A genome-wide analysis of human pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels compared to standard 2D or 3D cell culture platforms. Homo sapiens

The influence of 2D and 3D cell culture platforms on vascular function was investigated by comparing gene expression for human pluripotent stem cell-derived endothelial cells (H1-ECs), primary human brain vascular pericytes (pericytes), and human umbilical vein endothelial cells (HUVECs) cultured on tissue culture polystyrene (TCP, “2D”), on or in poly(ethylene glycol) (PEG) hydrogels formed via “thiol-ene” photopolymerization, and on or in gelled Matrigel. ECs cocultured with pericytes in PEG formed vascular networks with global gene expression that was highly correlated to a standard 3D Matrigel assay (Spearman’s coefficients ≥ 0.98). H1-ECs, HUVECs, and pericytes were characterized gene expression signatures associated with the cell cycle and mitosis when cultured on TCP surfaces compared to cells cultured on top of or encapsulated in PEG hydrogels or Matrigel. The proliferative signature was not necessarily a function of the 2D format, since endothelial cells cultured on PEG hydrogels were not characterized by increased proliferation or a proliferative gene signature compared to cells encapsulated in PEG hydrogels. The proliferative phenotype for H1-ECs on TCP was regulated by FAK-ERK activity, and inhibition or knockdown of ERK pathway signaling decreased proliferation and cell cycle genes while increasing expression of “3D-like” vasculature development genes. Our results suggest that cells in 2D culture adopt a highly proliferative state that interferes with normal vascular function and provides unique insight into the importance of cellular and extracellular context for in vitro tissue modeling. Overall design: 30 samples for RNA-Seq Experiment #1 ("RNAseq1": H1 human embryonic stem cells, “H1-ESC-1”; H1 ES cells-derived endothelial cells, "H1-EC1"; HUVECs, "HUV1"; pericytes, "PC"; and H1-EC1 cocultured with PC, H1-EC1-PC cocultures). Poly(ethylene glycol) (PEG) hydrogels : 4 million endothelial cells/mL (H1-EC1 or HUVECs) and/or 2 million pericytes/mL were encapsulated in 30 µL PEG hydrogels in the bottom of 24-well transwell inserts . Matrigel (MG): 2 million endothelial cells/mL and 1 million pericytes/mL were encapsulated in 30 µL gelled Matrigel (4.5 mg/mL). Endothelial cells, pericytes, and cocultures encapsulated in PEG or Matrigel were maintained under hypoxic conditions (1.5% O2) in E7V medium supplemented with 1% bovine serum albumin. H1-ECs were also seeded on gelled Matrigel (4.5 mg/mL, “2D on Matrigel”) at a density of 60,000 cells/cm2 and incubated for one day in E7V medium with 1% BSA under normoxic conditions. Tissue culture polystyrene (TCP): TCP surfaces were coated with Matrigel at a dilute concentration that does not form a gel (H1-ES cells), recombinant vitronectin (H1-ECs or HUVECs), or poly-l-lysine (pericytes, as per manufacturer’s instructions) and cultured under normoxic conditions. 18 samples for RNA-Seq Experiment #2 ("RNAseq2": H1-ECs, “H1-EC2”; or HUVECs “HUV2”; cultured using PEG hydrogels, gelled Matrigel (6.4 mg/mL), or on Matrigel-coated TCP surfaces. For 3D culture, 4 million endothelial cells/mL were encapsulated in 30 µL PEG hydrogels (50% crosslinking, 2 mM CRGDS) or gelled Matrigel formed in single wells of 24-well TCP plates. For 2D culture, 200,000 endothelial cells per well of a 12-well TCP plates were seeded on Matrigel-coated surfaces (“MG-TCP”) or on 300 µL PEG hydrogels (50% crosslinking, 2 mM CRGDS) or gelled Matrigel formed in the bottom of the well. H1-ES cells were included as a control for both experiments. Duplicate samples are provided for each condition.