An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Abstract

Induced pluripotent stem cells are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors, which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

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

“…Herein, we report the creation of a new IPN of NorHA and collagen, providing control over matrix degradability and stiffness, independent of total protein and polysaccharide density (Figure 1). We demonstrate the formation of a viable vascular network from iPSC-EPs in this platform, quantified by an improved open-source computational pipeline [34]. These data represent a significant advance toward engineered vascular tissue from a patient-specific cell type.…”

“…The resin was filtered out, and the solution was titrated to pH 7.05 with tetrabutylammonium hydroxide, frozen, and lyophilized. Next, HA-TBA (2 wt%), 5- Collagen hydrogels were fabricated as described previously [34,36] −ln ( A e − A t A e − A 0 ) = kt Where Ae is the absorbance at equilibrium, At is the absorbance at a given time t, and A0…”

“…iPSCs were differentiated into CD34 + iPSC-EPs following an established low-serum medium protocol [39]. Five days after CHIR induction (LC Technologies), cells were purified by methods we have described previously [34]. Briefly, the resulting confluent monolayer of differentiated cells was dispersed into a single-cell suspension with Accutase, tagged with a CD34-PE antibody (Miltenyi Biotec), and purified on a fluorescence-activated cell sorting (FACS) instrument (S3e Cell Sorter, Bio-Rad).…”

“…To analyze the microvascular networks' topology (i.e., length and connectivity), we improved a computational pipeline that we had previously developed in ImageJ and MATLAB [34]. Briefly, confocal z-stacks that contained a cobblestone morphology on the surface of the gel were manually removed.…”

Perivascular Secretome Influences Hematopoietic Stem Cell Maintenance in a Gelatin Hydrogel

“…As this approach generates a 3D skeleton of all vessels within an experimental volume, it is able to accurately resolve both the number of branches and vessel segments. 17…”