Isolating and defining cells to engineer human blood vessels

Abstract

A great deal of attention has been recently focused on understanding the role that bone marrow-derived putative endothelial progenitor cells (EPC) may play in the process of neoangiogenesis. However, recent data indicate that many of the putative EPC populations are comprised of various haematopoietic cell subsets with proangiogenic activity, but these marrow-derived putative EPC fail to display vasculogenic activity. Rather, this property is reserved for a rare population of circulating viable endothelial cells with colony-forming cell (ECFC) ability. Indeed, human ECFC possess clonal proliferative potential, display endothelial and not haematopoietic cell surface antigens, and display in vivo vasculogenic activity when suspended in an extracellular matrix and implanted into immunodeficient mice. Furthermore, human vessels derived became integrated into the murine circulatory system and eventually were remodelled into arterial and venous vessels. Identification of this population now permits determination of optimal type I collagen matrix microenvironment into which the cells should be embedded and delivered to accelerate and even pattern number and size of blood vessels formed, in vivo. Indeed, altering physical properties of ECFC-collagen matrix implants changed numerous parameters of human blood vessel formation, in host mice. These recent discoveries may permit a strategy for patterning vascular beds for eventual tissue and organ regeneration.

Figure 1

Colony‐forming unit‐Hill (CFU‐Hill) and endothelial colony‐forming cell (ECFC) colonies as they appear in vitro. (a) Representative phase‐contrast image of CFU‐Hill colony, which can be identified as an aggregate of phase‐contrast bright round cells with stellate‐shaped phase‐contrast dark‐appearing adherent cells emerging from the base of the round cell aggregate. (b) ECFC depicted with edges of the flattened colony highlighted by arrows, and cells within the colony forming cobblestone pattern. Scale bars represent 500 μm. Image is reproduced with permission from Yoder et al. (5).

Figure 2

Three‐dimensional matrix fibril density varies with collagen concentration. Fibril density increased linearly with increasing collagen concentration from 0.5 mg/ml (a) to 2.5 mg/ml (b) as imaged by confocal reflectance microscopy (scale bar = 10 μm). Image is reproduced with permission from Critser et al. (52).

Figure 3

Three‐dimensional matrix physical properties vary with collagen concentration. Shear storage modulus (G′ measured in Pascal [Pa] – a measure of stiffness) increased with greater collagen concentration (a), δ significantly decreased with greater collagen concentration (indicative of shear response being dominated by solid collagen fibril phase of the matrix) (b), and compressive modulus (E _c) significantly increased with greater collagen concentration (indicative of increased compressive resistance) (c). Asterisks highlight significant differences P < 0.05. Image is reproduced with permission from Critser et al. (52).

Figure 4

Histochemical analysis of explanted cell matrices. Removal of matrices after 14 days revealed significant differences in size and apparent vascularity of the matrices. Representative histological sections from control matrix with no added ECFC (a), or ECFC suspended in gels of 0.5 (b), 1.5 (c), 2.5 (d), or 3.5 (e) mg/ml collagen concentration. Given that the same amount of gel was implanted in each animal, significant differences in extent of remodelling are apparent. Scale bar represents 1 mm. Image is reproduced with permission from Critser et al. (52).

Figure 5

Analysis of red blood cell‐containing blood vessel areas within explanted matrices. Light micrographs of explant histological sections reveal percentage of human CD31‐expressing vessels as distribution of various area subsets (a) and total human CD31 vascular area as a function of varying collagen concentration matrices (b). Asterisks indicate significant differences P < 0.05 among the different collagen matrices. Image is reproduced with permission from Critser et al. (52).