Inverse agonism of RARs augments mesoderm induction. (A-H)
Representative images of reporter expression under separate and combined
treatment conditions for Wnt3a/CHIR99021, AGN193109, and Noggin after 2 days of
treatment (day 4 of differentiation). (A’-H’) FACS analyses of
Tbx6 and Brachyury reporter expressing
cells after 3 days of treatment (day 5 of differentiation) under the same
treatment combinations as shown in (A). RAR inhibition resulted in substantial
increases in the percentage of Tbx6+, T+, and
Tbx6+/T+ cells. Gates were set based on
undifferentiated control stem cells. (I) Gene expression analyses on day 5 for
paraxial mesoderm gene markers Tbx6, Msgn1, Meox1, and
Hoxb1 using indicated combinations of Wnt3a/CHIR99021,
AGN193109, and Noggin. Dramatic up- regulation of all paraxial gene markers was
observed when AGN193109 was added with Wnt3a/CHIR99021 with or without Noggin
relative to their respective individual treatments. Data shown as mean ±
SEM (n = 3, *p < .05, **p < .05,
where * and ** denotes the comparison of treatments between Wnt3a, CHIR99021 to
Wnt3a, CHIR99021 with AGN193109).