Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

Abstract

Introduction: Human induced pluripotent stem cells (iPSCs) are a promising source of endothelial cells (iPSC-ECs) for engineering three-dimensional (3D) vascularized cardiac tissues. To mimic cardiac microvasculature, in which capillaries are oriented in parallel, we hypothesized that endothelial differentiation of iPSCs within topographically aligned 3D scaffolds would be a facile one-step approach to generate iPSC-ECs as well as induce aligned vascular organization.

Methods: Human iPSCs underwent endothelial differentiation within electrospun 3D polycaprolactone (PCL) scaffolds having either randomly oriented or parallel-aligned microfibers. Using transcriptional, protein, and endothelial functional assays, endothelial differentiation was compared between conventional two-dimensional (2D) films and 3D scaffolds having either randomly oriented or aligned microfibers. Furthermore, the role of parallel-aligned microfiber patterning on the organization of vessel-like networks was assessed.

Results: The cells in both the randomly oriented and aligned 3D scaffolds demonstrated an 11-fold upregulation in gene expression of the endothelial phenotypic marker, CD31, compared to cells on 2D films. This upregulation corresponded to >3-fold increase in CD31 protein expression in 3D scaffolds, compared to 2D films. Concomitantly, other endothelial phenotypic markers including CD144 and endothelial nitric oxide synthase also showed significant transcriptional upregulation in 3D scaffolds by >7-fold, compared to 2D films. Nitric oxide production, which is characteristic of endothelial function, was produced 4-fold more abundantly in 3D scaffolds, compared to on 2D PCL films. Within aligned scaffolds, the iPSC-ECs displayed parallel-aligned vascular-like networks with 70% longer branch length, compared to cells in randomly oriented scaffolds, suggesting that fiber topography modulates vascular network-like formation and patterning.

Conclusion: Together, these results demonstrate that 3D scaffold structure promotes endothelial differentiation, compared to 2D substrates, and that aligned topographical patterning induces anisotropic vascular network organization.

Keywords: anisotropy; endothelial cell; induced pluripotent stem cell; three-dimensional scaffolds; tissue engineering; topography; vascularization.

Figure 1

Fabrication and characterization of electrospun 3D PCL scaffolds. (a) Schematic of concurrent electrospinning of PCL and PEO; (b) Macroscopic view of 3D scaffold; (c–d) SEM images of randomly oriented (c) and aligned (d) scaffold structure; (e) Quantification of average microfiber diameter (n = 3); (f) Quantification of average pore size (n = 3). Scale bar: 1 mm (b); 50 µm (c–d).

Figure 2

Characterization of 3D scaffolds using 2D Fast Fourier Transform (FFT) analysis. (a–b) Representative reflectance microscopy image of randomly oriented scaffold (a) and corresponding alignment histogram (b); (c–d) Representative reflectance microscopy image of an aligned scaffold (c) and corresponding alignment histogram (d). Insets in B and D depict frequency plots.

Figure 3

Cell viability in 3D micofibrous scaffolds after 5 days of differentiation using Live/Dead cytotoxicity vital dyes. Representative fluorescent images depicting viable (green) and dead (red) cells are shown in samples consisting of 2D tissue culture polystyrene (TCPS) (a), 2D PCL (b), 3D randomly oriented scaffold (c), or 3D aligned scaffold (d). Depicted in the inset is the cellular morphology after 5 days of differentiation as imaged by SEM (e). Quantification of cell viability (n = 3). Scale bar: 100 µm.

Figure 4

Analysis of the depth of cellular penetration into 3D scaffolds. TO-PRO-3 (red) nuclear labeling of cells in transverse sections of randomly oriented (a) or aligned scaffolds (b); (c) Quantification of penetration depth (n = 3). Top side refers to the air-facing side of the scaffold. Corresponding microfibers are visualized using reflectance microscopy (white). Scale bar: 100 µm.

Figure 5

Quantification of endothelial differentiation after 5 days of differentiation. (a) Comparison of normalized relative CD31 intensity between 2D PCL and 3D randomly oriented or aligned scaffolds (n = 3); (b) Normalized relative fold change in gene expression for endothelial-related genes (n = 3). * indicates p < 0.05 (n = 3).

Figure 6

Nitric oxide production in 3D microfibrous scaffolds after 5 days of differentiation. (a–c) Representative confocal images of nitric oxide fluorescent probe (green) are depicted in the 2D PCL film (a), 3D randomly oriented scaffold (b), and 3D aligned scaffold (c); (d) Quantification of normalized fluorescence signal (n = 3). Total nuclei were labeled using Hoechst 33342 (blue). * Denotes statistically significant relationship, compared to 2D PCL group (p < 0.05). Scale bar = 100 µm.

Figure 7

Vascular network-like formation in 3D microfibrous scaffolds. (a) 3D stacked confocal images of CD31 staining in 2D PCL film, 3D randomly oriented scaffold, and 3D aligned scaffold. (b) Transformation of CD31 expression into skeletonized filaments; (c–d) Quantification of branch length (c) and branch points (d). * indicates statistically significant relationship to 2D PCL film, and # indicates statistically significant comparison between 3D groups. p < 0.05 (n = 5).

Figure 8

iPSC-EC morphology and focal adhesion assembly in 3D microfibrous scaffolds. (a–b) Confocal microscopy images depict paxillin expression in iPSC-ECs within 3D randomly oriented (a) or aligned (b) microfibrous scaffolds. CD31 (red), paxillin (green), and nuclei (blue). Arrows point to a representative CD31⁺ iPSC-EC. (c–d) Confocal microscopy images of iPSC-ECs cultured within randomly oriented or aligned scaffold depict elongated cellular morphology and wrapping of cell bodies around microfibers. The iPSC-ECs were visualized by CD31 (green) expression. The microfibers were visualized by autofluorescence (blue) near the 480 nm emission wavelength. Arrow denotes bulk microfiber alignment direction. Scale bar: 100 µm.