Quantifying the Vasculogenic Potential of Induced Pluripotent Stem Cell-Derived Endothelial Progenitors in Collagen Hydrogels

Abstract

Induced pluripotent stem cell-derived endothelial progenitors (iPSC-EPs) have emerged as a promising candidate cell source for patient-specific ischemic therapies. Before these cells can be appropriately deployed in a clinical setting, it is imperative to study their assembly into functional vascular networks in extracellular matrix (ECM)-mimicking, three-dimensional (3D) microenvironments. To elucidate the interactions of iPSC-EPs with the ECM, we examined how in vitro modulation of structural protein density, the presence of angiogenic growth factors, and relative proteolytic activity affected the vasculogenic potential of these progenitors, that is, their ability to self-assemble into vessel-like networks. We found that the addition of a ROCK pathway inhibitor and exogenous vascular endothelial growth factor (VEGF) are imperative for inducing robust iPSC-EP vasculogenesis in collagen hydrogels. Under these conditions, 3D vascular-like networks containing VE-cadherin-expressing lumens formed within a week of culture. To quantify this 3D vessel-like network, we developed a computational pipeline to analyze network length, connectivity, and average lumen diameter. Increasing the concentration of collagen in the hydrogels abrogated network formation and encouraged the formation of disconnected, largediameter lumens. This phenomenon was in part related to the cells' proteolytic capacity and the hydrogels' properties, specifically hydrogel deformability and pore size. In conclusion, we demonstrate that the vasculogenic potential of iPSC-EPs is regulated by cell-matrix interactions and the matrix properties of collagen hydrogels.

Quantification of iPSC-derived vascular networks in novel phototunable angiogenic hydrogels

“…Previously, we had quantified iPSC-EP vasculogenesis in type I collagen hydrogels and confirmed that the network topology was dependent on the density of the surrounding matrix, which may have influenced the mean diffusion distance and degradability [18,34]. However, our experiments were limited to a single week due to severe collagen compaction.…”

“…To this end, biomaterials provide an excellent means to study this process in vitro, due to their high tunability and ability to mimic native tissue [17]. We have previously utilized rat tail collagen to demonstrate the high vasculogenic potential of iPSC-EPs [18].…”

Perivascular Secretome Influences Hematopoietic Stem Cell Maintenance in a Gelatin Hydrogel

“…The networks were quantified for metrics of total network length, and number of branches and junctions, using a previously established computational pipeline implemented in ImageJ and MATLAB. 16 Briefly, a custom plugin in ImageJ filters and binarizes the z-stacks so that the vessels are extracted from the image. In MATLAB, the vessels are skeletonized and then converted into a quantifiable nodal network.…”

“…This could indicate possible indirect effects on cell types in the niche, not necessarily the HSCs themselves. 16…”

Structural control of fibrin bioactivity by mechanical deformation

“…Fibrin morphology was imaged using an upright laser scanning confocal microscope (LSM-510, Zeiss) under reflectance using a 40X, 1.0 NA water-dipping objective (Zeiss). Fibrin network samples were illuminated with a 488 nm laser and reflected light was collected by setting the Meta detector channel between 475-500 nm wavelength in accordance to previous literature [22]. The diameter of fibrin fibers was measured on raw images using ImageJ.…”