Research ArticleDEVELOPMENTAL BIOLOGY

Compliant substratum guides endothelial commitment from human pluripotent stem cells

See allHide authors and affiliations

Science Advances  31 May 2017:
Vol. 3, no. 5, e1602883
DOI: 10.1126/sciadv.1602883
  • Fig. 1 PDMS substrates are permissive to hiPSC differentiation.

    (A) Representative images of water droplets on collagen IV–coated and uncoated PDMS and E ~ 3 GPa substrates for contact angle measurements. (B) Contact angle quantification across substrates. (C) FTIR analysis of substrates with and without collagen IV coating. a.u., arbitrary units. (D) Attachment efficiency across substrates. (E) Sparse seeding elicits varied YAP (green) localization and spreading [filamentous actin (F-actin) in gray] dependent on substrates with corresponding (F) quantification. All data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. At least three replicates were performed.

  • Fig. 2 YAP/β-catenin signaling as a function of substrate stiffness along mesoderm induction.

    (A) (i) Sample of image quantification of the ratio between nuclear and cytoplasmic YAP intensity. (ii) YAP nuclear-to-cytoplasmic (nuc/cyto) ratios on days 2, 4, and 6 of differentiation on the various substrates. NS, not significant. (B) (i) Representative immunofluorescence images (red, β-catenin; blue, nuclei) and (ii) quantification of the junctional-to-cytosolic (junc/cyto) ratio of β-catenin expression on days 2, 4, and 6 of differentiation on the various substrates. DAPI, 4′,6-diamidino-2-phenylindole. (C) Comparison between YAP (green) and β-catenin (red) localization as a function of time on the various substrates (comparison between relative YAP and β-catenin localization reported as *; changes in the relative YAP nuclear-to-cytoplasmic ratio across days of differentiation reported as #). Data are represented as means ± SEM. */#P < 0.05, **/##P < 0.01, ***/###P < 0.001, two-way analysis of variance (ANOVA). At least three biological replicates were performed.

  • Fig. 3 Stiffness-primed mesoderm induction in the presence of serum enhances EC differentiation.

    (A) Schematic of stiffness-primed mesoderm induction followed by EC differentiation on E ~ 3 GPa substrates. α-MEM, α-minimum essential medium; FBS, fetal bovine serum; EGM, endothelial growth medium. (B) Left: Gene expression of mesodermal markers for cells differentiated on soft 3-kPa substrates, normalized to expression from E ~ 3 GPa surfaces. Right: Gene expression analysis of cells differentiated on stiff 1.7-MPa substrates, normalized to expression from E ~ 3 GPa surfaces. Color key is presented in log10 scale. (C) Bright-field images of cobblestone endothelial colonies (white arrows) on day 12 EVCs. (D) Day 12 EVC flow cytometry plots of VECad expression in red, with corresponding HUVEC VECad expression in green. Black font, VECad+ cells; green font, highly expressing VECad+ cells. Data are presented as means ± SEM. (E) Representative immunofluorescence images of VECad expression on day 12 EVCs: Low-magnification (top) and high-magnification (bottom) images are shown (green, VECad; red, phalloidin; blue, nuclei). At least three biological replicates were performed.

  • Fig. 4 Stiffness-primed mesoderm induction in serum-free conditions results in robust EC differentiation.

    (A) Schematic of stiffness-primed mesoderm induction followed by EC differentiation on polystyrene plates. (B) Left: Gene expression of mesodermal markers for cells differentiated on soft 3-kPa substrates, normalized to expression from E ~ 3 GPa surfaces. Right: Gene expression analysis of cells differentiated on stiff 1.7-MPa substrates, normalized to expression from E ~ 3GPa surfaces. Color key is presented in log10 scale. (C) Bright-field images of cobblestone endothelial colonies (white arrows) on day 12 EVCs. (D) Representative day 12 EVC flow cytometry plots of VECad expression in red, with corresponding HUVEC VECad expression in green. (E) (i) Total VECad expression as a function of substrate stiffness. (ii) Percentage of VECad expression relative to HUVEC control samples. (F) Representative immunofluorescence images of day 12 EVC expression (top: green, eNOS; red, F-actin; blue, nuclei, bottom: green, vWF; red, CD31; blue, nuclei). Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, paired Student’s t test. At least three biological replicates were performed.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602883/DC1

    fig. S1. Development of compliant PDMS substrates.

    fig. S2. Differentiation and proliferation are supported on compliant silicone substrates.

    fig. S3. PDGFR-β expression from mesodermal differentiation in serum.

    fig. S4. Immunofluorescence microscopy of mature EC markers after mesoderm stiffness priming.

    fig. S5. Differentiation and proliferation are supported on compliant silicone substrates in serum-free conditions.

    fig. S6. PDGFR-β expression from mesodermal differentiation in serum-free conditions.

    fig. S7. Immunofluorescence microscopy of EC markers after chemically defined mesoderm stiffness priming.

    fig. S8. Immunofluorescence microscopy of EC markers after chemically defined mesoderm stiffness priming.

    table S1. Literature review of techniques to induce mesodermal specification from hiPSCs.

    table S2. Antibodies used in this study.

    References (41, 42)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Development of compliant PDMS substrates.
    • fig. S2. Differentiation and proliferation are supported on compliant silicone substrates.
    • fig. S3. PDGFR-β expression from mesodermal differentiation in serum.
    • fig. S4. Immunofluorescence microscopy of mature EC markers after mesoderm stiffness priming.
    • fig. S5. Differentiation and proliferation are supported on compliant silicone substrates in serum-free conditions.
    • fig. S6. PDGFR-β expression from mesodermal differentiation in serum-free conditions.
    • fig. S7. Immunofluorescence microscopy of EC markers after chemically defined mesoderm stiffness priming.
    • fig. S8. Immunofluorescence microscopy of EC markers after chemically defined mesoderm stiffness priming.
    • table S1. Literature review of techniques to induce mesodermal specification from hiPSCs.
    • table S2. Antibodies used in this study.
    • References (41, 42)

    Download PDF

    Files in this Data Supplement:

Navigate This Article