Microenvironment influences vascular differentiation of murine cardiovascular progenitor cells

Abstract

We examined the effects of the microenvironment on vascular differentiation of murine cardiovascular progenitor cells (CPCs). We isolated CPCs and seeded them in culture exposed to the various extracellular matrix (ECM) proteins in both two-dimensional (2D) and 3D culture systems. To better understand the contribution of the microenvironment to vascular differentiation, we analyzed endothelial and smooth muscle cell differentiation at both day 7 and day 14. We found that laminin and vitronectin enhanced vascular endothelial cell differentiation while fibronectin enhanced vascular smooth muscle cell differentiation. We also observed that the effects of the 3D electrospun scaffolds were delayed and not noticeable until the later time point (day 14), which may be due to the amount of time necessary for the cells to migrate to the interior of the scaffold. The study characterized the contributions of both ECM proteins and the addition of a 3D culture system to continued vascular differentiation. Additionally, we demonstrated the capability bioengineer a CPC-derived vascular graft.

Keywords: cell differentiation; extracellular matrix; progenitor cells; scaffolds; stem cells; vascular.

FIGURE 1.

Electrospun tubular scaffold fabrication. Electrospun fibers were collected on a rotating mandrel (A). The electrospun tubular scaffolds were placed in a bioreactor chamber and seeded with Flk-1⁺ CPCs. Media from the reservoir was pumped through the lumen of the tubular scaffold at a controlled and sustained pressure of 120 mmHg.

FIGURE 2.

Experimental Design. Schematic depicting experimental design. mES cells were expanded in initial differentiation media and then sorted for Flk-1⁺ CPCs. Isolated CPCs were then exposed to various microenvironments in both 2D and 3D and analyzed at day 7 or 14.

FIGURE 3.

Vascular endothelial cell differentiation. mES cell-derived Flk-1⁺ CPCs were exposed to different microenvironments for both 7 and 14 days. Cells were analyzed for vWF (red) and VE-cadherin (green).

FIGURE 4.

Vascular smooth muscle cell differentiation. mES cell-derived Flk-1⁺ CPCs were exposed to different microenvironments for both 7 and 14 days. Cells were analyzed for SM-myosin (green).

FIGURE 5.

FACS analysis of vascular endothelial cells. Flk-1⁺ CPCs were exposed to different microenvironments in both 2D (grey bars) and 3D (black bars) for both 7 (A) and 14 (B) days. CD31 was used as a cell surface marker for endothelial cells for FACS analysis. There was a significant difference increase in CD31 expression on day 14 compared to day 7. N = 3 *p < 0.05 for pairwise statistical comparison.

FIGURE 6.

FACS analysis of vascular smooth muscle cells. Flk-1⁺ CPCs were exposed to different microenvironments in both 2D (grey bars) and 3D (black bars) for both 7 (A) and 14 (B) days. SM-myosin was used as a marker for FACS analysis. There was a significantly higher expression of myosin for day 7 compared to day 14. Likewise, at each time point there was a significant difference between 2D and 3D culture system. N = 3 *p < 0.0001 for pairwise statistical comparison.

FIGURE 7.

Physical properties of electrospun tubular scaffold. Representative SEM micrographs show the fiber morphology of the outer most layer of fibers (A) and inner most layer (D). Fiber diameter distributions were calculated for the outer most layer fibers (B) and the inner most layer (E). Pore size distributions were also calculated for the outer fibers (C) and inner fibers (F).

FIGURE 8.

Immunofluorescent imaging of the electrospun tubular scaffold after 30 days in the bioreactor chamber. Cells were found evenly distributed throughout the cross-section of the tubular scaffold. The lumen of the scaffold is denoted by “L” and the lines represent the boundaries of the scaffold (A). The existence of CPC-derived cell layers is found by the higher concentration of green fluorescence—more GFP⁺ cells (shown with *) which were seeded second are found in the inner most circumference (denoted as “I”) of the tubular scaffold (B); more CD31⁺ cells (shown with *) which were seeded second are found in the inner most circumference or lumen (C); more SM-myosin⁺ cells (shown with *) which were seeded first are found on the outer most circumference of the tubular scaffold (denoted as “O”) (D).