like cells expressing functional drug metabolizing enzymes open up possibilities for novel, more relevant human cell based toxicity models. The present study aimed to investigate the use of hiPSC derived hepatocytes for conducting mechanistic toxicity testing by image based high content analysis (HCA). The hiPSC derived hepatocytes were exposed to drugs known to cause hepatotoxicity through steatosis and phospholipidosis, measuring several endpoints representing different mechanisms involved in drug induced hepatotoxicity. The hiPSC derived hepatocytes were benchmarked to the HepG2 cell line and generated robust HCA data with low imprecision between plates and batches. The different parameters measured were detected at subcytotoxic concentrations and the order of which the compounds were categorized (as severe, moderate, mild, or nontoxic) based on the degree of injury at isomolar concentration corresponded to previously published data. Taken together, the present study shows how hiPSC derived hepatocytes can be used as a platform for screening drug induced hepatotoxicity by HCA

Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models

Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue

Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use