With an autologous origin, high proliferation capacity and potentialities to differentiate into matures endothelial cells (EC), endothelial progenitor cells (EPC) have raised huge interest and offer new opportunities in vascular engineering. Currents protocols for isolation and differentiation of EPC requires at least 1 month to observe an endothelium-like morphology and about 2 months for confluent EC monolayer. We report here a new method to differentiate and expand EPC on polyelectrolyte multilayer films (PEM) which promotes rapid differentiation of EPC into EC.

Rabbit EPC isolated from leukocytes fraction were cultivated on a PEM or on Fibronectin (Fn) coated glass surfaces and jugular vein EC (JVEC) were used as control. Phase-contrast microscopy observations allowed to follow differentiation and morphological changes of the cells after 4, 14, 21 and 60 days. The evolution of the phenotype during the progressive differentiation of EPC into mature EC was also checked by confocal microscopy, verifying the expression of CD34, CD31, vWF and LDL uptake. The grey level quantification was used for the quantitative analyses of CD31, vWF and Dil-Ac-LDL expression.

After 4 days, the cells appeared to be round shaped without noticeable differences between the Fn and the PEM coated surface. Moreover, confocal microscopy observations have showed different stage of EPC going from early (CD34+/-) to late EPC (CD31+/- and vWF+/-) on both surfaces. After 14 days on PEM, EPC formed already a confluent monolayer and exhibited an endothelium-like morphology similar to those found in JVEC whereas on Fn, the similar cellular morphology was observed after 60 days of culture. Confocal microscopy observations after 14 days have indicated the full disappearance of CD34 expression, specific of haematopoietic phenotype whereas CD31, vWF and Dil-Ac-LDL, specific markers of matures EC, are expressed on both surfaces, signature of cellular differentiation. The grey levels quantification for these markers indicates an expression significatively higher only on PEM close to matures EC compare to Fn.

The present study describes an easy method for expansion and differentiation of EPC seeded on PEM and demonstrate that this strategy accelerate the differentiation into EC.