Notch/Delta4 signaling inhibits human megakaryocytic terminal differentiation.

The effects of Notch signaling on human megakaryocytic and erythroid differentiation were investigated by exposing human CD34(+) progenitor cells to an immobilized chimeric form of the Notch ligand, Delta-like4 (Dll4Fc). Exposure of human cord blood CD34(+) cells to Dll4Fc induced a modest enhancement of erythroid cell production. Conversely, under megakaryocytic culture conditions, Dll4Fc strongly impaired platelet production by reducing the generation of mature CD41a(+)CD42b(+) megakaryocytes (MKs) and platelet-forming cells. The inhibitory activity of Dll4 on terminal MK differentiation was confirmed by culturing CD34(+) cells onto Dll-4-expressing stroma cells (engineered to express the membrane-anchored form of Dll4). The reduced production of mature CD41a(+)CD42(+) cells was rescued by inhibiting Notch signaling either with the N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester γ-secretase inhibitor or the dominant-negative version of Mastermind. Dll4 impaired the generation of mature CD41a(+)CD42b(+) cells and proplatelet formation without affecting earlier steps of MK differentiation, such as production of megakaryocytic/erythroid progenitors and colony-forming units-MKs. This blockade was accompanied by a modulation of the transcriptional program of megakaryocytic differentiation. All these results indicate that Dll4/Notch signaling inhibits human terminal MK differentiation.

Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies.

Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR(4131)] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR(4131)] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

Porous polysaccharide-based scaffolds for human endothelial progenitor cells.

Human ECFCs contribute to vascular repair. For this reason, they are considered as valuable cell therapy products in ischemic diseases. Porous scaffolds are prepared that are composed of natural polysaccharides, pullulan and dextran, by chemical crosslinking without use of organic solvents. These porous scaffolds, which have pores with an average size of 42 µm and a porosity of 21%, preserve the viability and the proliferation of cord-blood ECFCs. After 7 d of culture in porous scaffolds, ECFCs express endothelial markers (CD31 and vWf) and maintain endothelial functions. The cultured cells can be easily retrieved by enzymatic degradation of the porous scaffolds. In vitro results suggest that the porous scaffold could allow cell delivery of ECFCs for treatment of vascular diseases.