Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays

Abstract

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events during angiogenesis in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by undefined substrates and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). We rapidly encapsulated iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres using thiol-ene chemistry and subsequently encapsulated cell-dense hydrogel spheres in a cell-free hydrogel layer. The hydrogel sprouting array supported pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. iPSC-ECs in the sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin, which confirms their functional role in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds from the US Environmental Protection Agency's ToxCast library identified six compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs at a single concentration. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment.

Statement of significance: Angiogenesis assays that are commonly used for drug screening and toxicity assessment applications typically utilize natural substrates like Matrigel(TM) that are difficult to spatially pattern, costly, ill-defined, and may exhibit lot-to-lot variability. Herein, we describe a novel angiogenic sprouting assay using chemically-defined, bioinert poly(ethylene glycol) hydrogels functionalized with biomimetic peptides to promote cell attachment and degradation in a reproducible format that may mitigate the need for natural substrates. The quantitative assay of angiogenic sprouting here enables precise control over the initial conditions and can be formulated into arrays for screening. The sprouting assay here was dependent on key angiogenic signaling axes in a screen of angiogenesis inhibitors and a blinded screen of putative vascular disrupting compounds from the US-EPA.

Keywords: Angiogenic sprouting; Chemically-defined assay; Endothelial cells; Extracellular matrix; Poly(ethylene glycol) hydrogels; Thiol-ene chemistry; ToxCast.

Figure 1. iPSC-ECs exhibit sprouting behavior in synthetic hydrogel arrays over time

A: Schematic of double encapsulation procedure. (1) iPSC-ECs were initially encapsulated at 4 × 10⁷ cells/mL in PEG hydrogel spheres, and resulting cell-dense hydrogel spheres were plated on microscope slides with a physically-adhered 64-well PDMS stencil within the 16-well ProPlate manifold. (2) iPSC-EC cell-dense hydrogel spheres were cultured for 24 h in GM in ProPlates, and subsequently medium was aspirated and replaced (3) with an overlay PEG hydrogel. (4) iPSC-ECs were encapsulated in cell-free hydrogel post upon UV exposure. (5) 64-well PDMS stencil was replaced with 16-well stencil, and the 16-well ProPlate was re-assembled and subsequently filled with medium representing each respective condition (300 μL/well). B.i: Schematic of 64-well PDMS stencil that was physically adhered to a glass slide for step (1) of the procedure. B.ii: Schematic of fully-assembled iSM array with the 16-well silicone stencil in place of the 64-well PDMS stencil and medium covering each of the four posts per well. B.iii: Color photograph (top view) of fully assembled iSM array with 4 hydrogel posts per well of 16-well ProPlate before addition of medium. C: Color photograph (top view) of fully-assembled iSM array with 300 μL medium per well. Two iSM arrays were cultured at a time in OmniTray plates. D: Representative brightfield z-slices of iPSC-EC sprouting in iSM at Day 1 (D.i), Day 4 (D.ii), Day 6 (D.iii), and Day 8 (D.iv) of experimental protocol. iPSC-ECs exhibited both single-cell invasion and coordinated, multicellular cord-like structure formation (consistent in morphology with sprouting behavior) at Day 4 which rapidly progressed in extent through Day 8. Quantification in subsequent data was performed at Day 6 because of the enhanced ability to distinguish cells at Day 6 versus Day 8 and beyond (data not shown). Scale bars represent 100 μm.

Figure 2. Influence of growth factors on iPSC-EC structure formation and proliferation in synthetic hydrogel arrays

A–B: Representative maximum intensity projections of confocal z-stacks of iPSC-ECs in the absence (No GFs) or presence (GM) of growth factors. iPSC-ECs exhibited minimal invasion in the No GF control (A) and exhibited both sprouting behavior and primitive tubule network formation in the presence of GFs (B). Images were merged from red (F-Actin) and blue (DAPI) channels. Scale bar represents 100 μm. C: Quantification of iPSC-EC structure formation in the absence (No GFs) and presence (GM) of growth factors, presented as the cumulative length of skeletonized maximum intensity projections. D–E: Representative epifluorescence and phase contrast z-slices of encapsulated iPSC-ECs stained for EdU in the absence (D) or presence (E) of growth factors. Images were merged from phase contrast and red (EdU+ nuclei) channels. Scale bar represents 100 μm. F: Quantification of fraction proliferating iPSC-ECs, given as the fraction of EdU+/DAPI+ iPSC-ECs in the cell-free hydrogel post. Statistical analysis was performed using Student’s t-test at a significance level of α=0.05 (*). Data in C and D represent mean +/− standard error about the mean for 8 replicates per condition.

Figure 3. iPSC-ECs exhibit endothelial phenotype in sprouting arrays

A–C: Immunofluorescence images of iPSC-ECs stained and imaged using confocal laser scanning microscopy. iPSC-ECs were stained with primary antibodies for CD31 (A), CD144 (B), or PODXL (C) and secondary AF488-tagged antibodies (green) and were counter-stained with DAPI (to visualize nuclei) and TRITC-Phalloidin (to visualize F-Actin). The outline of the border of the original cell-dense sphere is shown with a dotted white line. Scale bars represent 100 μm.

Figure 4. Influence of cell adhesion peptide and MMP-labile crosslink concentration on growth factor-dependent iPSC-EC sprouting

Quantified iPSC-EC sprouting given as # of invading Calcein+ cells in cell-free hydrogel overlayer at the end of 6-day culture. A: iPSC-EC invasion in the presence of varying concentrations of CRGDS (0, 2, 4 mM) and MMP-labile crosslinker concentration (45, 50, 55% crosslinking, or C.L.%). Two-way analysis of variance was performed (CRGDS concentration p-value =0.0002, C.L.% p-value=0.005, interaction p-value>0.05) with post-hoc Student’s t-test comparing between conditions. Asterisk (*) denotes statistical significance compared to each respective crosslink density at 0 mM CRGDS or comparing conditions shown in brackets at α=0.05. B: Normalized iPSC-EC invasion tabulated from the data in ‘A’. Invasion in each respective crosslink % and CRGDS condition was normalized to the 0 mM CRGDS condition for each crosslink %. Normalized invasion was compared using Student’s t-test, and asterisks denote statistical significance compared to the 55% C.L. condition within each CRGDS concentration (α=0.05). C: iPSC-EC invasion in the presence of varying concentrations of CRGDS in the presence of GM without (white bars) and with (black bars) SU5416. Two-way analysis of variance was performed (CRGDS concentration p-value<0.0001, +/− SU5416 p-value>0.05, interaction p-value>0.05) with post-hoc Student’s t-test. Asterisk (*) denotes statistically different from condition without SU5416 at each respective CRGDS concentration at α=0.05. Data in A, B, and C represent the mean +/− standard deviation for 8 replicates per condition.

Figure 5. Influence of VEGF and FGF2 on iPSC-EC sprouting and viability

A: iPSC-EC sprouting quantified as # of invading Calcein+ cells in the absence of growth factors (No GFs), in the presence of particular growth factors, or in the presence of full growth factor supplementation (GM) without (−) or with VEGF inhibitors SU5416 or v114*. B: Fraction of viable iPSC-ECs in the conditions described above. For both A and B, asterisk (*) denotes statistical significance compared to No GF control at α=0.05 using Student’s t-test, and dagger (^†) denotes stascal signicance compared to the GM no inhibitor (−) control at α=0.05 using Student’s t-test. Data in A and B represent mean +/− standard deviation for 8 replicates per condition.

Figure 6. Influence of pharmacological angiogenesis inhibitors on iPSC-EC sprouting and viability

Quantified iPSC-EC sprouting given as # of invading Calcein+ cells (represented as black circles), and fraction viable iPSC-ECs given as blue squares with interconnected lines. iPSC-ECs were cultured in the presence of GM without inhibitor (DMSO control shown as lowest concentration in each figure) or with vatalanib (A), sunitinib malate (B), semaxinib (SU5416) (C), combretastatin A4 (D), withaferin A (E), thalidomide (F), SB-3CT (G), nilotinib (H), or temsirolimus (I) at the concentrations shown on the x-axis. Black asterisks (*) denote statistically significant difference in iPSC-EC invasion at each indicated level of inhibitor (α=0.05) using one-way ANOVA and Dunnett’s post-hoc test relative to lowest concentration (DMSO control) for each inhibitor, and blue asterisks (*) denote statistically significant difference in iPSC-EC viability at each indicated level of inhibitor relative to DMSO control at α=0.05 using one-way ANOVA and Dunnett’s post-hoc test. Data in A–I represent mean +/− standard deviation for 4 replicates per condition.