A comparison of methods for quantifying angiogenesis in the Matrigel assay in vitro.

Angiogenesis is of major interest because of its involvement in numerous pathologies or for promoting tissue repair. It is often assessed by the ability of endothelial cells to sprout, migrate, and form vascular tubules in Matrigel in vitro. Matrigel contains a mixture of basement membrane components, which stimulate endothelial cells to form capillary-like hexagonal structures, and is often preferred over other in vitro assays because of its ease of use, rapidity and the ability to measure key steps in angiogenesis, including migration, protease activity, and tubule formation. Various methods have been used to quantitate tubule formation, yet no consensus has been reached regarding the best quantification method for evaluating the efficacy of angiogenic stimulants or inhibitors in this Matrigel assay. Here, we have measured the ability of umbilical cord blood endothelial colony-forming cell-derived cells to form tubules in growth factor reduced Matrigel in the presence or absence of two angiogenic inhibitors, suramin and SU6668, to compare the benefits and limitations of two quantification methods-Angiosys and Wimasis. These comparative analyses revealed that both Angiosys and Wimasis are easy to use, accurately quantify angiogenesis, and will suit the needs of different types of users.

Characterization of endothelial progenitor cells in the NOD mouse as a source for cell therapies.

BACKGROUND: Endothelial progenitor cells (EPCs) in bone marrow (BM) and peripheral blood (PB) contribute to tissue repair in various pathological conditions via the formation of new blood vessels. Previous studies indicate that diabetic patients have reduced EPC number and deregulated EPC function, although the regenerative properties of EPCs in diabetes are unknown. We wish to characterize and compare EPCs from pre-diabetic and diabetic non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D), in order to delineate the role of these cells in the pathogenesis of autoimmune diabetes. METHODS: Whole BM was obtained by flushing femurs, tibias and illiac crests from pre-diabetic and diabetic NOD mice (5-30 weeks) in which the diabetic status was confirmed by measuring blood glucose levels (> or =11.5 mmol/L); PB was collected in heparin-coated tubes and lysed after incubation with antibodies directed against EPCs. RESULTS: FACS analyses revealed a significant decrease in EPC number (CD31(+), c-Kit(+), Sca-1(+), Lin(-)) in BM from diabetic compared to pre-diabetic mice (P = 0.02). Conversely, EPC number was significantly increased in PB from diabetic compared to pre-diabetic mice (P = 0.01). CONCLUSIONS: These data suggest that at the onset of diabetes, BM-derived EPCs are stimulated to enter the systemic circulation likely in response to signals from the pancreas. Further studies are required to elucidate whether EPCs home the damaged pancreas, thus representing a prospective source of autologous cells for beta-cell regeneration therapy.

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 beta-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 beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-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 beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.