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.

Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration

“…Using three confocal images per sample stained with CD31 antibody ( n = 4 each group) the vessel organization was quantified, whereby a score of 0 denotes the absence of spatial patterning, and 1 denotes parallel alignment of the vessels. To further substantiate the spatial organization of the vessels, two-dimensional Fast Fourier Transform analysis was performed using an ImageJ plugin to generate frequency plots 59,60 . In addition, longitudinal sections from the interface between the host and transplanted tissue were imaged by confocal microscopy for MHC, GFP, and Hoechst 33342 nuclear dye.…”

Fiber Scaffold Patterning for Mending Hearts: 3D Organization Bringing the Next Step

“…Thus topographical patterning can also induce vascular network organization and maturation. [ 182 ] Deepthi et al also used SES, but they obtained a trilayer scaffold mimicking the fiber orientation and mechanical properties of a vascular network. They spun a mixture of poly(hydroxy butyrate co -hydroxy valerate) (PHBV) and poly(vinyl alcohol) (PVA) for the intima layer and PHBV and elastin for the tunica media and adventitia.…”

“…Reproduced with permission. Copyright [ 182 ] 2017, Springer Science+Business Media. E) SEM pictures of a random electrospun fibrous scaffold and F) a patterned electrospun scaffold with the ridge/groove width of 300/100 μm, G) phalloidin staining of endothelial cells loaded on the random electrospun scaffold and H) smooth muscle cells on the patterned scaffold showing a preferred direction of smooth muscle cell actin fiber morphology on the patterned scaffold.…”

iPSCs: A powerful tool for skeletal muscle tissue engineering

“…Likewise, the phenomenon of angiogenesis requires a 3D microenvironment to achieve the correct scale of complexity of the different blood vessels. Thus, the engineered tissue in a 3D structure can be directly implanted for muscle repair, being tough to achieve with 2D micropatterned surfaces …”