Outgrowing endothelial and smooth muscle cells for tissue engineering approaches

Abstract

In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identified in the peripheral blood. In our study, we evaluated the utilization of both cell types isolated and differentiated from peripheral porcine blood in terms for their use for tissue engineering purposes. By means of density gradient centrifugation, the monocyte fraction from porcine blood was separated, split, and cultivated with specific culture media with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 for the differentiation of endothelial cells or smooth muscle cells. Obtained cells were characterized at an early stage of cultivation before the first passage and a late stage (fourth passage) on the basis of the expression of the antigens CD31, CD34, CD45, nitric oxide synthase, and the contractile filaments smooth-muscle alpha-actin (sm-alpha-actin) and smoothelin. Functional characterization was done based on the secretion of nitric oxide, the formation of a coherent monolayer on polytetrafluoroethylene, and capillary sprouting. During cultivation in both endothelial cell growth medium-2 and smooth muscle cell growth medium-2, substantially two types of cells grew out: early outgrown CD45-positive cells, which disappeared during further cultivation, and in 85% (n = 17/20) of cultures cultivated with endothelial cell growth medium-2 colony-forming late outgrowth endothelial cells. During cultivation with smooth muscle cell growth medium-2 in 80% (n = 16/20) of isolations colony-forming late outgrowth smooth muscle cells entered the stage. Cultivation with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 had selective effect on the late outgrown cells to that effect that the number of CD31-positive cells increased from 34.8% ± 13% to 83.9% ± 8% in cultures cultivated with endothelial cell growth medium-2 and the number of sm-α-actin+ cells increased from 52.6% ± 18% to 88% ± 5% in cultures cultivated with smooth muscle cell growth medium-2, respectively. Functional analyses revealed significantly higher levels of nitric oxide secretion, endothelialization capacity, and capillary formation in not expanded cultures cultivated with endothelial cell growth medium-2 in comparison to later stages of cultivation and mature aortic cells. Blood seems to be a reliable and feasible source for the isolation of both endothelial and smooth muscle cells for application in tissue engineering approaches. Whereas, early co-cultures of early and late outgrowth cells provide functional advantages, the differentiation of cells can be directed selectively by the used culture medium for the expansion of highly proliferative late outgrowth endothelial cells and late outgrowth smooth muscle cells, respectively.

Keywords: Circulating progenitor cells; early outgrowth endothelial cells; endothelial progenitor cells; late outgrowth endothelial cells; late outgrowth smooth muscle cells; tissue engineering.

Figure 1.

This figure shows a schematic overview of the experimental setting. (a) Blood was collected from pigs’ jugular vein. (b) By means of density gradient centrifugation, the monocyte fraction was separated, aspirated, and divided in two equal parts, which were cultivated with either EGM-2 or SGM-2. (c and d) In both culture settings after 4–5 days, small round-shaped EOEC appeared. During further cultivation in both settings, colony-forming clusters of late outgrowth cells appeared 9 ± 5 days after isolation: predominantly LOEC in cultures cultivated with EGM-2 and predominantly LOSMC in cultures cultivated with SGM-2 (c and d). Colony-forming late outgrown cells formed a coherent monolayer and overgrew the early outgrown cells.

Figure 2.

This figure shows representative immunofluorescence staining and FACS analysis of outgrown cells cultivated with (a) EGM-2 and (b) SGM-2. In both settings, most cells expressed CD34 (red, upper row in both columns), whose portion decreased slightly during further cultivation. During cultivation with EGM-2, colony-forming cells positive for CD31 (green, middle row) and NOS (red, bottom both in the left column) became the predominant cell type. During cultivation with SGM-2, colony-forming LOSMC positive for sm-alpha-actin-positive (green, middle row) and about 57% positive for smoothelin (red, bottom row) were the predominant cell type. Compare Table 3 for antigen expression.

Figure 3.

(a) First appearance of capillary sprouting was seen when colony-forming LOEC proliferated and surrounded EOEC (arrow). Formation of long tubular capillaries was found only in the presence of LOSMC in cultures cultivated with SGM-2. During cultivation, (b) these structures became more and more complex and (c) LOSMC participated in the tube formation. These cultures could be cultivated for about 4 weeks without signs of overgrowth. (d) In this time, capillaries grew up to a length of up to 4.5 cm.

Figure 4.

Cells which were cultivated with EGM-2 and taken from (a) not expanded cultures and (b) from the fourth passage, seeded on PTFE after 7 days of cultivation in a bioreactor (staining with calcein AM and ethidiumhomodimer-1, magnification 100×). Not expanded cells cultivated with EGM-2 formed a nearly coherent monolayer (a) in contrast to cells taken from the fourth passage (b). The percentage of the surface covered with LOEC and EC is shown in the both graphs (c under static conditions and d under dynamic conditions). (c) Under static conditions no significant differences were found between the different cell types. After perfusion in a bioreactor under dynamic conditions, coverage of the surface was significantly highest in setting with not expanded cells cultivated with EGM-2 in comparison to all other groups (p < 0.05), while no significant differences were found between the other settings.

Figure 5.

Significant higher levels of NO metabolites were measured in not expanded cell cultures cultivated with EGM-2 (EOEC and LOEC) in comparison to expanded cultures cultivated with EGM-2 in the fourth passage and aortic EC. The concentration of expanded cultures cultivated with EGM-2 was comparable to those of not expanded and expanded aortic EC. Almost no NO formation was found in cultures cultivated with SGM-2 and aortic SMC.