Integration of induced pluripotent stem cell-derived endothelial cells with polycaprolactone/gelatin-based electrospun scaffolds for enhanced therapeutic angiogenesis

Abstract

Background: Induced pluripotent stem-cell derived endothelial cells (iPSC-ECs) can be generated from any somatic cell and their iPSC sources possess unlimited self-renewal. Previous demonstration of their proangiogenic activity makes them a promising cell type for treatment of ischemic injury. As with many other stem cell approaches, the low rate of in-vivo survival has been a major limitation to the efficacy of iPSC-ECs to date. In this study, we aimed to increase the in-vivo lifetime of iPSC-ECs by culturing them on electrospun polycaprolactone (PCL)/gelatin scaffolds, before quantifying the subsequent impact on their proangiogenic function.

Methods: iPSC-ECs were isolated and stably transfected with a luciferase reporter to facilitate quantification of cell numbers and non-invasive imaging in-vivo PCL/gelatin scaffolds were engineered using electrospinning to obtain woven meshes of nanofibers. iPSC-ECs were cultured on scaffolds for 7 days. Subsequently, cell growth and function were assessed in vitro followed by implantation in a mouseback subcutaneous model for 7 days.

Results: Using a matrix of conditions, we found that scaffold blends with ratios of PCL:gelatin of 70:30 (PG73) spun at high flow rates supported the greatest levels of iPSC-EC growth, retention of phenotype, and function in vitro. Implanting iPSC-ECs seeded on PG73 scaffolds in vivo improved their survival up to 3 days, compared to cells directly injected into control wounds, which were no longer observable within 1 h. Enhanced engraftment improved blood perfusion, observed through non-invasive laser Doppler imaging. Immunohistochemistry revealed a corresponding increase in host angiogenic mechanisms characterized by the enhanced recruitment of macrophages and the elevated expression of proangiogenic cytokines vascular endothelial growth factor and placental growth factor.

Conclusions: Knowledge of these mechanisms combined with a deeper understanding of the scaffold parameters influencing this function provides the groundwork for optimizing future iPSC-EC therapies utilizing engraftment platforms. The development of combined scaffold and iPSC-EC therapies could ultimately improve therapeutic angiogenesis and the treatment of ischemic injury.

Keywords: Angiogenesis; Biomaterial scaffolds; Endothelial cells; Induced pluripotent stem cells; Regenerative medicine.

Fig. 1

a Schematic of iPSC-EC differentiation and characterization. b iPSC-EC seeding protocol on electrospun scaffolds. c SEM images of PCL/gelatin scaffolds at varying flow rates and PCL/gelatin blends; quantification of fiber thickness (inset), scale bar represents 50 μm. ****p < 0.0001. n = 5 scaffolds/group and n = 100 fibers/scaffold. PCL polycaprolactone, PG73 PCL/gelatin (70:30 ratio), PG55 PCL/gelatin (50:50 ratio)

Fig. 2

a Bioluminescence standard curve of iPSC-EC cell number, representative cell bioluminescence images (inset). b iPSC-EC growth at 3 and 7 days on electrospun scaffolds of varying flow rate and PCL/gelatin blends. ***p < 0.001, **p < 0.01 compared to PCL (8 ml/h) control scaffolds within the same time point. n = 5 scaffolds/group. c CD31 quantification of iPSC-EC cultures on scaffolds at 7 days in vitro. *p < 0.05, **p < 0.01. n = 5 scaffolds/group. d Representative photographs of iPSC-EC cultures stained with CD31 (red) and counterstained with DAPI (blue) at 5×; magnified images at 10× (inset). Scale bar represents 100 μm, inset scale bar represents 10 μm. iPSC-EC induced pluripotent stem cell-derived endothelial cell, PCL polycaprolactone, PG73 PCL/gelatin (70:30 ratio), PG55 PCL/gelatin (50:50 ratio), DAPI 4',6-diamidino-2-phenylindole

Fig. 3

a Angiogenic cytokine gene expression of iPSC-ECs cultured on tissue culture plastic (TCP) and PG73 (16 ml/h scaffolds) in normoxia vs hypoxia. Values presented as fold change normalized to normoxia. *p < 0.05, **p < 0.01. n = 5 samples/group. b In-vivo engraftment curves measured through IVIS bioluminescence measurement of iPSC-ECs and iPSC-EC-seeded PG73 scaffolds over 5 days. ***p < 0.001, *p < 0.05. n = 5 samples (animals)/group. c Area under the curve quantification of engraftment bioluminescence curves. *p < 0.05. n = 5 samples (animals)/group. d Representative bioluminescence photographs of animals implanted with experimental groups over 5 days. EGF epidermal growth factor, PlGF placental growth factor, VEGF vascular endothelial growth factor, PG73 polycaprolactone/gelatin (70:30 ratio), iPSC-EC induced pluripotent stem cell-derived endothelial cell, AUC area under the curve

Fig. 4

a Laser Doppler blood perfusion curves of areas encompassing wound site, representative photographs and Doppler images (inset). *p < 0.05, **p < 0.01, ***p < 0.001. n = 5 samples (animals)/group. b Representative macroscopic photographs of wound explants at day 2 and day 9, scale bar represents 3 mm. PG73 polycaprolactone/gelatin (70:30 ratio), iPSC-EC induced pluripotent stem cell-derived endothelial cell

Fig. 5

a Functional angiogenesis quantification of arteriole density (vessels double stained for CD31⁺ and SMC-α actin⁺). *p < 0.05, ***p < 0.001. n = 5 samples/group. b Quantification of arteriole location in surrounding tissue vs inside scaffold. **p < 0.01. n = 5 samples/group. c Quantification of average arteriole diameter. ***p < 0.001, **p < 0.01, *p < 0.05. n = 5 samples/group. d Representative photographs of DAPI/CD31/SMC-α actin merge, CD31, and SMC-α actin, respectively, at the wound site or tissue implant interface (dotted lines: above represents tissue, below represents scaffold) at day 2, scale bar represents 100 μm. SMC-α smooth muscle cell-actin, CD31 cluster of differentiation 31 (endothelial marker), PG73 polycaprolactone/gelatin (70:30 ratio), iPSC-EC induced pluripotent stem cell-derived endothelial cell, DAPI 4',6-diamidino-2-phenylindole

Fig. 6

a Quantification of total neutrophil levels at wound site. **p < 0.01. n = 5 samples/group. b Quantification of neutrophil location in surrounding tissue vs within scaffold. c Quantification of total macrophage levels at wound site. **p < 0.01, ****p < 0.0001. n = 5 samples/group. d Quantification of macrophage location in the surrounding tissue vs within the scaffold. p < 0.05 comparing macrophages within scaffolds only. n = 5 samples/group. Tissue implant interface marked by dotted lines: above represents tissue, below represents scaffold. NE neutrophil elastase, iPSC-EC induced pluripotent stem cell-derived endothelial cell, PG73 polycaprolactone/gelatin (70:30 ratio), DAPI 4',6-diamidino-2-phenylindole

Fig. 7

a Quantification of total PlGF expression at the wound site. *p < 0.05. n = 5 samples/group. b Quantification of PlGF expression location in surrounding tissue vs within the scaffold. *p < 0.05 comparing PlGF expression within the surrounding tissue only. n = 5 samples/group. c Representative photographs of PlGF staining with DAPI counterstain, scale bar represents 100 μm. Tissue implant interface marked by dotted lines: above represents tissue, below represents scaffold. PlGF placental growth factor, iPSC-EC induced pluripotent stem cell-derived endothelial cell, PG73 polycaprolactone/gelatin (70:30 ratio), DAPI 4',6-diamidino-2-phenylindole

Fig. 8

a Quantification of total VEGF expression at the wound site. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5 samples/group. b Quantification of VEGF expression location in surrounding tissue vs within the scaffold. *p < 0.05 comparing VEGF expression within the surrounding tissue only, #p < 0.05 comparing VEGF expression within scaffolds only. n = 5 samples/group. c Representative photographs of VEGF staining with DAPI counterstain, scale bar represents 100 μm. Tissue implant interface marked by dotted lines: above represents tissue, below represents scaffold. VEGF vascular endothelial growth factor, iPSC-EC induced pluripotent stem cell-derived endothelial cell, PG73 polycaprolactone/gelatin (70:30 ratio), DAPI 4',6-diamidino-2-phenylindole