EPCs (also known as CD34+
cell) were first isolated from peripheral blood by Asahara et al in 1997 [1
], and have the ability to differentiate into mature endothelial cells and take part in angiogenesis. EPCs and hematopoietic stem cells originate from angioblasts and normally form blood islands [17
]. EPCs are usually isolated from umbilical cord blood or peripheral blood, however, approximately 3% of EPCs are found in bone marrow mononuclear cells [18
]. In this study, we characterized EPCs isolated from chicken bone marrow.
The isolation methods of EPCs in vitro
include flow cytometry, MASC (Magnetic Activated Cell Sorting) and density centrifugation. Density centrifugation is usually used for mononuclear cells such as hematopoietic cells and EPCs in blood. Percoll is made of saccharose and characterized by high density, low osmotic pressure and non-toxicity. For the culture conditions, Medium B used in conjunction with fibronectin as a matrix, has previously been reported to provide the best proliferative conditions for mammalian cell types in vitro
]. However, it was not the best for our chicken-derived cell cultures. In comparison to other mammal-derived EPCs, the proliferation of chicken EPCs was slower and not easy to rapidly passage. Because DMEM/F12 (Medium A) has greater nutritive constituents than M199 (Medium B), it was used to maintain chicken EPCs in culture. We speculate that the different media preference is related to species-specific characteristics.
The identification criteria of EPCs include cell morphology, specific antigen markers and self-renewal ability. The morphology of EPCs changes from short spindle-shaped cells, cell colonies and linear arrays to a typical cobblestone-like shape during differentiation to mature endothelial cells [19
cells are considered putative EPCs by most researchers [20
]. CD133 (also known as AC133) is a marker for hematopoietic stem cells and EPCs, and is gradually lost as EPCs differentiate into mature endothelial cells. CD34 is also an antigen marker for hematopoietic and endothelial cell lines. VEGFR-2 (also known as FLK-1 and KDR) is a specific marker for endothelial cells. vWF also functions as a specific marker for endothelial cell lineage. It is produced by endothelial cells and located in the cytoplasm of Weibel-Palade bodies. Thus vWF and Weibel-Palade bodies serve as specific markers for endothelial identification [22
]. Moreover, the specific ability of endothelial cells to uptake both Dil-ac-LDL and FITC-UEA-1 can be used to identify endothelial cell types. In this experiment, EPCs were identified through detection of the markers CD34, CD133 and VEGFR-2 using immunofluorescence. Uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1also revealed that the EPCs were double positive for these indicators. Moreover, Weibel-Palade bodies were observed by transmission electron microscopy. Thus the chicken EPCs retained characteristics typical of EPCs in vitro
EPCs are responsible for the construction of the blood vessel lumen, and are able to differentiate into mature endothelial cells and smooth muscle cells. There are many factors influencing the differentiation of EPCs into mature endothelial cells, among which the central factor is VEGF. Apart from inducing the differentiation of EPCs, VEGF also induces the differentiation of other stem/progenitor cells into mature endothelial cells [19
]. The underlying mechanisms are far from clear, except that the VEGF receptor (R)-2 is located on endothelial cell membranes, and that activation of protein kinase B by VEGF initiates various intracellular signaling pathways, which promote the growth and differentiation of cells through the activation of inositol triphosphate [24
]. In our study, the concentration of VEGF used to induce differentiation was five-fold higher than that contained within the growth medium, thus greatly promoting differentiation of EPCS into mature endothelial cells. Through RT-PCR, CD34 and VEGFR-2 mRNA expression were found to be significantly different (P
0.05) before and after VEGF-induced differentiation of EPCs into mature endothelial cells. Thus, cultured chicken EPCs can develop into mature endothelial cells in response to VEGF as identified by morphological and phenotypic characteristics.
EPCs are also known to develop into smooth muscle cells. Smooth muscle cells are the main functional cells of the vascular mesosphere and are involved in the synthesis of the vascular matrix and vasomotor function. Platelet-derived growth factor-BB (PDGF-BB) was used to induce EPC differentiation into smooth muscle cells. Seven days post-induction, cells with myoid morphology were observed. In accordance with the morphology, immunolabelling for the stem cell marker CD133 was negative in the inducted cells, while expression of the smooth muscle cell specific marker α-MSA was positive as determined using immunofluorescence and RT-PCR. Together, these data indicate that chicken EPCs can be differentiated into both mature endothelial cells and smooth muscle cells by stimulation with VEGF and PDGF-BB, respectively, in vitro.