Figure 1. Integrin α6 interaction with LN-1 is required for differentiation of mESCs into Flk1⁺VE-cadherin⁺ECs.

(A) Cell surface expression of α6 and β1 integrin subunits in mESCs cultured on gelatin. Plot is representative of 3 separate experiments.

(B) Cell surface expression of β1 and β4 integrin subunits as determined by flow cytometry of mESCs cultured on gelatin. Plot is representative of 3 separate experiments.

(C) Culture of mESCs in the presence of integrin α6 blocking antibody GoH3 (50 µg/ml) did not affect adhesion to gelatin, whereas the antibody significantly reduced adhesion to LN-1. Error bars, mean ± S.E.M.; * P<0.05, n=3.

(D) mESCs were induced to differentiate in the presence of VEGF165 on Col-IV or LN-1. After 6 days, cell surface expression of EC markers Flk1 and VE-cadherin was determined by flow cytometry. LN-1-differentiated cells were more efficient in giving rise to double positive ECs (25% on LN-1 vs. 8% on Col-IV). Plot is representative of at least 5 separate experiments. Bar graph shows quantitation of Flk1⁺/VE-cadherin⁺ ECs grown on the two substrates. Error bars, mean ± S.E.M.; * P<0.05.

(E) Transfection of mESC with α6 siRNA led to significant impairment of cell proliferation when compared to scrambled siRNA control as determined by BrdU incorporation. Error bars, mean ± S.E.M.; * P<0.05, n=3.

(F) Flow cytometric analysis of Annexin V was used as indicator of apoptosis in mESC transfected with α6 siRNA and scrambled control. Knockdown of α6 resulted in enhancement of cell death as determined by Annexin V expression 96 hours after transfection. Plot is representative of 3 separate experiments.

(G) Impaired mesodermal differentiation of mESC treated with anti-α6 blocking antibody, as demonstrated by quantification of Flk1 transcript and flow cytometry for Flk1 expression three days after onset of mesodermal differentiation compared to non-blocked controls. Error bars, mean ± S.E.M.; * P<0.05, n=3. Representative plot of Flk1 cell surface expression is shown, (n=3).

(H) Use of anti-α6 blocking antibody resulted in reduced cell surface expression of EC markers Flk1 and VE-cadherin after 6 days of differentiation as determined by flow cytometry. Plot is representative of 3 separate experiments (n=3). Bar graph shows quantitation of Flk1⁺/VE-cadherin⁺ ECs differentiated from blocked mESC and non-blocked controls. Error bars, mean ± S.E.M.; * P<0.05, n=3.