PAX4 Enhances Beta-Cell Differentiation of Human Embryonic Stem Cells

Background Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of β-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. Methods and Findings Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a β-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca2+ homeostasis of β-cells developing in embryoid bodies produced from such HESC. Cells expressing key β-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. Conclusion Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative β-cells. Our findings provide a novel foundation to study the mechanism of pancreatic β-cells differentiation during early human development and to help evaluate strategies for the generation of purified β-cells for future clinical applications

A novel function for the haemopoietic supportive murine bone marrow MS-5 mesenchymal stromal cell line in promoting human vasculogenesis and angiogenesis

The bone marrow contains specific microenvironmental stem cell niches that maintain haemopoiesis. CXCL12-expressing mesenchymal stromal cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin+ cell line, MS-5, provides a clonal surrogate bone marrow stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor cell (HSC/HPC) fate in vitro, we hypothesized that MS-5 cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin+MS-5 cells, including CXCL12 (SDF-1), NOV (CCN3), HGF, Angiopoietin-1 and CCL2 (MCP-1). Concentrating on CXCL12, we confirmed its presence in MS-5 conditioned media and demonstrated that its antagonist in receptor binding, AMD-3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS-5 mediated human vasculogenesis in vitro, principally by regulating human endothelial cell migration. Thus, the clonal nestin+MS-5 murine bone marrow stromal cell line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes

Association of Maternal Antiangiogenic Profile at Birth With Early Postnatal Loss of Microvascular Density in Offspring of Hypertensive Pregnancies

Offspring of hypertensive pregnancies are more likely to have microvascular rarefaction and increased blood pressure in later life. We tested the hypothesis that maternal angiogenic profile during a hypertensive pregnancy is associated with fetal vasculogenic capacity and abnormal postnatal microvascular remodeling. Infants (n=255) born after either hypertensive or normotensive pregnancies were recruited for quantification of postnatal dermal microvascular structure at birth and 3 months of age. Vasculogenic cell potential was assessed in umbilical vein endothelial cells from 55 offspring based on in vitro microvessel tube formation and proliferation assays. Maternal angiogenic profile (soluble fms-like tyrosine kinase-1, soluble endoglin, vascular endothelial growth factor, and placental growth factor) was measured from postpartum plasma samples to characterize severity of pregnancy disorder. At birth, offspring born after hypertensive pregnancy had similar microvessel density to those born after a normotensive pregnancy, but during the first 3 postnatal months, they had an almost 2-fold greater reduction in total vessel density (−17.7±16.4% versus −9.9±18.7%; P=0.002). This postnatal loss varied according to the vasculogenic capacity of the endothelial cells of the infant at birth (r=0.49; P=0.02). The degree of reduction in both in vitro and postnatal in vivo vascular development was proportional to levels of antiangiogenic factors in the maternal circulation. In conclusion, our data indicate that offspring born to hypertensive pregnancies have reduced vasculogenic capacity at birth that predicts microvessel density loss over the first 3 postnatal months. Degree of postnatal microvessel reduction is proportional to levels of antiangiogenic factors in the maternal circulation at birth.Hypertension is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited

Association of Maternal Antiangiogenic Profile at Birth With Early Postnatal Loss of Microvascular Density in Offspring of Hypertensive Pregnancies

Offspring of hypertensive pregnancies are more likely to have microvascular rarefaction and increased blood pressure in later life. We tested the hypothesis that maternal angiogenic profile during a hypertensive pregnancy is associated with fetal vasculogenic capacity and abnormal postnatal microvascular remodeling. Infants (n=255) born after either hypertensive or normotensive pregnancies were recruited for quantification of postnatal dermal microvascular structure at birth and 3 months of age. Vasculogenic cell potential was assessed in umbilical vein endothelial cells from 55 offspring based on in vitro microvessel tube formation and proliferation assays. Maternal angiogenic profile (soluble fms-like tyrosine kinase-1, soluble endoglin, vascular endothelial growth factor, and placental growth factor) was measured from postpartum plasma samples to characterize severity of pregnancy disorder. At birth, offspring born after hypertensive pregnancy had similar microvessel density to those born after a normotensive pregnancy, but during the first 3 postnatal months, they had an almost 2-fold greater reduction in total vessel density (−17.7±16.4% versus −9.9±18.7%; P=0.002). This postnatal loss varied according to the vasculogenic capacity of the endothelial cells of the infant at birth (r=0.49; P=0.02). The degree of reduction in both in vitro and postnatal in vivo vascular development was proportional to levels of antiangiogenic factors in the maternal circulation. In conclusion, our data indicate that offspring born to hypertensive pregnancies have reduced vasculogenic capacity at birth that predicts microvessel density loss over the first 3 postnatal months. Degree of postnatal microvessel reduction is proportional to levels of antiangiogenic factors in the maternal circulation at birth.Hypertension is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited