Figure 3: Mesp1-lineage is contingent upon BMP2/4, canonical Wnt and Nodal/Activin signals.

(A) BMP2/4 were the most potent growth factors in driving the appearance/expansion of Mesp1-CPCs cells. Of the eight growth factors surveyed, BMP2/4 were most potent in driving the appearance/expansion of the EYFP+ population, while Activin and Wnt3a were also effective though to a much less extent. FACS was performed for day 5 EBs receiving growth factor treatments from day 0 to 4. (B) Nodal/Activin inhibitor SB431542(SB) and canonical Wnt inhibitor IWR1 blocked the induction/expansion of Mesp1-CPCs. Cells cultured as EBs were exposed to the treatment as indicated, then the EFYP signals were captured on day 5 by FACS. While SB431542 and IWR1 effectively blocked the induction/expansion of the EFYP+ cells when given at all-time intervals, they were more potent at the later stage (day 2–4). (C) Mesp1-CPCs cells represent a specialized population out of the Flk1+/PDGFR1+. Cells from day 4 EBs were dissociated, stained for both Flk1 and PDGFR1, and analyzed by tri-color FACS. Left side: cells were first analyzed for Flk1 and PDGFR1 expression, next the double negative cells (Flk1-/PDGFR1-) or double positive cells (Flk1+/PDGFR1+) were analyzed for EFYP signal. EYFP+ cells are almost exclusively from Flk1+/PDGFR1+ cells. Right side: cells were first analyzed for EYFP signal; next the EYFP- or EYFP+ cells were analyzed for Flk1 and PDGFR1 signals. EYFP- cells were close to evenly distributed into Flk1-/PDGFR1- and Flk1+/PDGFR1+ cells (35.2% vs. 39.5%). EYFP+ cells were mainly Flk1+/PDGFR1+ (71.1%).