Dissecting the role of human embryonic stem cell-derived mesenchymal cells in human umbilical vein endothelial cell network stabilization in three-dimensional environments

Abstract

The microvasculature is principally composed of two cell types: endothelium and mural support cells. Multiple sources are available for human endothelial cells (ECs) but sources for human microvascular mural cells (MCs) are limited. We derived multipotent mesenchymal progenitor cells from human embryonic stem cells (hES-MC) that can function as an MC and stabilize human EC networks in three-dimensional (3D) collagen-fibronectin culture by paracrine mechanisms. Here, we have investigated the basis for hES-MC-mediated stabilization and identified the pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) as a putative hES-MC-derived regulator of EC network stabilization in 3D in vitro culture. Pharmacological inhibition of the HGF receptor (Met) (1 μm SU11274) inhibits EC network formation in the presence of hES-MC. hES-MC produce and release HGF while human umbilical vein endothelial cells (HUVEC) do not. When HUVEC are cultured alone the networks collapse, but in the presence of recombinant human HGF or conditioned media from human HGF-transduced cells significantly more networks persist. In addition, HUVEC transduced to constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked immunosorbent assay, the coculture media were enriched in both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), but at significantly different levels (Ang1=159±15 pg/mL vs. Ang2=30,867±2685 pg/mL) contributed by hES-MC and HUVEC, respectively. Although the coculture cells formed stabile network architectures, their morphology suggests the assembly of an immature plexus. When HUVEC and hES-MC were implanted subcutaneously in immune compromised Rag1 mice, hES-MC increased their contact with HUVEC along the axis of the vessel. This data suggests that HUVEC and hES-MC form an immature plexus mediated in part by HGF and angiopoietins that is capable of maturation under the correct environmental conditions (e.g., in vivo). Therefore, hES-MC can function as microvascular MCs and may be a useful cell source for testing EC-MC interactions.

FIG. 1.

Cell characterization. (A) Flow cytometry analysis of marker expression on human embryonic stem cells–mesenchymal cells (hES-MC). (B) hES-MC express HGF protein at a level of 5303±1022 pg/mL as detected by ELISA. (C) Flow cytometry analysis of Met expression on human umbilical vein endothelial cells (HUVEC) and hES-MC. HGF, hepatocyte growth factor. Color images available online at www.liebertpub.com/tea

FIG. 2.

Met blockade disrupts hES-MC-stabilized EC networks. (A) HUVEC and hES-MC coculture alone (Control, left) or treated with either dimethyl sulfoxide (DMSO) (middle) or Met inhibitor SU11274 at 1 μM (right). (B) Quantification of mean network length and one-way analysis of variance (ANOVA) indicated a significant effect of SU11274 (61±2 μm) treatment compared with DMSO control (658±200 μm) (*p=0.001, n=3; 4×, scale bar=250 μm). Color images available online at www.liebertpub.com/tea

FIG. 3.

Exogenous recombinant human HGF rescues EC networks. (A) HUVEC cultured in collagen-Fn gel were exposed to no treatment (left, 209±16 μm), 50 or 100 ng/mL of human HGF (middle, 453±56 μm and 382±23 μm, respectively), or 100 ng/mL mouse HGF (right, 191±12 μm). (B) Mean network length and one-way ANOVA indicate significant effect of treatment with human HGF on network formation (*p<0.05, n=3; 4×, scale bar=250 μm). EC, endothelial cell. Color images available online at www.liebertpub.com/tea

FIG. 4.

HGF-conditioned media stabilize EC networks. (A) Non-HGF-expressing HepG2 were stably transduced with empty (Empty) or human HGF (HGF) retrovirus and transcription was confirmed by polymerase chain reaction. (B) HepG2 transduced to constitutively express HGF produced 53,072 pg/mL as detected by ELISA. (C) HUVEC cultured in collagen-Fn gel were cultured in HepG2-CM-expressing empty vector (left, 164±4 μm), human HGF (middle, 503±48 μm), or non-CM (right). (D) Mean network length and one-way ANOVA indicate significant effect of conditioning media with HepG2 expressing human HGF on network formation (*p<0.001, n=3; 4×, scale bar=250 μm). Color images available online at www.liebertpub.com/tea

FIG. 5.

HUVEC autocrine expression of HGF stabilizes networks. (A) HUVEC were stably transduced with empty (left, 116±6 μm) or human HGF (right, 325±10 μm) and cultured in collagen-Fn gel. (B) HUVEC transduced to constitutively express HGF produced 81,680 pg/mL HGF as detected by ELISA. (C) Mean network length and one-way ANOVA indicate significant effect of HUVEC autocrine expression of human HGF on network formation (*p<0.001, n=3; 4×, scale bar=250 μm). Color images available online at www.liebertpub.com/tea

FIG. 6.

PI3K/Akt inhibitors disrupt hES-MC-stabilized EC networks. (A) HUVEC and hES-MC were cocultured in collagen-Fn gels and treated with the PI3K/Akt inhibitors Wortmannin (10 μM, top), LY294002 (50 μM, bottom), or equivalent volumes of DMSO (n=3; 4×, scale bar=250 μm). (B) HUVEC exposed to HGF showed phosphorylation of Akt that was blocked by addition of Wortmannin (10 μM) or LY294002 (50 μM). Total Akt and β-actin loading controls. Color images available online at www.liebertpub.com/tea

FIG. 7.

Angiopoietin1/2 expression by HUVEC and hES-MC. ELISA analysis for expression of (A) Ang1 and (B) Ang2 for EM control, conditioned EM from coculture of HUVEC and hES-MC, HUVEC alone, or hES-MC alone. EM registered background levels of 26±5 pg/mL and 622±101 pg/mL, while coculture (Co) measured 159±15 pg/mL and 30,867±2685 pg/mL, which were both significantly greater than EM control (*p=0.004 and 0.01). Conditioned media from HUVEC alone were not different compared with EM control for Ang1 (25±4 pg/mL) but showed significantly more expression of Ang2 than EM control (30,867±2685 pg/mL, ^#p=0.01). In contrast, conditioned media from hES-MC alone were significantly enriched in Ang1 (100±18 pg/mL, ^#p=0.02) but not in Ang2 (600±144 pg/mL). (C) HUVEC were seeded in collagen-Fn gels at 10⁶/mL and cultured in EM only (left), CM incubated with IgG-Fc (middle), or CM incubated with Tie2-Fc (right). (D) Mean network length quantification (±SEM) and one-way ANOVA indicate that exposure to hES-MC CM significantly increased network formation compared with EM control (*p>0.05), but no significant difference was detected by treatment with Tie2-Fc (494±58 μm) compared with IgG-Fc (505±59 μm) control (p=0.4) (HUVEC UEA-fluorescein, n=3; 10×, scale bar=200 μm). Color images available online at www.liebertpub.com/tea

FIG. 8.

hES-MC contact with EC networks in vivo. (A) HUVEC-DsRed and hES-MC-GFP (2×10⁶:0.4×10⁶/mL) were cocultured in GFR-Matrigel for 3 days in vitro before subcutaneous implantation in the back of Rag1 immune compromised mice for 1 week. Constructs were harvested and images were acquired by confocal microscopy and stack volume rendering. hES-MC integrate into EC networks that were formed in vivo (arrows). (B) Quantification of the percentage of direct cell contact with respect to HUVEC (cell overlap/HUVEC volume) and direct cell contact with respect to hES-MC (cell overlap/hES-MC volume) (40×, scale bar=50 μm). Color images available online at www.liebertpub.com/tea